CN114384180B - Method for rapidly screening antiparasitic active compounds - Google Patents
Method for rapidly screening antiparasitic active compounds Download PDFInfo
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- CN114384180B CN114384180B CN202210000231.7A CN202210000231A CN114384180B CN 114384180 B CN114384180 B CN 114384180B CN 202210000231 A CN202210000231 A CN 202210000231A CN 114384180 B CN114384180 B CN 114384180B
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Abstract
The embodiment of the invention discloses a method for rapidly screening antiparasitic active compounds, which comprises the steps of pre-treating acetylcholinesterase and lactate dehydrogenase with a chinaberry plant, incubating the mixture, and performing chromatographic-mass spectrometry analysis on a solution after ultrafiltration and centrifugation according to the bioaffinity effect between enzyme and small molecule ligand after incubation to obtain chromatographic peak information after the chinaberry plant acts with the acetylcholinesterase and the lactate dehydrogenase, and calculating the binding degree of a target enzyme and the active compounds in the chinaberry plant according to the chromatographic peak information, namely the antiparasitic active compounds; the method provided by the embodiment of the invention has the advantages of high sensitivity and accuracy in screening active compounds, high analysis speed, capability of rapidly screening and identifying specific target compounds from a complex system based on protein-ligand interaction, small required sample amount, capability of efficiently identifying ligands combined with receptor target enzymes, realization of rapid screening of active ingredients in the complex system, and application in research of interaction between drug small molecules and biological macromolecules.
Description
Technical Field
The invention relates to the technical field of natural product analysis, in particular to a method for rapidly screening antiparasitic active compounds.
Background
The diversity of parasite species and host prevalence severely impact human and livestock health and cause significant economic losses. Acetylcholinesterase is a neurotransmitter enzyme, and its inhibitors have been used in many applications including parasite control. Lactate dehydrogenase is widely distributed in microorganisms, is one of the key enzymes for anaerobic fermentation to explain energy release, and is closely related to parasite survival. Inhibiting activity of acetylcholinesterase and lactate dehydrogenase, and can effectively control parasitic diseases. Current acetylcholinesterase inhibitors for controlling parasitic diseases include organic phosphorus, carbamates, and the like. Praziquantel, artemisinin and its derivatives and mebendazole cause the parasite to stop developing or die by inhibiting its lactate dehydrogenase. Although these drugs have remarkable insect repellent effects, their side effects such as drug resistance and toxicity are becoming serious.
In the related art, the screening of active ingredients may also lead to loss of trace and unstable active ingredients during the process through complicated steps such as separation, purification, structure identification, activity verification and the like.
Disclosure of Invention
In view of this, embodiments of the present invention provide a highly sensitive, highly accurate method for rapid screening of antiparasitic active compounds.
Embodiments of the present invention provide a method for rapid screening of antiparasitic active compounds comprising the steps of: (1) Dissolving a proper amount of chinaberry plant in a buffer solution to obtain a sample solution to be detected;
(2) Respectively dissolving a proper amount of acetylcholinesterase and lactate dehydrogenase powder in a buffer solution to obtain an acetylcholinesterase solution and a lactate dehydrogenase solution;
(3) Taking a proper amount of sample solution to be tested, respectively incubating the sample solution to be tested in an acetylcholinesterase solution and a lactic dehydrogenase solution, respectively ultrafiltering and centrifuging the incubated solutions, eluting the ligand trapped after centrifugation, collecting eluent, drying the eluent, and dissolving to obtain an experimental group solution; meanwhile, setting an equal concentration inactivated enzyme solution as a control group, and obtaining a control group solution from the rest of the same experiment groups;
(4) Taking the sample solution to be detected, the experimental group and the control group solution to be detected for chromatographic-mass spectrometry analysis, and collecting peak data;
(5) And respectively calculating the binding degree of the chinaberry plant, acetylcholinesterase and lactate dehydrogenase according to the collected peak data, and screening out the antiparasitic active compounds.
Further, the concentration of the sample solution to be detected is 5.0-10.0 mg/mL.
Further, 121.1g of tris was taken, 800mL of double distilled water was added to the solution to be mixed and dissolved, pH was adjusted to 8.0 (25 ℃ C.) with concentrated hydrochloric acid, and the volume was set to 1L to obtain the buffer solution.
Further, the incubation temperature is 37 ℃; and (3) during ultrafiltration and centrifugation, eluting the incubated solution by using a buffer solution, centrifuging for 3 times, collecting the centrifugally trapped ligand, eluting by using an organic solvent, and incubating and centrifuging again to obtain an experiment group solution.
Further, the degree of binding is calculated using a formula
BD(％)＝(A Experiment -A Control )/A Experiment ×100％
Wherein BD is the degree of binding of the enzyme to the active compound, A Experiment For the peak area of the chromatographic peak after the interaction of the sample to be tested and the enzyme, A Control The peak area of the chromatographic peak after the interaction of the sample to be tested and the control group is shown.
The technical scheme provided by the embodiment of the invention has the beneficial effects that: according to the method for rapidly screening the antiparasitic active compound, acetylcholinesterase and lactic dehydrogenase are incubated together after pretreatment of the plants in the family of chinaberry, chromatographic-mass spectrometry is carried out on the solution after ultrafiltration and centrifugation according to the bioaffinity effect between enzyme and small molecule ligand after incubation, chromatographic peak information after the actions of the plants in the family of chinaberry and acetylcholinesterase and lactic dehydrogenase is obtained, the binding degree of the active compounds in the plants in the family of chinaberry and acetylcholinesterase and lactic dehydrogenase is calculated according to the chromatographic peak information, and the active compounds capable of inhibiting acetylcholinesterase and lactic dehydrogenase in the plants in the family of chinaberry are determined according to the value of the binding degree, so that the antiparasitic active compound is obtained; the method provided by the embodiment of the invention has the advantages of high sensitivity and high accuracy in screening active compounds, high analysis and screening speed, and can be used for rapidly screening and identifying specific target compounds from a complex system based on protein-ligand interaction, the required sample amount is small, the antiparasitic active compounds can be effectively screened out, and the active compounds capable of inhibiting acetylcholinesterase and lactate dehydrogenase can be screened out from the chinaberry plants at the same time, so that the screening efficiency and accuracy are greatly improved.
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FIG. 1 is a flow chart of a method according to an embodiment of the invention.
FIG. 2 is a liquid chromatogram of neem incubated with acetylcholinesterase according to an embodiment of the present invention.
FIG. 3 is a liquid chromatogram of neem incubated with lactate dehydrogenase according to an embodiment of the invention.
Detailed Description
For the purpose of making the objects, technical solutions and advantages of the present invention more apparent, embodiments of the present invention will be further described with reference to the accompanying drawings.
Referring to fig. 1, an embodiment of the present invention provides a method for rapid screening of antiparasitic active compounds, comprising the steps of: (1) Dissolving a proper amount of chinaberry plant in a buffer solution to obtain a sample solution to be detected; specifically, 121.1g of tris (hydroxymethyl) aminomethane is taken, 800mL of double distilled water is added for mixing and dissolution, the pH is regulated to 8.0 (25 ℃) by concentrated hydrochloric acid, and the buffer solution is obtained by constant volume to 1L; the plant of Meliaceae adopts Neem; the concentration of the sample solution to be measured is 5.0-10.0 mg/mL;
(2) Respectively dissolving proper amounts of acetylcholinesterase and lactate dehydrogenase powder in the buffer solution to obtain acetylcholinesterase solution with the concentration of 0.25U/mu L and lactate dehydrogenase solution with the concentration of 0.30U/mu L;
(3) Taking a proper amount of sample solution to be tested, respectively incubating the sample solution to be tested in an acetylcholinesterase solution and a lactic dehydrogenase solution, respectively ultrafiltering and centrifuging the incubated solutions, eluting the ligand trapped after centrifugation, collecting eluent, drying the eluent, and dissolving to obtain an experimental group solution; meanwhile, setting an equal concentration inactivated enzyme solution as a control group, and obtaining a control group solution from the rest of the same experiment groups;
specifically, the incubation temperature was 37 ℃; during ultrafiltration and centrifugation, eluting with buffer solution and centrifuging for 3 times, collecting the centrifugally trapped ligand, eluting with organic solvent, collecting eluent, drying the eluent, and dissolving in methanol-water solution (9:1, v/v) to obtain experimental group solution;
(4) Taking the sample solution to be detected, the experimental group and the control group solution to be detected for chromatographic-mass spectrometry analysis, and collecting peak data; specifically, the chromatographic-mass spectrometry adopts an ultra-high performance liquid chromatograph and a high resolution mass spectrometer; wherein, the high performance liquid chromatograph uses formic acid-water and acetonitrile as mobile phases and uses a chromatographic column to carry out gradient elution; the high-resolution mass spectrometer performs analysis and identification under the condition that the molecular weight scanning range is 100-1500 daltons;
(5) And respectively calculating the binding degree of the chinaberry plant, acetylcholinesterase and lactate dehydrogenase according to the collected peak data, and screening out the antiparasitic active compounds.
The degree of binding is calculated by a formula
BD(％)＝(A Experiment -A Control )/A Experiment ×100％
Wherein BD is the degree of binding of the enzyme to the active compound, A Experiment For the peak area of the chromatographic peak after the interaction of the sample to be tested and the enzyme, A Control The peak area of the chromatographic peak after the interaction of the sample to be detected and the control group;
referring to FIG. 2, the calculated degrees of binding of acetylcholinesterase to peaks 1, 3, 5, 7, 9-10, 15-16, 20-21 were 40.97%, 28.09%, 38.43%, 22.84%, 38.04%, 26.23%, 24.03%, 39.24%, 29.84%, 42.01%, and 32.94%, respectively; wherein the active compounds corresponding to peak compounds 1, 5, 9, 16 and 21 are gallic acid, D- (+) catechin, (-) -epicatechin, resveratrol 5-O-beta-D-glucoside and carnosol in sequence, and the binding degree of the active compounds with acetylcholinesterase is higher than that of other compounds, which indicates that the active compounds have better potential antiparasitic activity.
Referring to FIG. 3, the calculated binding degree of lactate dehydrogenase to the active compounds corresponding to peaks 1, 3, 5, 7 to 10, 13, 14 to 15, 20 to 21 is 4.03%, 23.18%, 15.01%, 14.45%, 38.61%, 19.06%, 14.35%, 17.08%, 20.03%, 3.96%, 21.27%, 26.02% and 20.53%, respectively; wherein, the combination degree of the compounds corresponding to peaks 3, 8 and 21 and lactic dehydrogenase is higher than that of other compounds, and the compounds corresponding to peaks 3 and 21 are protocatechuic acid and carnosol in sequence, which shows that the compounds have better potential antiparasitic activity.
Acetylcholinesterase and lactate dehydrogenase are closely related to the survival of parasites in humans and domestic animals, and are very attractive therapeutic targets; the method of the embodiment of the invention adopts the nim to be respectively incubated with acetylcholinesterase and lactate dehydrogenase, and active compounds for resisting parasites are screened out; the invention is based on the biological affinity between enzyme and small molecule ligand, carries out screening of antiparasitic active compounds, adopts acetylcholinesterase and lactic dehydrogenase for screening, and screens out various antiparasitic active compounds, wherein the screened carnosol is a compound capable of simultaneously inhibiting the activities of acetylcholinesterase and lactic dehydrogenase. The method provided by the embodiment of the invention can be used for effectively predicting the binding site between the enzyme and the inhibitor, can be widely applied to the research between the drug ligand and the biomacromolecule receptor, and can be used for improving the screening efficiency of the active ingredients in a complex system; it can also be used to evaluate the degree of in vitro binding of acetylcholinesterase and lactate dehydrogenase inhibitors in natural products such as Meliaceae plants.
In this document, terms such as front, rear, upper, lower, etc. are defined with respect to the positions of the components in the drawings and with respect to each other, for clarity and convenience in expressing the technical solution. It should be understood that the use of such orientation terms should not limit the scope of the protection sought herein.
The embodiments described above and features of the embodiments herein may be combined with each other without conflict.
The foregoing description of the preferred embodiments of the invention is not intended to limit the invention to the precise form disclosed, and any such modifications, equivalents, and alternatives falling within the spirit and scope of the invention are intended to be included within the scope of the invention.
Claims (6)
1. A method for rapid screening of antiparasitic active compounds, comprising the steps of: (1) Dissolving a proper amount of chinaberry plant in a buffer solution to obtain a sample solution to be detected; (2) Respectively dissolving a proper amount of acetylcholinesterase and lactate dehydrogenase powder in a buffer solution to obtain an acetylcholinesterase solution and a lactate dehydrogenase solution; (3) Taking a proper amount of sample solution to be tested, respectively incubating the sample solution to be tested in an acetylcholinesterase solution and a lactic dehydrogenase solution, respectively ultrafiltering and centrifuging the incubated solutions, eluting the ligand trapped after centrifugation, collecting eluent, drying the eluent, and dissolving the eluent to obtain an experimental group solution; meanwhile, setting the corresponding inactivated enzyme solution with equal concentration as a control group, and obtaining a control group solution from the rest of the same experiment groups; (4) Taking the sample solution to be detected, the experimental group and the control group solution to be detected for chromatographic-mass spectrometry analysis, and collecting peak data; (5) Respectively calculating the combination degree of the chinaberry plant, acetylcholinesterase and lactate dehydrogenase according to the collected peak data, and screening out an antiparasitic active compound;
in the steps (1) and (2), 121.1g of tris (hydroxymethyl) aminomethane is taken, 800mL of double distilled water is added for mixing and dissolving, the pH is regulated to 8.0 and 25 ℃ by using concentrated hydrochloric acid, and the volume is fixed to 1L to obtain the buffer solution;
in the step (5), the binding degree is calculated by using a formula BD (%) = (a) Experiment -A Control )/A Experiment X 100%, where BD is the degree of binding of the enzyme to the active compound, A Experiment For the peak area of the chromatographic peak after the interaction of the sample to be tested and the enzyme, A Control The peak area of the chromatographic peak after the interaction of the sample to be tested and the control group is shown.
2. The method for rapid screening of antiparasitic active compounds according to claim 1, wherein the concentration of the sample solution to be tested is 5.0-10.0 mg/mL.
3. A method for rapid screening of antiparasitic active compounds according to claim 1, wherein in step (2) the concentration of acetylcholinesterase solution is 0.25U/μl and lactate dehydrogenase solution is 0.30U/μl.
4. A method for rapid screening of antiparasitic active compounds according to claim 1, wherein in step (3) the incubation temperature is 37 ℃; and (3) during ultrafiltration and centrifugation, eluting by adopting a buffer solution, centrifuging for 3 times, collecting the centrifugally trapped ligand, eluting by using an organic solvent, and incubating and centrifuging again to obtain an experimental group solution.
5. The method for rapid screening of antiparasitic active compounds according to claim 1, wherein the antiparasitic active compounds screened by the Meliaceae plant and acetylcholinesterase are gallic acid, D- (+) catechin, (-) -epicatechin, resveratrol 5-O-beta-D-glucoside, carnosol.
6. The method for rapid screening of antiparasitic active compounds according to claim 1, wherein the antiparasitic active compounds screened by the Meliaceae plant and lactate dehydrogenase are protocatechuic acid, carnosol.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102818868A (en) * | 2012-08-27 | 2012-12-12 | 浙江大学 | Screening method of active ingredients in complex natural product and its application |
| CN109596749A (en) * | 2019-01-08 | 2019-04-09 | 西安科技大学 | Based on ultrafiltration-liquid chromatography-mass spectrography quick screening drugs active constituent method |
| CN111721870A (en) * | 2020-06-24 | 2020-09-29 | 兰州大学 | Method for high-throughput screening of acetylcholinesterase inhibitor |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102818868A (en) * | 2012-08-27 | 2012-12-12 | 浙江大学 | Screening method of active ingredients in complex natural product and its application |
| CN109596749A (en) * | 2019-01-08 | 2019-04-09 | 西安科技大学 | Based on ultrafiltration-liquid chromatography-mass spectrography quick screening drugs active constituent method |
| CN111721870A (en) * | 2020-06-24 | 2020-09-29 | 兰州大学 | Method for high-throughput screening of acetylcholinesterase inhibitor |
Non-Patent Citations (1)
| Title |
|---|
| Comparative study of different solvents seed extracts of Azadirachta indica (neem) and its in-vitro acaricidal properties against Rhipicephalus (B.) microplus;Dr. Arvind Kumar Das 等;《The Pharma Innovation Journal》;第9卷(第11期);281-284 * |
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