CN114395612A - Method for rapidly screening multi-target active ligand in dysosma versipellis - Google Patents

Method for rapidly screening multi-target active ligand in dysosma versipellis Download PDF

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CN114395612A
CN114395612A CN202210029382.5A CN202210029382A CN114395612A CN 114395612 A CN114395612 A CN 114395612A CN 202210029382 A CN202210029382 A CN 202210029382A CN 114395612 A CN114395612 A CN 114395612A
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dysosma versipellis
topoisomerase
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郭明全
冯慧霞
陈桂林
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Wuhan Botanical Garden of CAS
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Abstract

The invention discloses a method for rapidly screening multi-target active ligands in Dysosma versipellis, wherein the Dysosma versipellis total extract is incubated with DNA topoisomerase I, topoisomerase II, cyclooxygenase-2 and angiotensin converting enzyme 2, micromolecule ligands with affinity activity in Dysosma versipellis and the four biological target molecules form receptor-ligand complexes, the complexes are intercepted on one side of an ultrafiltration membrane by mixed liquid after incubation and combination through an ultrafiltration tube with a certain interception aperture, then the micromolecule ligands are released after treatment, and the active ligands are screened out by utilizing an instrument for analysis; the high-throughput screening and rapid identification of bioactive components or lead compounds in complex natural products are realized, the research on the action mechanism of multiple targets and multiple components is realized, and a new thought and a new method are provided for the field of new drug development.

Description

Method for rapidly screening multi-target active ligand in dysosma versipellis
Technical Field
The invention relates to the technical field of analysis of active compounds of natural products, in particular to a method for rapidly screening multi-target active ligands in Dysosma versipellis.
Background
Dysosma versipellis is a perennial herb of Podophyllum of berberidaceae, is a Chinese herbal medicine with high medicinal value, and can be used for treating traumatic injury, rheumatalgia, venomous snake bite, menoxenia, etc. Hitherto, chemical components separated and identified from dysosma versipellis are mainly lignans and flavonoids.
In the related art, the screening method of multi-target active ligand generally uses a spectroscopic method or a nuclear magnetic resonance method. However, the inventor finds that the methods meet the screening requirements to a certain extent, but have limitations, such as the spectrum method is easy to be interfered by the background to generate false positive or false negative phenomena, and the nuclear magnetic resonance method is not suitable for quantitatively researching the binding parameters; and the methods can only screen various target enzymes one by one, and the screening process is complicated and the workload is large.
Disclosure of Invention
In view of this, the embodiment of the present invention provides a method for rapidly screening multiple target active ligands in dysosma versipellis, which has high sensitivity, is not easily interfered by background signals, and can simultaneously screen multiple target enzymes quantitatively.
The embodiment of the invention provides a method for rapidly screening multi-target active ligands in Dysosma versipellis, which comprises the following steps: (1) preparing an enzymatic reaction buffer solution;
(2) preparing a sample solution to be detected; dissolving a proper amount of dysosma versipellis total extract in the enzymatic reaction buffer solution to obtain a sample solution to be detected;
(3) carrying out affinity reaction on a sample solution to be detected and a multi-target enzyme; respectively taking a proper amount of a sample solution to be tested, DNA topoisomerase I, topoisomerase II, cyclooxygenase-2 and angiotensin converting enzyme 2 into a centrifugal tube, uniformly mixing, incubating at a constant temperature, eluting the centrifugal tube for multiple times by using an enzymatic buffer solution after the reaction is finished, eluting an elution retention ligand by using an organic solvent, and collecting an eluent to obtain an experimental group solution; taking the inactivated enzyme as a control group, and obtaining a control group solution by using the rest of the inactivated enzyme as the same as the experimental group;
(4) collecting peak data; taking a proper amount of the sample solution to be tested, the experimental group solution and the control group solution, freeze-drying, adding an organic solution for dissolving, analyzing by an instrument, and collecting peak data;
(5) calculating the enrichment rate to screen out active ligands in the dysosma versipellis; calculating the enrichment ratio according to the peak data to obtain the active ligand of DNA topoisomerase I, topoisomerase II, cyclooxygenase-2 and angiotensin converting enzyme 2 after screening Dysosma versipellis, wherein the formula of the enrichment ratio is
EF(％)＝(A1-A2)/A0×100％
Wherein EF is an enrichment factor between multi-target enzyme and multi-target active ligand screened by Dysosma versipellis, A0、A1And A2Respectively represents the peak areas of the chromatographic peaks of the Dysosma versipellis treated by the enzyme without addition and the active enzyme and the inactivated enzyme.
Preferably, in the step (1), the enzymatic reaction buffer is 0.1mol/L sodium phosphate solution or 0.1mol/L tris hydrochloride; uniformly mixing 50mL of 0.1mol/L tris solution with 34.5mL of 0.1mol/L hydrochloric acid, and diluting the mixture to 100mL by using deionized water to obtain 0.1mol/L tris hydrochloride enzymatic buffer with the pH value of 7.8; 1mol/L disodium hydrogen phosphate solution and 1mol/L sodium dihydrogen phosphate solution are respectively taken and dissolved by deionized water until the total volume is 1L, and the solution is fully mixed to obtain 0.1mol/L sodium phosphate enzymatic buffer solution with the pH value of 6.8.
Preferably, in the step (2), a proper amount of dysosma versipellis total extract is fully dissolved in an enzymatic reaction buffer solution to obtain a dysosma versipellis sample solution to be detected with a concentration of 8.0 mg/mL.
Preferably, in the step (3), 100 μ L of a sample solution to be tested, 10 μ L of 2U or 5U DNA topoisomerase I, 10 μ L of 2U or 4U DNA topoisomerase II, 10 μ L of 2U or 4U cyclooxygenase-2, and 0.5 μ g angiotensin converting enzyme 2 are respectively and uniformly mixed in a 0.2mL centrifuge tube, incubated in a 37 ℃ constant temperature incubator for 40min, transferred to ultrafiltration with a cut-off molecular weight of 10KD after the reaction is finished, centrifuged at 10000rpm for 10min, 200 μ L of an enzymatic buffer solution is added to repeatedly elute and centrifuge, a solvent is added to a ligand intercepted in the centrifuge tube to elute, and an experimental group solution is obtained by collecting an eluent; the control group was prepared by heating inactivated DNA topoisomerase I, topoisomerase II, cyclooxygenase-2, and angiotensin converting enzyme 2 in boiling water, and the rest was the same as the experimental group.
The technical scheme provided by the embodiment of the invention has the following beneficial effects: the invention relates to a method for rapidly screening multi-target active ligands in Dysosma versipellis, which comprises the steps of incubating Dysosma versipellis total extract, DNA topoisomerase I, DNA topoisomerase II, cyclooxygenase-2 and angiotensin converting enzyme 2 in an enzymatic buffer solution at constant temperature, ultrafiltering, centrifuging, eluting, forming a receptor-ligand compound according to micromolecule ligands with affinity activity in Dysosma versipellis and the four biological target molecules, intercepting the compound on one side of an ultrafiltration membrane by an ultrafiltration tube with a certain interception aperture in the incubated and combined mixed solution, treating or changing the pH value of the solution by an organic solvent to release the micromolecule ligands, analyzing by an ultra-high performance liquid chromatography and a mass spectrometer, and calculating the enrichment rate by peak output data to obtain the active ligands in Dysosma versipellis combined with the multi-target enzymes; the method of the embodiment of the invention adopts the simultaneous screening of the multi-target enzymes, and compared with the single successive screening, the workload is greatly reduced, and the screening efficiency is improved; in the embodiment of the invention, the multi-target enzyme and the dysosma versipellis total extract sample are incubated in a solution state, so that the change of the property of a receptor-ligand complex can be avoided, and the natural conformations of the multi-target enzyme and the active ligand are maintained, thereby truly reflecting the actual conditions of the interaction between the multi-target enzyme and the active small molecular ligand to the maximum extent; directly utilizing the interception of an ultrafiltration membrane with a certain aperture to realize the selection of an active micromolecule ligand combined by the dysosma versipellis total extract and the target enzyme under the condition of not carrying out pretreatment; the method provided by the embodiment of the invention has the important characteristics of rapidness, high flux, high sensitivity, high specificity and the like, the used sample amount is small, and a large amount of spectral information can be improved to identify the screened active ligand; the multi-target enzyme, namely the receptor in the method of the embodiment of the invention can recover the activity and be recycled, which is important for some target enzymes which are expensive or not easily obtained; based on the sample extract and the change of chromatographic peak after the multi-target enzyme acts, the method provided by the embodiment of the invention can evaluate the strength of the combination of the multi-target enzyme and the active ligand by utilizing the enrichment rate, further determine the potential active ligand with stronger combination, and provide a screening basis for the subsequent pharmacological and pharmacodynamic verification; the potential ligand matrix in the embodiment of the invention does not influence the screening result, and can be widely applied to screening active ingredients or lead compounds from complex natural products.
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FIG. 1 is a schematic flow chart of a method for rapidly screening multi-target active ligands in Dysosma versipellis according to an embodiment of the present invention;
FIG. 2 is an ultrafiltration screening chromatogram of the chemical components of the Dysosma versipellis total extract in the embodiment of the present invention;
FIG. 3 is an ultrafiltration screening chromatogram of the chemical components of the Dysosma versipellis total extract in the embodiment of the present invention;
FIG. 4 is an ultrafiltration screening chromatogram of the chemical components of the Dysosma versipellis total extract in the embodiment of the present invention;
FIG. 5 is an ultrafiltration screening chromatogram of the chemical components of the Dysosma versipellis total extract in the embodiment of the present invention;
FIG. 6 is an extraction chromatogram of multiple reaction detection positive ion patterns of peaks 8 and 12 of a liquid chromatogram of an embodiment of the invention;
FIG. 7 is an extraction chromatogram of multiple reaction detection positive ion mode of liquid chromatogram peak 9 according to an embodiment of the present invention;
FIG. 8 is an extraction chromatogram of the multiple reaction detection positive ion mode of liquid chromatogram peak 10 of the example of the invention;
FIG. 9 is an extraction chromatogram of multiple reaction detection positive ion mode of liquid chromatogram peak 11 in the example of the present invention.
Wherein, the detection wavelength is 292nm in FIG. 2; line a represents the HPLC chromatogram of the Dysosma versipellis total extract; lines b and c represent the liquid chromatogram after the interaction with topoisomerase I and inactivated topoisomerase I, respectively; detection wavelength 292nm in FIG. 3; line d represents the HPLC chromatogram of the Dysosma versipellis total extract; line e and line f represent the liquid chromatogram after the interaction with topoisomerase II and inactivated topoisomerase II, respectively; detection wavelength 292nm in FIG. 4; line g represents the HPLC chromatogram of the total extract of Dysosma versipellis; lines h and i represent the reaction with cyclooxygenase-2 and inactivated cyclooxygenase-2, respectivelyA liquid chromatogram map; detection wavelength 292nm in FIG. 5; line j represents the HPLC chromatogram of the Dysosma versipellis total extract; line k and line l represent the liquid chromatogram after the action with angiotensin converting enzyme 2 and angiotensin converting enzyme 2 respectively; in FIG. 6, the extracted ion in positive ion mode for multi-reaction detection of active ligand beta-anhydropicropodophyllin corresponding to peak 8 of liquid chromatography is parent ion 397.17, and the signal intensity is 3.05X 106(ii) a The multi-reaction detection of the podophyllotoxin as the active ligand corresponding to the liquid chromatography peak 12 has the extracted ion in the positive ion mode of mother ion 397.18 and the signal intensity of 1.68 × 106(ii) a In FIG. 7, the multiple reaction detection of the active ligand deoxypodophyllotoxin corresponding to the liquid chromatography peak 9 shows that the extracted ion in the positive ion mode is the parent ion 399.15, and the signal intensity is 4.38 × 105(ii) a Peaks 8 and 12 of the liquid chromatography are the same as in FIG. 5; in FIG. 8, the extracted ion in positive ion mode for multi-reaction detection of quercetin as active ligand corresponding to peak 10 of liquid chromatography is parent ion 303.00, and the signal intensity is 5.64 × 105(ii) a In FIG. 9, the extracted ion in positive ion mode for multi-reaction detection of kaempferol as active ligand corresponding to peak 11 of liquid chromatography is parent ion 287.00, and signal intensity is 3.05X 106。
Detailed Description
In order to make 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.
With the cross fusion and continuous development of multiple disciplines, numerous new targets for preventing and treating diseases are developed and utilized, and the research focus of natural medicines gradually turns to the research of novel medicines aiming at specific targets or mechanisms. The enzyme maintains the balance in the organism by regulating the activity of cells and catalyzing metabolic reaction, and provides a new idea for searching new drugs for preventing and treating diseases by taking the enzyme as a target. Enzyme inhibitors can correct the metabolic imbalance in the body by regulating the activity of enzymes, making them one of the important routes in natural drug development. The interaction of drugs with various related biological enzymes in the disease development process is one of the main ways that drugs exert corresponding pharmacological actions in the body. Most of natural medicines prevent and treat certain diseases by inhibiting the expression of specific enzymes related to certain diseases in vivo, such as tyrosine kinase, DNA topoisomerase, cyclooxygenase-2, anaplastic lymphoma kinase and the like which are widely applied to anti-tumor research; alpha-glucosidase, aldose reductase and the like can be used as targets of diabetes; xanthine oxidase can be used for treating gout and hyperuricemia. Based on the traditional application and pharmacological activity of dysosma versipellis, four accepted drug targets, namely DNA topoisomerase I, DNA topoisomerase II, cyclooxygenase-2 and angiotensin converting enzyme 2, are adopted in the embodiment of the invention. DNA topoisomerase plays a key role in the process of cell life by transiently inducing DNA chain disconnection and combination to further catalyze DNA topoisomerase mutual transformation. DNA topoisomerases can be classified into type I and type II according to the mode of isomerization. Type I topoisomerases alter the topology by cleaving one strand of a DNA double strand, while type II topoisomerases alter the topology by cleaving both strands of a DNA double strand. Topoisomerase I and topoisomerase II belong to ribozymes, can maintain normal topoisomerase of DNA double strands, and are highly expressed in tumor cells, so that rapid proliferation of tumor cells can be retarded by inhibiting the activities of topoisomerase I and topoisomerase II, thereby inducing necrosis and apoptosis of tumor cells. Cyclooxygenase-2 is a key enzyme in the synthesis of prostaglandins and plays an important role in the pathological process of various diseases such as chronic inflammation, cancer and the like. Therefore, the synthesis of prostaglandin can be blocked by inhibiting the activity of cyclooxygenase-2, thereby inducing the apoptosis of tumor cells and inhibiting the angiogenesis, invasion and metastasis of tumors. The cyclooxygenase-2 inhibitor is a new generation of antitumor drugs, has the advantages of small side effect and high curative effect, can be used as an auxiliary drug for treating cancers, and the design and synthesis of the cyclooxygenase-2 inhibitor are the hot spots which are always concerned by researchers. In addition, cyclooxygenase-2 inhibitor has antiinflammatory and analgesic effects. Angiotensin converting enzyme 2 is a type I transmembrane metallocarboxypeptidase comprising a signal peptide, a transmembrane domain and a metalloprotease active site comprising a zinc binding domain of HEMGH. Angiotensin converting enzyme 2 is expressed in a variety of different tissues, including the human lung, heart, liver, kidney, and gastrointestinal mucosa. In the whole process of infection and pathogenesis of the new coronavirus, angiotensin converting enzyme 2 plays an important role and can be used as a receptor to be combined with virus S protein so as to mediate virus infection. Many adults with hypertension, diabetes and chronic kidney disease take angiotensin converting enzyme inhibitors and angiotensin II receptor blockers, and these drug up-regulated angiotensin converting enzyme 2 receptors are the main binding receptor for new coronaviruses to enter host cells, and down-regulation of angiotensin converting enzyme 2 plays a key role in the pathogenesis of severe lung failure following viral infection; can be used as a strong negative regulation factor for regulating renin-angiotensin system and participating in blood pressure lowering process; is involved in the acute lung injury process and can be used as a key regulatory factor for protecting acute lung injury. When new coronavirus is infected, the level of angiotensin-converting enzyme 2 is significantly down-regulated, thereby causing acute respiratory distress syndrome; can participate in the immune regulation process, leads to the down regulation of angiotensin converting enzyme 2 after the infection of new coronavirus, and leads to the initiation of cytokine storm by a large amount of inflammatory signals, thereby aggravating inflammation; the neutral amino acid transporter expressed on the surface of epithelial cells can be combined with the amino acid transporter and plays an important role in the absorption of amino acids in the intestinal tract and the kidney. Based on the traditional application of Dysosma versipellis, the search for DNA topoisomerase I, topoisomerase II, cyclooxygenase-2 and angiotensin converting enzyme 2 inhibitors is of great significance for the development of new drugs.
Referring to the attached figure 1, the embodiment of the invention provides a method for rapidly screening multi-target active ligands in dysosma versipellis, which comprises the following steps: (1) preparing an enzymatic reaction buffer solution;
specifically, the enzymatic reaction buffer solution is 0.1mol/L sodium phosphate solution or 0.1mol/L tris hydrochloride; uniformly mixing 50mL of 0.1mol/L tris solution with 34.5mL of 0.1mol/L hydrochloric acid, and diluting the mixture to 100mL by using deionized water to obtain 0.1mol/L tris hydrochloride enzymatic buffer with the pH value of 7.8; respectively taking 46.3mL and 53.7mL of 1mol/L disodium hydrogen phosphate solution and 1mol/L sodium dihydrogen phosphate solution, dissolving the solutions by using deionized water until the total volume is 1L, and fully mixing to obtain 0.1mol/L sodium phosphate enzymatic buffer solution with the pH value of 6.8;
(2) preparing a sample solution to be detected; dissolving a proper amount of dysosma versipellis total extract in the enzymatic reaction buffer solution to obtain a sample solution to be detected; the concentration of the sample solution to be detected is 8.0 mg/mL;
(3) performing affinity reaction on a sample to be detected and the multi-target enzyme; respectively taking a proper amount of a sample solution to be tested, DNA topoisomerase I, topoisomerase II, cyclooxygenase-2 and angiotensin converting enzyme 2 into a centrifugal tube, uniformly mixing, incubating at a constant temperature, eluting the centrifugal tube for multiple times by using an enzymatic buffer solution after the reaction is finished, eluting an elution retention ligand by using an organic solvent, and collecting an eluent to obtain an experimental group solution; taking the inactivated enzyme as a control group, and obtaining a control group solution by using the rest of the inactivated enzyme as the same as the experimental group;
specifically, 100 μ L of sample solution to be tested, 10 μ L of 2U or 5U DNA topoisomerase I, 10 μ L of 2U or 4U DNA topoisomerase II, 10 μ L of 2U or 4U cyclooxygenase-2, and 0.5 μ g angiotensin converting enzyme 2 are respectively taken and uniformly mixed in a 0.2mL centrifuge tube, incubated in a 37 ℃ constant temperature incubator for 40min, transferred to an ultrafiltration centrifuge tube with the molecular weight cutoff of 10KD after the reaction is finished, centrifuged at 10000rpm for 10min, 200 μ L of enzymatic buffer solution is added for repeated elution and centrifugation, a solvent is added to the ligand intercepted in the centrifuge tube for elution, and the eluent is collected to obtain an experimental group solution; the control group adopts DNA topoisomerase I, topoisomerase II, cyclooxygenase-2 and angiotensin converting enzyme 2 which are heated and inactivated in boiling water, and the rest is the same as the experimental group;
(4) collecting peak data; taking a proper amount of the sample solution to be tested, the experimental group solution and the control group solution, freeze-drying, adding an organic solution for dissolving, analyzing by an instrument, and collecting peak data;
specifically, after freeze drying, adding 50 or 60 μ L of pure methanol solution or pure acetonitrile solution for dissolving, and performing high performance liquid chromatography-mass spectrometry;
wherein, the high performance liquid chromatograph model: agilent 1100, Agilent 1220 Infinity or Agilent 1290 Infinity II; a chromatographic column: waters Symmetry RP-C18 (specification 4.6mm × 250mm, 5 μm), Hypersil BDS C18 (specification 4.6mm × 150mm, 3 μm) or Athena C18-WP (specification 4.6mm × 150mm, 5 μm); mobile phase: a-0.1% formic acid-water and B-acetonitrile; gradient elution procedure: 0-40 min, 5-95% B, 0-40 min, 8-70% B, 0-40 min, 5-75% B, 0-6 min, 5-23% B; 6-25 min, 23% -40% B; 25-40 min, 40-75% B, 0-4 min and 5-20% B; 4-35 min, 20-90% B or 0-5 min, 5-20% B; 5-35 min, 20% -88% of B; the percentages indicated are volume percentages; detection wavelength: 254nm, 280nm or 292 nm; flow rate: 0.6mL/min, 0.8mL/min or 1 mL/min; sample introduction amount: 5 μ L or 10 μ L; column temperature: 25 ℃ or 30 ℃;
high performance liquid chromatography mass spectrometer: thermo Fisher Scientific (access max) or Agilent 1290 definition II 6530C; an ion source: electrospray (ESI) ionization source positive or negative ion mode; spraying voltage: 3000V; mass scan range: 150 to 1000m/z or 100 to 1500 m/z; secondary spectrum scanning mode: a data dependent scan; vaporization temperature: 250 ℃, 300 ℃ or 350 ℃; capillary temperature: 250 ℃, 300 ℃ or 350 ℃; sheath gas pressure: 40lb/in2(ii) a Auxiliary gas pressure: 10lb/in2；
(5) Calculating the enrichment rate to screen out active ligands in the dysosma versipellis; calculating the enrichment ratio according to the peak data to obtain the active ligand of DNA topoisomerase I, topoisomerase II, cyclooxygenase-2 and angiotensin converting enzyme 2 after screening Dysosma versipellis, wherein the formula of the enrichment ratio is
EF(％)＝(A1-A2)/A0×100％
Wherein EF is an enrichment factor between multi-target enzyme and multi-target active ligand screened by Dysosma versipellis, A0、A1And A2Respectively represents the peak areas of the chromatographic peaks of the Dysosma versipellis treated by the enzyme without addition and the active enzyme and the inactivated enzyme.
Further, in the step (2), 200 μ L or 300 μ L of 90% methanol solution or 90% acetonitrile solution is added to the ligand trapped in the centrifuge tube, the centrifuge tube is kept standing at room temperature for 10min, then the centrifuge tube is centrifuged at 10000rpm or 13000rpm for 8min or 10min, the elution is repeated for 2-3 times, and the eluent is collected to obtain the experimental group solution.
Referring to the attached figures 2-8, the Dysosma versipellis extract contains 12, 9 and 12 potential active ingredients which respectively show affinity activity with different degrees on DNA topoisomerase I, topoisomerase II, cyclooxygenase-2 and angiotensin converting enzyme 2. For topoisomerase I, the EF value of peak 12 is 5.39%, the EF value of the second peak is 4.91%, the 11 peak is 4.74%, the 8 peak is 4.15%, the 9 peak is 3.63%, the 7 peak is 2.59%, and the like, which are calculated according to the enrichment ratio formula; for topoisomerase II, peak 12 had the largest EF value of 9.08%, followed by 6.64% for peak 10, 6.15% for peak 8, 4.49% for peak 9, and so on; for cyclooxygenase-2, peak 11 has the largest EF value of 8.71%, followed by peak 12 of 2.45%, peak 8 of 1.79%, peak 9 of 1.13%, and peak 7 of 0.94%, etc.; for angiotensin converting enzyme 2, peak 10 had a maximum EF value of 2.98%, followed by 2.86% of peak 12 and 2.83% of peak 8, etc. The EF value of each peak is obviously different, and the peak area of the active ligand after the action with the corresponding target enzyme is larger than that of the ligand after inactivation, which indicates that competitive interaction exists between the active ligands and the corresponding target enzyme.
According to different EF values of all active ingredients screened from the dysosma versipellis total extract, the affinity capability of different potential active ingredients to different targets is reflected, and the enzyme inhibitor with strong corresponding activity can be determined. For the in vitro antiproliferative activity, peaks 8 (beta-anhydropicropodophyllin), 9 (deoxypodophyllotoxin), 10 (quercetin), 11 (kaempferol) and 12 (podophyllotoxin) with larger EF value have strong affinity binding force with topoisomerase I, so that more peaks are reserved in the reaction process; while the active ligands corresponding to peaks 8, 9, 10 and 12 show a strong affinity for topoisomerase II. That is, the affinity of peaks 8, 9, 10 and 12 to topoisomerase I and topoisomerase II is relatively strong, indicating that there is a synergistic effect between these active ligands, which can exert a targeted anti-tumor effect together. While the other components with lower EF values are less retained during the reaction due to the lower affinity binding to topoisomerase I or topoisomerase II. Wherein, the active ligands kaempferol and podophyllotoxin corresponding to the peaks 11 and 12 have large EF value to cyclooxygenase-2, namely, the affinity between the active ligands is high, which indicates that effective anti-inflammatory active ingredient groups exist in the dysosma versipellis extract. The EF value of quercetin and angiotensin converting enzyme 2 corresponding to peak 10 is large, and the affinity binding force is stronger, which indicates that effective antiviral active ingredients exist in the dysosma versipellis extract. Therefore, the method provided by the embodiment of the invention is adopted to screen a plurality of Dysosma versipellis active ligands which can act on a plurality of targets and jointly play the role of antitumor, anti-inflammatory or antiviral active ligands, and the synergistic effect of the plurality of active ligands plays a unique role in the disease treatment field compared with that of a single ligand, and meanwhile, the method provided by the embodiment of the invention can be widely applied to identification and screening of multi-active ligands in other natural products.
Example two
The embodiment of the invention provides a method for rapidly screening multi-target active ligands in Dysosma versipellis, which comprises the following steps: (1) preparing an enzymatic reaction buffer solution;
(2) preparing a sample solution to be detected;
(3) carrying out affinity reaction on a sample solution to be detected and a multi-target enzyme;
specifically, 100 μ L of sample solution to be tested, 20 μ L of 2U or 5U DNA topoisomerase I, 20 μ L of 2U or 4U DNA topoisomerase II, 20 μ L of 2U or 4U cyclooxygenase-2, and 1 μ g angiotensin converting enzyme 2 are respectively taken and mixed uniformly in a 0.2mL centrifuge tube, incubated for 60min in a 37 ℃ constant temperature incubator, transferred to an ultrafiltration centrifuge tube with molecular weight cut-off of 30KD after the reaction is finished, centrifuged for 80min at 13000rpm, and 300 μ L of enzymatic buffer solution is added for repeated elution and centrifugation.
The rest is the same as the first embodiment.
In this document, the terms front, back, upper and lower are used to define the components in the drawings and the positions of the components relative to each other, and are used for clarity and convenience of the technical solution. It is to be understood that the use of the directional terms should not be taken to limit the scope of the claims.
The features of the embodiments and embodiments described herein above may be combined with each other without conflict.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.

Claims (7)

1. A method for rapidly screening multi-target active ligands in Dysosma versipellis is characterized by comprising the following steps:
the method comprises the following steps: (1) preparing an enzymatic reaction buffer solution;
(2) preparing a sample solution to be detected; dissolving a proper amount of dysosma versipellis total extract in the enzymatic reaction buffer solution to obtain a sample solution to be detected;
(3) performing affinity reaction on a sample to be detected and the multi-target enzyme; respectively taking a proper amount of a sample solution to be tested, DNA topoisomerase I, topoisomerase II, cyclooxygenase-2 and angiotensin converting enzyme 2 into a centrifugal tube, uniformly mixing, incubating at a constant temperature, eluting the centrifugal tube for multiple times by using an enzymatic buffer solution after the reaction is finished, eluting an elution retention ligand by using an organic solvent, and collecting an eluent to obtain an experimental group solution; taking the inactivated enzyme as a control group, and obtaining a control group solution by using the rest of the inactivated enzyme as the same as the experimental group;
(4) collecting peak data; taking a proper amount of the sample solution to be tested, the experimental group solution and the control group solution, freeze-drying, adding an organic solution for dissolving, analyzing by an instrument, and collecting peak data;
(5) calculating the enrichment rate to screen out active ligands in the dysosma versipellis; and (3) calculating an enrichment rate according to the peak data, and obtaining the active ligand of the screened dysosma versipellis by using the DNA topoisomerase I, the topoisomerase II, the cyclooxygenase-2 and the angiotensin converting enzyme 2 according to the enrichment rate.
2. The method for rapidly screening the multi-target active ligand in Dysosma versipellis as claimed in claim 1, wherein the method comprises the following steps: the multi-target active ligand in Dysosma versipellis obtained according to the enrichment rate is beta-anhydropodophyllotoxin, deoxypodophyllotoxin, quercetin, kaempferol and podophyllotoxin.
3. The method for rapidly screening the multi-target active ligand in Dysosma versipellis as claimed in claim 1, wherein the method comprises the following steps: in the step (1), the enzymatic reaction buffer solution is 0.1mol/L sodium phosphate solution or 0.1mol/L tris hydrochloride.
4. The method for rapidly screening the multi-target active ligand in Dysosma versipellis as claimed in claim 3, wherein: in the step (1), 50mL of 0.1mol/L tris solution and 34.5mL of 0.1mol/L hydrochloric acid are mixed uniformly and diluted to 100mL by deionized water, so as to obtain 0.1mol/L tris hydrochloride enzymatic buffer with the pH value of 7.8.
5. The method for rapidly screening the multi-target active ligand in Dysosma versipellis as claimed in claim 3, wherein: in the step (1), 46.3mL and 53.7mL of 1mol/L disodium hydrogen phosphate solution and 1mol/L sodium dihydrogen phosphate solution are respectively taken, deionized water is used for dissolving until the total volume is 1L, and the solutions are fully mixed to obtain 0.1mol/L sodium phosphate enzymatic buffer solution with the pH value of 6.8.
6. The method for rapidly screening the multi-target active ligand in Dysosma versipellis as claimed in claim 1, wherein the method comprises the following steps: and (3) in the step (2), taking a proper amount of dysosma versipellis total extract, and fully dissolving in an enzymatic reaction buffer solution to obtain a dysosma versipellis sample solution to be detected, wherein the concentration of the dysosma versipellis sample solution is 8.0 mg/mL.
7. The method for rapidly screening the multi-target active ligand in Dysosma versipellis as claimed in claim 1, wherein the method comprises the following steps: in the step (3), 100 μ L of sample solution to be tested, 10 μ L of 2U or 5U DNA topoisomerase I, 10 μ L of 2U or 4U DNA topoisomerase II, 10 μ L of 2U or 4U cyclooxygenase-2 and 0.5 μ g angiotensin converting enzyme 2 are respectively taken and uniformly mixed in a 0.2mL centrifuge tube, incubated in a 37 ℃ incubator for 40min, after the reaction is finished, the mixture is transferred to an ultrafiltration centrifuge tube with the molecular weight of 10KD, centrifuged at 10000rpm for 10min, 200 μ L of enzymatic buffer solution is added for repeated elution and centrifugation, a solvent is added to the ligand intercepted in the centrifuge tube for elution, and the eluent is collected to obtain an experimental group solution; the control group was prepared by heating inactivated DNA topoisomerase I, topoisomerase II, cyclooxygenase-2, and angiotensin converting enzyme 2 in boiling water, and the rest was the same as the experimental group.
CN202210029382.5A 2022-01-12 2022-01-12 Method for rapidly screening multi-target active ligand in dysosma versipellis Pending CN114395612A (en)

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