Anti-acetylcholinesterase active composition in Gongluomu and its screening method and application

A technology of acetylcholinesterase and screening method, which is applied in the field of medicine, can solve the problems of indeterminate conclusion, high cost, complicated operation, etc., and achieve the effect of developing drugs for the treatment of AD

Active Publication Date: 2021-07-27
SHENZHEN INST FOR DRUG CONTROL SHENZHEN TESTING CENT OF MEDICAL DEVICES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, whether the medicinal effects of genuine gongluomu and gongluomu substitutes are the same, there is no definite conclusion
[0005] Preliminary studies have shown that the extract of Gongluomu has a good activity of inhibiting acetylcholinesterase (AChE), but the specific types of active substances need to be further studied
Due to the complex and diverse components of Gongluomu extract, it greatly increases the difficulty of screening and research of active substances
At present, the methods for studying the active substances of traditional Chinese medicine mainly include tracking separation method, ultrafiltration method and magnetic microsphere fishing method. Although these methods can effectively find active ingredients, they have defects such as long cycle, high cost, complicated operation, and low degree of automation.

Method used

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  • Anti-acetylcholinesterase active composition in Gongluomu and its screening method and application
  • Anti-acetylcholinesterase active composition in Gongluomu and its screening method and application
  • Anti-acetylcholinesterase active composition in Gongluomu and its screening method and application

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0064] The preparation of embodiment 1 acetylcholinesterase reactor

[0065] (1) Capillary pretreatment

[0066] Take a quartz capillary with a length of about 2 m and an inner diameter of 400 μm, wash it with NaOH (1moL / L) solution for 20 min; seal both ends of the capillary filled with NaOH with rubber stoppers, and react in a water bath at 100°C for 2 h; take out the capillary, Rinse the capillary with distilled water until the effluent is neutral (pH test paper test), rinse the capillary with MeOH for 30 min; dry with nitrogen for 2 h, then perfuse the mixture of γ-MAPS / MeOH (50 / 50, v / v) until it is dry The capillary was sealed with a rubber stopper and placed in a water bath at 60°C for 12 h; the capillary was rinsed with methanol and distilled water for 30 min each, and finally the capillary was fully dried with nitrogen.

[0067] As shown in Figure 1(A), by activating the inner wall of the quartz capillary, alkenyl groups are modified on the inner wall of the capillary...

Embodiment 2

[0077] The characterization of embodiment 2 acetylcholinesterase reactor

[0078] (1) Infrared spectrum characterization

[0079] The AChE, poly(GMA-co-EDMA) monolithic matrix material without immobilized enzyme and the AChE reactor AChE-ICER were characterized by infrared spectrometer.

[0080] Such as figure 2 As shown, 1725cm -1 Stretching vibration absorption for poly(GMA-co-EDAM) integral matrix C=O, 1544cm -1 For the bending vibration of the N-H bond of the peptide bond, 1666cm -1 For the C=O stretching vibration absorption in the peptide bond, FT-IR results show that AChE has been successfully modified on the poly(GMA-co-EDAM) monolithic matrix carrier.

[0081] (2) SEM characterization

[0082] In this example, an Ultra-55 field emission scanning electron microscope was used to visually characterize the morphology and structure of the poly(GMA-co-EDMA) monolithic matrix material and the cross-section of the AChE reactor AChE-ICER.

[0083] As shown in Figure 3(A)...

Embodiment 3

[0085] Example 3 Ligand Fishing Method

[0086] (1) Preparation of extract

[0087] Accurately weigh 2.0g of gonglao wood powder, place it in a stoppered conical flask, add 50mL of hydrochloric acid-methanol (1:100, v / v) mixed solution, seal the weight and then cold soak for 30min, ultrasonic treatment (power 300W, frequency 40kHz) 60min, vacuum rotary steaming until dry extract is obtained;

[0088] Accurately weigh 15 mg of Gonglao Mugan extract, place it in a 5 mL volumetric flask, add ammonium acetate buffer (10 mM, pH=7.4) to dissolve to the scale, and make the concentration 3 mg / mL, and carry out ligand fishing analysis;

[0089] (2) Elution method of functional AChE-ICER monolithic column:

[0090] The first step: use the mobile phase ammonium acetate buffer (10mM, pH=7.4) to provide it through a peristaltic pump, the flow rate is 5 μL / min, the sample injection volume is 100 μL, and last for 40 minutes to collect fractions;

[0091] Step 2: Change the mobile phase fr...

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Abstract

The present invention provides anti-acetylcholinesterase active composition and its screening method and application in Gongluomu. Object, based on the principle of affinity chromatography, combined with immobilized enzyme technology, prepared an acetylcholinesterase reactor, constructed an active compound fishing platform, successfully screened and verified 4 kinds of anti-acetylcholinesterase active ingredients, and the anti-acetylcholinesterase of Gongluomu extract The discovery of active substances provides an efficient screening method. When the compound Tetrandrine and Jatrorrhizine are fixed, other compounds are added to mix with it. When the concentration of Tetrandrine or Jatrorrhizine is lower than the IC 60 or IC 70 When there is an obvious synergistic or additive effect, this study first discovered and verified the anti-AChE activity of the compound components in the Gonggongo wood extract, and the obtained combination has a synergistic or additive effect, which can undoubtedly be developed into a potential anti-AChE. Inhibitor.

Description

technical field [0001] The invention belongs to the technical field of medicine, and relates to a composition with anti-acetylcholinesterase activity in gonglaomu and its screening method and application. Background technique [0002] Alzheimer's disease (AD) is a primary degenerative brain disease of unknown etiology, which has brought a huge burden to society and families and has become a global health problem. Acetylcholinesterase inhibitors, as the only AD drug approved by the US FDA, have a certain therapeutic effect, but the action time is short and often accompanied by side effects. In recent years, traditional Chinese medicine has shown good prospects in the prevention and treatment of AD. It has many potential advantages such as low toxicity and side effects, and overall effect. treatment is particularly important. [0003] Gonggongmu is recorded as its name, and its source is the dried stems of Mahonia bealei (Fort.) Carr. and Mahoniafortunei (Lindl.) Fedde., whi...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K31/4375A61P25/28G01N30/02
CPCA61K31/4375A61P25/28G01N30/02A61K2300/00G01N2030/8813A61K36/29A61K31/4704A61K31/4745C12Q1/46C12Y301/01007G01N33/6896G01N2800/2821G01N2560/00G01N33/573
Inventor 王铁杰黄洋王珏殷果江坤江正瑾
Owner SHENZHEN INST FOR DRUG CONTROL SHENZHEN TESTING CENT OF MEDICAL DEVICES
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