CN113376287A - Method for detecting activity of cyclic nucleotide phosphodiesterase inhibitor based on high performance liquid chromatography - Google Patents
Method for detecting activity of cyclic nucleotide phosphodiesterase inhibitor based on high performance liquid chromatography Download PDFInfo
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Abstract
The invention relates to a method for detecting the activity of a cyclic nucleotide phosphodiesterase inhibitor based on high performance liquid chromatography, which is characterized in that the activity of the nucleotide phosphodiesterase inhibitor is rapidly detected by means of the existing HPLC technology, cyclic nucleotide is used as a reaction substrate, the cyclic nucleotide is decomposed into GMP after being catalyzed by the cyclic nucleotide phosphodiesterase, the GMP is further metabolized into nucleoside and base, and the cyclic nucleotide can be detected by ultraviolet absorption light (254 nm). During detection, the residual amount of the cyclic nucleotide can be judged by the peak area generated by the reaction substrate cyclic nucleotide, and the activity of the nucleotide phosphodiesterase inhibitor can be calculated.
Description
Technical Field
The invention relates to the technical field of bioanalytical chemistry, in particular to a method for detecting activity of a cyclic nucleotide phosphodiesterase inhibitor by HPLC.
Background
The working principle of High Performance Liquid Chromatography (HPLC) is that each component of a mixture is separated and purified in a chromatographic column through the interaction between each component and a stationary phase and a mobile phase.
The stationary phase is a very small porous particulate material present in the column and the mobile phase is a solvent or mixture of solvents forced through the column under high pressure. The sample is injected by a syringe, mixed with the mobile phase through a valve connected to the sample circuit, and then the components in the sample pass through the column at different rates, which in turn flow out of the column due to the different adsorption capacities between the components and the stationary phase, and the component concentrations are converted into electrical signals by a suitable detector and transmitted to the HPLC software of the computer. After the run was completed, the chromatogram was obtained in the HPLC software.
Cyclic nucleotide Phosphodiesterase (PDE) is an enzyme that hydrolyzes cyclic guanylic acid (cGMP), of which PDE5a1 is a subtype.
Cyclic nucleotide (cGMP) is an important active substance widely existing in the body. The cyclic nucleotides are important second messengers in the human body and participate in various signal pathways of the body. The cyclic nucleotide can regulate energy metabolism, can regulate neurotransmitter transmission, gene expression, hormone regulation, immune response, cell proliferation and differentiation and the like, and has regulation and control effects on vasodilation of blood vessels, neurotransmitter transmission and kidney functions.
Disclosure of Invention
The invention aims to provide a method for detecting the activity of a cyclic nucleotide phosphodiesterase inhibitor based on high performance liquid chromatography, and solve the problem that the semen cassiae extract is difficult to distinguish.
The technical scheme adopted by the invention for solving the technical problems is as follows: a method for detecting the activity of a cyclic nucleotide phosphodiesterase inhibitor based on high performance liquid chromatography comprises the following steps:
step one, dissolving cyclic nucleotide in Tris-HCl buffer solution to obtain cyclic nucleotide solution;
dissolving cyclic nucleotide phosphodiesterase in a Tris-HCl buffer solution to obtain a cyclic nucleotide phosphodiesterase solution, incubating in a water bath at 35 ℃ for 3-10 min in advance, and uniformly mixing with cyclic nucleotide phosphodiesterase inhibitors with different concentrations to obtain a mixed solution;
step three, pre-incubating the cyclic nucleotide solution in the step one in a water bath at 35 ℃ for 3-10 min, and then adding the mixed solution in the step two into the cyclic nucleotide solution to form a reaction system;
step four, incubating the reaction system in the step three in a water bath at 35 ℃, and then heating in a water bath at 100 ℃ to obtain a final solution;
and step five, injecting the final solution into a high performance liquid chromatograph, and detecting the concentration of the cyclic nucleotide, thereby calculating the activity of the cyclic nucleotide phosphodiesterase inhibitor.
Preferably, the Tris-HCl buffer solution has a concentration of 40-60mM and a pH of 8.
Preferably, the cyclic nucleotide phosphodiesterase inhibitor is sildenafil.
Preferably, the reaction system comprises a cyclic nucleotide phosphodiesterase solution with the concentration of 0.01-0.5 mug/mL, a sildenafil solution with the concentration of 0-750 nM and a cyclic nucleotide solution with the final concentration of 0.5-150 mug/mL.
Preferably, in the fifth step, the high performance liquid chromatograph uses a Kromasil & C18 chromatographic column, 4.6mm × 250mm, and the particle size is 5 μm.
Preferably, in the fifth step, the mobile phase used is 90% buffer solution and 10% methanol.
Preferably, the buffer solution is 6.8mg/mL potassium dihydrogen phosphate in water.
Preferably, the flow rate of the mobile phase in the high performance liquid chromatography apparatus is 1 ml/min.
The invention has the following beneficial effects: the method adopts cyclic nucleotide as a reaction substrate, the cyclic nucleotide is decomposed into GMP after being catalyzed by cyclic nucleotide phosphodiesterase, the GMP is further metabolized into nucleoside and base, and the cyclic nucleotide can be detected by ultraviolet absorption light (254 nm).
The addition of the cyclic nucleotide phosphodiesterase inhibitor can effectively inhibit the activity of the cyclic nucleotide phosphodiesterase, the content of the cyclic nucleotide phosphodiesterase inhibitor can cause the change of the inhibition efficiency, the content change of the cyclic nucleotide can be obtained by analyzing the peak area change of the cyclic nucleotide in a reaction substrate through high performance liquid chromatography, the inhibition efficiency of the inhibitor is calculated, the traditional serum detection method is simplified, and the method has the advantages of simplicity, convenience, rapidness and low cost.
Drawings
FIG. 1 is a graph of cyclic nucleotide concentration versus peak area size;
FIG. 2 is a graph of the inhibition efficiency of sildenafil on cyclic nucleotide phosphodiesterase in example 1;
FIG. 3 is a graph of the inhibition efficiency of the assay of epimedin A on cyclic nucleotide phosphodiesterase in example 2;
FIG. 4 is a graph of the inhibition efficiency of the assay of epimedin B on cyclic nucleotide phosphodiesterase in example 3;
FIG. 5 is a graph of the inhibition efficiency of the assay of epimedin C on cyclic nucleotide phosphodiesterase in example 4;
FIG. 6 is a graph of the inhibition efficiency of baohuoside I on cyclic nucleotide phosphodiesterase in example 5;
FIG. 7 is a graph of the inhibition efficiency of cyclic nucleotide phosphodiesterase by icariin detected in example 6;
FIG. 8 is a graph of the inhibition efficiency of detecting 2 "-O-rhamnosyl icariside against cyclic nucleotide phosphodiesterase in example 7.
Detailed Description
Example 1
A method for detecting the activity of a cyclic nucleotide phosphodiesterase inhibitor based on high performance liquid chromatography comprises the following steps:
step one, 8 centrifuge tubes of 0.5mL were taken, and a cyclic nucleotide solution of 7.5 μ g/mL was added thereto, and the cyclic nucleotide was dissolved in a Tris-HCl buffer solution (100mM MgCl) of 50mMPH 82) Diluting, pre-dilutingFirstly, incubating in 35 ℃ water bath for 10 min;
step two, mixing cyclic nucleotide phosphodiesterase with the final concentration of 0.0625 mu g/mL and sildenafil solutions with different concentrations to obtain eight groups of different mixed solutions, wherein the final concentration of sildenafil is 0, 0.183105, 0.732422, 2.929688, 11.71875, 46.875, 187.5 and 750nM, and the total reaction volume is 60 mu L;
step three, respectively adding the mixed solution in the step two into the 8 centrifugal tubes in the step one to form a reaction system;
step four, incubating the reaction system in the step three in a 35 ℃ water bath for 90min, and then heating in a 100 ℃ water bath for 2min to obtain a final solution;
injecting the final solution into a high performance liquid chromatograph, and detecting cyclic nucleotide to obtain the total peak area of the cyclic nucleotide in the final solution;
the liquid chromatographic analysis conditions were:
a chromatographic column: kromasil & C18 column, 4.6mm × 250mm, particle size 5 μm;
column temperature: 30 ℃;
mobile phase: volume ratio: the concentration of the buffer solution is 90 percent, the concentration of the methanol is 10 percent, wherein the buffer solution is potassium dihydrogen phosphate aqueous solution with the concentration of 6.8 mg/mL;
flow rate: 1 mL/min;
sample loading amount: the final solution was 50. mu.L.
Preparing cyclic nucleotide solutions with different concentrations, drawing chromatograms of the solutions by using a high performance liquid chromatograph, recording peak areas of the cyclic nucleotides, and finally obtaining a relation curve of the concentration of the cyclic nucleotides and the peak areas as shown in figure 1.
The inhibition efficiency of sildenafil with different concentrations can be converted by comparing the cyclic nucleotide peak area in the final solution with that in fig. 1, and the final result is shown in fig. 2.
Example 2
A method for detecting the activity of a cyclic nucleotide phosphodiesterase inhibitor based on high performance liquid chromatography comprises the following steps:
step one, taking 7 centrifuge tubes with the volume of 0.5mL, and respectively adding cyclic nucleoside with the concentration of 7.5 mu g/mLAcid solution, cyclic nucleotide using 50mMPH ═ 8 Tris-HCl buffer solution (100mM MgCl)2) Diluting, and incubating in 35 deg.C water bath for 10 min;
step two, mixing epimedin A solutions with different concentrations of cyclic nucleotide phosphodiesterase, the final concentration of which is 0.0625 mu g/mL, to obtain seven groups of different mixed solutions, wherein the final concentration of the epimedin A is 0.002637, 0.010547, 0.042188, 0.16875, 0.675, 2.7 and 10.8mg/mL, and the total reaction volume is 60 mu L;
step three, respectively adding the mixed solution in the step two into the 7 centrifugal tubes in the step one to form a reaction system;
step four, incubating the reaction system in the step three in a 35 ℃ water bath for 90min, and then heating in a 100 ℃ water bath for 2min to obtain a final solution;
injecting the final solution into a high performance liquid chromatograph, and detecting cyclic nucleotide to obtain the total peak area of the cyclic nucleotide in the final solution;
the liquid chromatographic analysis conditions were:
a chromatographic column: kromasil & C18 column, 4.6mm × 250mm, particle size 5 μm;
column temperature: 30 ℃;
mobile phase: volume ratio: the concentration of the buffer solution is 90 percent, the concentration of the methanol is 10 percent, wherein the buffer solution is potassium dihydrogen phosphate aqueous solution with the concentration of 6.8 mg/mL;
flow rate: 1 mL/min;
sample loading amount: the final solution was 50. mu.L.
The inhibition efficiency of epimedin A with different concentrations can be converted by comparing the cyclic nucleotide peak area in the final solution with that in figure 1, and the final result is shown in figure 3.
Example 3
A method for detecting the activity of a cyclic nucleotide phosphodiesterase inhibitor based on high performance liquid chromatography comprises the following steps:
step one, taking 7 0.5mL centrifuge tubes, adding cyclic nucleotide solution with concentration of 7.5 mug/mL, and using Tris-HCl buffer solution (100mM MgCl) with concentration of 50mMPH 8 for cyclic nucleotide2) Diluting, pre-incubating in 35 deg.C water bath10min；
Step two, mixing cyclic nucleotide phosphodiesterase with final concentration of 0.0625 mu g/mL and epimedin B solutions with different concentrations to obtain seven groups of different mixed solutions, wherein the final concentration of the epimedin B is 0.000903, 0.003613, 0.014453, 0.057813, 0.23125, 0.925 and 3.7mg/mL, and the total reaction volume is 60 mu L;
step three, respectively adding the mixed solution in the step two into the 7 centrifugal tubes in the step one to form a reaction system;
step four, incubating the reaction system in the step three in a 35 ℃ water bath for 90min, and then heating in a 100 ℃ water bath for 2min to obtain a final solution;
injecting the final solution into a high performance liquid chromatograph, and detecting cyclic nucleotide to obtain the total peak area of the cyclic nucleotide in the final solution;
the liquid chromatographic analysis conditions were:
a chromatographic column: kromasil & C18 column, 4.6mm × 250mm, particle size 5 μm;
column temperature: 30 ℃;
mobile phase: volume ratio: the concentration of the buffer solution is 90 percent, the concentration of the methanol is 10 percent, wherein the buffer solution is potassium dihydrogen phosphate aqueous solution with the concentration of 6.8 mg/mL;
flow rate: 1 mL/min;
sample loading amount: the final solution was 50. mu.L.
The inhibition efficiency of epimedin B with different concentrations can be converted by comparing the cyclic nucleotide peak area in the final solution with that in figure 1, and the final result is shown in figure 4.
Example 4
A method for detecting the activity of a cyclic nucleotide phosphodiesterase inhibitor based on high performance liquid chromatography comprises the following steps:
step one, taking 7 0.5mL centrifuge tubes, adding cyclic nucleotide solution with concentration of 7.5 mug/mL, and using Tris-HCl buffer solution (100mM MgCl) with concentration of 50mMPH 8 for cyclic nucleotide2) Diluting, and incubating in 35 deg.C water bath for 10 min;
step two, mixing cyclic nucleotide phosphodiesterase with final concentration of 0.0625 mu g/mL and epimedin C solutions with different concentrations to obtain seven groups of different mixed solutions, wherein the final concentration of epimedin C is 0.000891, 0.003564, 0.014258, 0.057031, 0.228125, 0.9125 and 3.65mg/mL, and the total reaction volume is 60 mu L;
step three, respectively adding the mixed solution in the step two into the 7 centrifugal tubes in the step one to form a reaction system;
step four, incubating the reaction system in the step three in a 35 ℃ water bath for 90min, and then heating in a 100 ℃ water bath for 2min to obtain a final solution;
injecting the final solution into a high performance liquid chromatograph, and detecting cyclic nucleotide to obtain the total peak area of the cyclic nucleotide in the final solution;
the liquid chromatographic analysis conditions were:
a chromatographic column: kromasil & C18 column, 4.6mm × 250mm, particle size 5 μm;
column temperature: 30 ℃;
mobile phase: volume ratio: the concentration of the buffer solution is 90 percent, the concentration of the methanol is 10 percent, wherein the buffer solution is potassium dihydrogen phosphate aqueous solution with the concentration of 6.8 mg/mL;
flow rate: 1 mL/min;
sample loading amount: the final solution was 50. mu.L.
The inhibition efficiency of epimedin C with different concentrations can be converted by comparing the cyclic nucleotide peak area in the final solution with that in figure 1, and the final result is shown in figure 5.
Example 5
A method for detecting the activity of a cyclic nucleotide phosphodiesterase inhibitor based on high performance liquid chromatography comprises the following steps:
step one, taking 7 0.5mL centrifuge tubes, adding cyclic nucleotide solution with concentration of 7.5 mug/mL, and using Tris-HCl buffer solution (100mM MgCl) with concentration of 50mMPH 8 for cyclic nucleotide2) Diluting, and incubating in 35 deg.C water bath for 10 min;
step two, mixing cyclic nucleotide phosphodiesterase with the final concentration of 0.0625 mu g/mL and baohuoside I solutions with different concentrations to obtain seven groups of different mixed solutions, wherein the final concentration of baohuoside I is 0.000393, 0.001572, 0.006289, 0.025156, 0.100625, 0.4025 and 1.61mg/mL, and the total reaction volume is 60 mu L;
step three, respectively adding the mixed solution in the step two into the 7 centrifugal tubes in the step one to form a reaction system;
step four, incubating the reaction system in the step three in a 35 ℃ water bath for 90min, and then heating in a 100 ℃ water bath for 2min to obtain a final solution;
injecting the final solution into a high performance liquid chromatograph, and detecting cyclic nucleotide to obtain the total peak area of the cyclic nucleotide in the final solution;
the liquid chromatographic analysis conditions were:
a chromatographic column: kromasil & C18 column, 4.6mm × 250mm, particle size 5 μm;
column temperature: 30 ℃;
mobile phase: volume ratio: the concentration of the buffer solution is 90 percent, the concentration of the methanol is 10 percent, wherein the buffer solution is potassium dihydrogen phosphate aqueous solution with the concentration of 6.8 mg/mL;
flow rate: 1 mL/min;
sample loading amount: the final solution was 50. mu.L.
The inhibition efficiency of baohuoside I with different concentrations can be converted by comparing the cyclic nucleotide peak area in the final solution with that in figure 1, and the final result is shown in figure 6.
Example 6
A method for detecting the activity of a cyclic nucleotide phosphodiesterase inhibitor based on high performance liquid chromatography comprises the following steps:
step one, taking 7 0.5mL centrifuge tubes, adding cyclic nucleotide solution with concentration of 7.5 mug/mL, and using Tris-HCl buffer solution (100mM MgCl) with concentration of 50mMPH 8 for cyclic nucleotide2) Diluting, and incubating in 35 deg.C water bath for 10 min;
step two, mixing cyclic nucleotide phosphodiesterase with the final concentration of 0.0625 mu g/mL and icariin solutions with different concentrations to obtain seven groups of different mixed solutions, wherein the final concentration of icariin is 0.00003, 0.000121, 0.000484, 0.01938, 0.00775, 0.031 and 0.124mg/mL, and the total reaction volume is 60 mu L;
step three, respectively adding the mixed solution in the step two into the 7 centrifugal tubes in the step one to form a reaction system;
step four, incubating the reaction system in the step three in a 35 ℃ water bath for 90min, and then heating in a 100 ℃ water bath for 2min to obtain a final solution;
injecting the final solution into a high performance liquid chromatograph, and detecting cyclic nucleotide to obtain the total peak area of the cyclic nucleotide in the final solution;
the liquid chromatographic analysis conditions were:
a chromatographic column: kromasil & C18 column, 4.6mm × 250mm, particle size 5 μm;
column temperature: 30 ℃;
mobile phase: volume ratio: the concentration of the buffer solution is 90 percent, the concentration of the methanol is 10 percent, wherein the buffer solution is potassium dihydrogen phosphate aqueous solution with the concentration of 6.8 mg/mL;
flow rate: 1 mL/min;
sample loading amount: the final solution was 50. mu.L.
Comparing the cyclic nucleotide peak area in the final solution with that in FIG. 1, the inhibitory efficiency of icariin with different concentrations can be converted, and the final result is shown in FIG. 7.
Example 7
A method for detecting the activity of a cyclic nucleotide phosphodiesterase inhibitor based on high performance liquid chromatography comprises the following steps:
step one, taking 7 0.5mL centrifuge tubes, adding cyclic nucleotide solution with concentration of 7.5 mug/mL, and using Tris-HCl buffer solution (100mM MgCl) with concentration of 50mMPH 8 for cyclic nucleotide2) Diluting, and incubating in 35 deg.C water bath for 10 min;
step two, mixing cyclic nucleotide phosphodiesterase with the final concentration of 0.0625 mu g/mL and 2 '-O-rhamnosyl icariin solutions with different concentrations to obtain seven groups of different mixed solutions, wherein the final concentration of the 2' -O-rhamnosyl icariin is 0.001538, 0.006152, 0.024609, 0.098438, 0.39375, 1.575 and 6.3mg/mL, and the total reaction volume is 60 mu L;
step three, respectively adding the mixed solution in the step two into the 7 centrifugal tubes in the step one to form a reaction system;
step four, incubating the reaction system in the step three in a 35 ℃ water bath for 90min, and then heating in a 100 ℃ water bath for 2min to obtain a final solution;
injecting the final solution into a high performance liquid chromatograph, and detecting cyclic nucleotide to obtain the total peak area of the cyclic nucleotide in the final solution;
the liquid chromatographic analysis conditions were:
a chromatographic column: kromasil & C18 column, 4.6mm × 250mm, particle size 5 μm;
column temperature: 30 ℃;
mobile phase: volume ratio: the concentration of the buffer solution is 90 percent, the concentration of the methanol is 10 percent, wherein the buffer solution is potassium dihydrogen phosphate aqueous solution with the concentration of 6.8 mg/mL;
flow rate: 1 mL/min;
sample loading amount: the final solution was 50. mu.L.
Comparing the cyclic nucleotide peak area in the final solution with that in FIG. 1, the inhibition efficiency of 2 "-O-rhamnosyl icariside with different concentrations can be converted, and the final result is shown in FIG. 8.
In summary, although the present invention has been described with reference to the preferred embodiments, the above-described preferred embodiments are not intended to limit the present invention, and those skilled in the art can make various changes and modifications without departing from the spirit and scope of the present invention, therefore, the scope of the present invention shall be determined by the appended claims.
Claims (7)
1. A method for detecting the activity of a cyclic nucleotide phosphodiesterase inhibitor based on high performance liquid chromatography is characterized in that: the method comprises the following steps:
step one, dissolving cyclic nucleotide in Tris-HCl buffer solution to obtain cyclic nucleotide solution;
dissolving cyclic nucleotide phosphodiesterase in a Tris-HCl buffer solution to obtain a cyclic nucleotide phosphodiesterase solution, incubating in a water bath at 35 ℃ for 3-10 min in advance, and uniformly mixing with cyclic nucleotide phosphodiesterase inhibitors with different concentrations to obtain a mixed solution;
step three, pre-incubating the cyclic nucleotide solution in the step one in a water bath at 35 ℃ for 3-10 min, and then adding the mixed solution in the step two into the cyclic nucleotide solution to form a reaction system;
step four, incubating the reaction system in the step three in a water bath at 35 ℃, and then heating in a water bath at 100 ℃ to obtain a final solution;
and step five, injecting the final solution into a high performance liquid chromatograph, and detecting the concentration of the cyclic nucleotide, thereby calculating the activity of the cyclic nucleotide phosphodiesterase inhibitor.
2. The method for detecting the activity of a cyclic nucleotide phosphodiesterase inhibitor according to claim 1, wherein the method comprises the steps of: the concentration of the Tris-HCl buffer solution is 40-60mM, and the pH value is 8.
3. The method for detecting the activity of a cyclic nucleotide phosphodiesterase inhibitor according to claim 1, wherein the method comprises the steps of: the reaction system comprises a cyclic nucleotide phosphodiesterase solution with the concentration of 0.01-0.5 mug/mL and a cyclic nucleotide solution with the final concentration of 0.5-150 mug/mL.
4. The method for detecting the activity of a cyclic nucleotide phosphodiesterase inhibitor according to claim 1, wherein the method comprises the steps of: in the fifth step, the high performance liquid chromatograph uses a Kromasil & C18 chromatographic column, 4.6mm × 250mm, and the particle size is 5 μm.
5. The method for detecting the activity of a cyclic nucleotide phosphodiesterase inhibitor according to claim 4, wherein the method comprises the steps of: in the fifth step, the mobile phase used is 90% buffer solution and 10% methanol.
6. The method for detecting the activity of a cyclic nucleotide phosphodiesterase inhibitor according to claim 5, wherein the method comprises the steps of: the buffer solution is 6.8mg/mL potassium dihydrogen phosphate water solution.
7. The method for detecting the activity of a cyclic nucleotide phosphodiesterase inhibitor according to claim 6, wherein the method comprises the steps of: the flow rate of the mobile phase in the high performance liquid chromatography is 1 ml/min.
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