Butyl bile esterase inhibitor activity detection kit

Instructions for Butyl Cholesterol Esterase Inhibition Activity Test Kit

Visible spectrophotometry

Item No.:BC5980

Specifications:50T/48S

Product composition: Before use, please carefully check whether the volume of the reagent matches the volume inside the bottle. If you have any questions, please contact the staff of Solaibao in a timely manner.

Preparation of solution:

1. Reagent 1: Take 1 vial of reagent 1 before use and add it1.8mLDistilled water, fully dissolved, unused reagents-20℃Can be stored after packaging4Zhou, avoid repeated freeze-thaw cycles (this reagent is a freeze-dried reagent, and there may be significant differences or even small amounts of reagent observed by the naked eye between different bottles, which does not affect its use and has the same actual quality).

2. Reagent 4: Add 17mL of reagent 2 before use, dissolve thoroughly, and use up any remaining reagents-20℃Can be packaged and stored separately4Zhou, avoid repeated freezing and thawing.

3. Reagent 5: 10mmol/L rivastigmine solution. Before use, take 15 μ L of 10mmol/L rivastigmine solution and add it to 985μLDistilled water, prepared into0.15Mmol/L rivastigmine solution, prepared on site (this reagent is used for positive tube experiments and is selected).

Product Description:

Butylcholinesterase,BchE，EC3.1.1.8）Plasma bile esterase, also known as pseudo bile esterase, is a type of serine acid hydrolase that is synthesized by the liver and enters the bloodstream. It is present in almost all animal tissues. Acetyl bile esterase（AchE）Compared to,BchEIt can effectively hydrolyze larger bile esters such as butyryl bile and benzoyl bile, and can also eliminate the toxic effects of nerve agents such as organophosphate pesticides and carbamate pesticides. Studies have shown that,BchECan serve as an important target for the treatment of Alzheimer's disease,BchEInhibitors are used to improve symptoms such as memory loss and cognitive impairment in Alzheimer's disease patients.

BchE catalyzes the hydrolysis of butyryl bile to produce bile, which reacts with dithionitrobenzoic acid (DTNB) to form bile5-sulfhydryl-Nitrobenzoic acid（TNB），BchEInhibitors inhibit by suppressingBchEActivity reduces the hydrolysis of butyryl bile *.TNBin412nmThere is an absorption peak at this location, which can be measured by412nmCalculate the activity of BchE inhibitors based on changes in absorbance.

Attention: It is recommended to select 2-3 samples with significant expected differences for pre experiments before the experiment.

Equipment and supplies to be provided:

Visible spectrophotometer, balance, oven, pulverizer/mortar/Homogenizer30-50Mesh sieve, ultrasonic cleaner, centrifuge, water bath pot/Constant temperature incubator1mLGlass colorimetric dish, adjustable pipette, ice and distilled water.

Operation steps: Please refer to the "Product Information" document for specific operation steps

Notes:

To ensure the accuracy and stability of the experimental results, please strictly control the reaction time and operation time.

2. Sample absorbance A '_{determination}Greater than 1.5 orΔA_{determination}approachΔA_blankIt is recommended to increase the proportion of tissue samples in the sample processing step or prepare powder samples into a higher concentration solution before conducting the measurement. WhenΔA_{determination}When it is less than 0.01, the sample can be diluted with extraction solution before measurement.

If used to compare the degree of inhibition of BchE by different reagents, extracts, drugs, or tissues, the reagents, extracts, drugs, or tissue homogenates must be prepared at the same concentration for comparison.

Experimental Example:

1. Take 0.1005g grapefruit peel sample (dried) and add it1mLUltrasonic extraction is performed on the extraction solution, and the supernatant is taken after centrifugation. Follow the measurement steps and use1mLCalculation of glass colorimetric dish measurement:ΔA_blank=（A’_{Blank 1}-A_{Blank 1}）-（A’_{Blank 2}-A_{Blank 2}）=（1.372-0.205）-（0.166-0.112）=1.113，ΔA_{determination}=（A’_{determination}-A_{determination}）-（A’_{Blank 2}-A_{Blank 2}）=（1.117-0.485）-（0.166-0.112）=0.578Calculate the inhibition rate as follows:

Inhibition rate of BchE inhibitor（%）=（1.113-0.578）÷1.113×100%=48.068%.

2. Take 0.1014g persimmon peel sample (dried) and add it1mLExtract the solution by ultrasonic extraction, centrifuge and collect the supernatant, dilute it16After doubling, follow the measurement steps and use1mLCalculation of glass colorimetric dish measurement:ΔA_blank=（A’_{Blank 1}-A_{Blank 1}）-（A’_{Blank 2}-A_{Blank 2}）=（1.372-0.205）-（0.166-0.112）=1.113，ΔA_{determination}=（A’_{determination}-A_{determination}）-（A’_{Blank 2}-A_{Blank 2}）=（0.706-0.149）-（0.166-0.112）=0.503Calculate the inhibition rate as follows:

Inhibition rate of BchE inhibitor（%）=（1.113-0.503）÷1.113×100%=54.807%.

3. Take 50 μ L of 0.15mmol/L rivastigmine solution, follow the measurement steps, and use1mLCalculation of glass colorimetric dish measurement:ΔA_blank=（A’_{Blank 1}-A_{Blank 1}）-（A’_{Blank 2}-A_{Blank 2}）=（1.372-0.205）-（0.166-0.112）=1.113，ΔA_{determination}=（A’_{determination}-A_{determination}）-（A’_{Blank 2}-A_{Blank 2}）=（0.838-0.171）-（0.166-0.112）=0.613Calculate the inhibition rate as follows:

Inhibition rate of BchE inhibitor（%）=（1.113-0.613）÷1.113×100%=44.924%.

References:

[1] Ellman GL, Courtney KD, Andres V Jr. et al. A new and rapid colorimetric determination of acetylcholinesterase activity [J]. Biochemical Pharmacology, 1961, 7(2): 88-95.

[2] Noor Atatreh, Sara Al Rawashdah, Shaikha S Al Neyadi. et al. Discovery of new butyrylcholinesterase inhibitors via structure-based virtual screening [J]. Journal of Enzyme Inhibition and Medicinal Chemistry, 2019, 34(1): 1373-1379.

[3] Li Q, Chen Y, Xing S. et al. Highly Potent and Selective Butyrylcholinesterase Inhibitors for Cognitive Improvement and Neuroprotection [J]. Journal of Medicinal Chemistry, 2021, 64(10): 6856-6876.

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