CN112057494A

CN112057494A - Application of swertia extract in preparing medicine for inhibiting glutathione S-transferase activity

Info

Publication number: CN112057494A
Application number: CN202010921479.8A
Authority: CN
Inventors: 覃艳; 王炜; 彭彩云; 刘斌; 杨维; 易攀; 范家龙
Original assignee: Hunan University of Chinese Medicine
Current assignee: Hunan University of Chinese Medicine
Priority date: 2020-09-04
Filing date: 2020-09-04
Publication date: 2020-12-11
Anticipated expiration: 2040-09-04
Also published as: CN112057494B

Abstract

The invention relates to the field of biochemical analysis and drug screening, and discloses application of swertia davidi extract in preparation of a drug for inhibiting activity of glutathione S-transferase. The swertia pseudochinensis extract is selected from:

and

at least one of (1). The invention also provides a pharmaceutical composition containing the swertia extract. The swertia pseudochinensis extract provided by the invention has a strong inhibition effect on the activity of glutathione S-transferase in cells, and can enhance the synergistic effect and remarkably improve the killing efficiency of tumor cells by combining the use of anti-tumor drugs.

Description

Application of swertia extract in preparing medicine for inhibiting glutathione S-transferase activity

Technical Field

The invention relates to the field of biochemical analysis and drug screening, in particular to application of swertia davidi extract in preparation of a drug for inhibiting activity of glutathione S-transferase.

Background

Glutathione S-transferases (GSTs), which are key enzymes in the glutathione binding reaction, catalyze the initial step of the glutathione binding reaction. GSTs can catalyze the combination of harmful polar compounds and glutathione in organisms, and can also discharge various potential carcinogens and lipophilic compounds from the bodies in a non-enzyme combination mode so as to achieve the aim of detoxification. Researches show that the enzyme activity level of GSTs is closely related to drug resistance of tumors and can be potential drug action targets for treating drug-resistant tumors.

It has been found that over-expressed GSTs can be observed in some tumor cells with strong drug resistance, therefore, the research of GSTs inhibitor has important significance for the research and development of anti-tumor drugs. In recent years, studies on GSTs inhibitors are increasing, and currently reported GSTs inhibitors mainly comprise ethacrynic acid and analogues thereof, TLK199 and analogues thereof, flavonoid compounds and bifunctional compounds, but most of the GSTs inhibitors are obtained by artificial synthesis, and have the defects of complex preparation process, high cost and the like.

Disclosure of Invention

The invention aims to overcome the problems in the prior art and provides application of a swertia extract in preparing a medicament for inhibiting the activity of glutathione S-transferase.

In order to achieve the above objects, the present invention provides, in a first aspect, use of an extract of swertia davidi in the preparation of a medicament for inhibiting glutathione S-transferase activity, the extract of swertia davidi being selected from the group consisting of:

at least one of (1).

Preferably, the screening method of the swertia davidi extract comprises the following steps:

(1) extracting, separating and purifying the swertia davidi to obtain an extract to be screened;

(2) mixing the extract to be screened with a probe in the presence of glutathione and glutathione S-transferase I, and screening out a compound which can not recover the fluorescence of the probe to obtain the swertia extract;

the probe contains a fluorescent group and a quenching group, and the glutathione S-transferase can catalyze the combination of the glutathione and an electron-deficient recognition site in the probe, so that the fluorescence of the probe is recovered.

Preferably, the extracting in step (1) comprises: extracting swertia davidii with an organic solvent I under reflux to obtain an extracting solution, and drying the extracting solution to obtain a total extract;

the separation process comprises the following steps: dispersing the total extract by using water, sequentially extracting by using petroleum ether, dichloromethane, ethyl acetate and n-butyl alcohol to obtain a petroleum ether extract, a dichloromethane extract, an ethyl acetate extract and an n-butyl alcohol extract, separating the petroleum ether extract by using a silica gel column, performing gradient elution by using a petroleum ether-ethyl acetate mixed solution to obtain a petroleum ether fraction, separating the ethyl acetate extract by using a silica gel column, and sequentially performing gradient elution by using a petroleum ether-acetone mixed solution and a dichloromethane-methanol mixed solution respectively to obtain an ethyl acetate fraction;

the purification process comprises the following steps: and respectively cleaning the petroleum ether fraction and the ethyl acetate fraction or performing silica gel column chromatography treatment to obtain the extract to be screened.

Preferably, the silica gel column separation adopts 200-300 mesh silica gel.

Preferably, the fluorescent group of the probe in step (2) is provided by resorufin and the quenching group is provided by 2, 4-dinitrobenzenesulfonyl chloride.

Preferably, the preparation method of the probe comprises the following steps: under the anaerobic condition, mixing a fluorescent agent with triethylamine in an organic solvent II, and then mixing with a quencher III; wherein the fluorescent agent is resorufin, and the quencher is 2, 4-dinitrobenzenesulfonyl chloride.

Preferably, the organic solvent II is selected from at least one of tetrahydrofuran, N-dimethylformamide, N-dimethylacetamide, and dimethylsulfoxide;

the mixture II at least satisfies the following conditions: stirring at 15-25 deg.C for 25-35 min;

the blend III satisfies at least the following conditions: the temperature is 15-25 ℃, and the stirring time is 10-15 h.

Preferably, the method for preparing the probe further comprises: and removing the organic solvent II from the mixed solution obtained by mixing the III, and purifying by silica gel chromatography to obtain the probe.

Preferably, in the step (2), the concentration of the probe is 3-8mg/L, the concentration of the glutathione is 180-220 μ M and the concentration of the extract to be screened is 15-25 μ M based on the glutathione S-transferase with the concentration of 10 mg/L;

the process of mixing I comprises the following steps: mixing the extract to be screened with the glutathione S-transferase I-1, and then mixing with the probe and the glutathione I-2;

the mixture I-1 at least satisfies the following conditions: the temperature is 0-40 deg.C, and the time is 10-20 min;

the mixture I-2 at least satisfies the following conditions: the temperature is 35-40 deg.C, and the time is 15-25 min.

The second aspect of the invention provides a pharmaceutical composition containing swertia extract, or pharmaceutically acceptable salt, hydrate or solvate of the swertia extract, wherein the swertia extract is selected from the group consisting of:

at least one of (1).

Through the technical scheme, the invention has the beneficial effects that:

the invention provides application of swertia extract in preparation of a medicament for inhibiting glutathione S-transferase activity, wherein multiple bonding effects exist between the swertia extract and the glutathione S-transferase, so that the swertia extract has a strong inhibition effect on intracellular glutathione S-transferase activity, and can enhance synergistic effect and remarkably improve killing efficiency of tumor cells by combining with the use of an anti-tumor medicament. Compared with the existing drugs for inhibiting the activity of glutathione S-transferase, the swertia extract provided by the invention is a natural compound, and has simple extraction process and high safety.

Drawings

FIG. 1 is a graph showing the fluorescence intensity of the reaction solution after the action of the extract to be screened (C1-C10) on GSTs in the present invention;

FIG. 2 is a graph of the inhibition of GSTs by C1 in accordance with the present invention versus C1 concentration;

FIG. 3 is a graph of the inhibition of GSTs by C2 in accordance with the present invention versus C2 concentration;

FIG. 4 is a graph of the inhibition of GSTs by C7 in accordance with the present invention versus C7 concentration;

FIG. 5 is a graph of HepG2 cell viability versus probe RP concentration in accordance with the invention;

FIG. 6 is a fluorescence spectrum of GSTs activity fluorescence assay in cell lysate extract after C7(10 μ M) treatment for 24h and EA (500 μ M) treatment for 1h, respectively, in the present invention, wherein A is probe RP blank control, B is control group, C is EA group, and D is C7 group;

FIG. 7 is a histogram of fluorescence intensity of GSTs activity in cell lysate extracts after 24h treatment with C7(10 μ M) and 1h treatment with EA (500 μ M), respectively, in accordance with the present invention;

FIG. 8 is a graph showing the expression of GSTs protein in cell lysate extracts after C7 (10. mu.M) treatment for 24h and EA (500. mu.M) treatment for 1h, respectively, in the present invention;

FIG. 9 is a confocal fluorescence microscopy image of HepG2 cells after 24h treatment with C7(10 μ M) and 1h treatment with EA (500 μ M) in accordance with the present invention;

FIG. 10 is a confocal fluorescence microscope image of HepG2 cells after 48h treatment with C7(10 μ M) and 1h treatment with EA (500 μ M) in accordance with the present invention.

FIG. 11 is a graph of HepG2 cell viability versus C7, cisplatin + C7 concentration in accordance with the present invention;

FIG. 12 is the IC of the present invention after combined treatment with C7, cisplatin + C7₅₀A value;

FIG. 13 is a graph of an apoptosis assay of C7, cisplatin + C7 combined treated HepG2 cells of the invention.

Detailed Description

The endpoints of the ranges and any values disclosed herein are not limited to the precise range or value, and such ranges or values should be understood to encompass values close to those ranges or values. For ranges of values, between the endpoints of each of the ranges and the individual points, and between the individual points may be combined with each other to give one or more new ranges of values, and these ranges of values should be considered as specifically disclosed herein.

In a first aspect, the invention provides an application of an swertia extract in preparing a medicament for inhibiting the activity of glutathione S-transferase, wherein the swertia extract is selected from the following components:

at least one of (1).

According to the invention, the screening method of the swertia extract comprises the following steps:

In the invention, the swertia davidii are extracted and treated to obtain corresponding extracts to be screened, and the extracts to be screened are screened to obtain corresponding swertia davidii extracts which can be applied to preparing medicines for inhibiting the activity of glutathione S-transferase (GSTs). Specifically, the screening method adopts a difunctional fluorescent probe containing a fluorescent group and a quenching group as a screening tool, GSTs are combined with electron-deficient recognition sites in the probe through catalysis of Glutathione (GSH), so that the fluorescent group is released, the fluorescent signal of the trigger probe is obviously enhanced, the change of the fluorescent signal is in positive correlation with the activity of the GSTs, and a compound with an inhibiting effect on the targeted GSTs in vitro can be screened by monitoring the change of the activity of the GSTs. The inventors have found that, in this preferred embodiment, an extract of swertia pseudochinensis having an inhibitory effect on GSTs can be rapidly and visually screened.

The extraction process of the extract to be screened is not particularly limited, and the target substance can be separated and extracted from the swertia. Preferably, the extraction process in step (1) comprises: extracting swertia davidii with an organic solvent I under reflux to obtain an extracting solution, and drying the extracting solution to obtain a total extract; the separation process comprises the following steps: dispersing the total extract by using water, sequentially extracting by using petroleum ether, dichloromethane, ethyl acetate and n-butyl alcohol to obtain a petroleum ether extract, a dichloromethane extract, an ethyl acetate extract and an n-butyl alcohol extract, separating the petroleum ether extract by using a silica gel column, performing gradient elution by using a petroleum ether-ethyl acetate mixed solution to obtain a petroleum ether fraction, separating the ethyl acetate extract by using a silica gel column, and sequentially performing gradient elution by using a petroleum ether-acetone mixed solution and a dichloromethane-methanol mixed solution respectively to obtain an ethyl acetate fraction; the purification process comprises the following steps: and respectively cleaning the petroleum ether fraction and the ethyl acetate fraction or performing silica gel column chromatography treatment to obtain the extract to be screened. The inventors have found that in this preferred embodiment it is advantageous to increase the separation efficiency of the extracts to be screened and the purity of the individual extracts. Preferably, the silica gel column separation adopts 200-300 mesh silica gel.

According to the invention, the fluorescent group and the quenching group contained in the probe can generate electron transfer between the fluorescent group and the quenching group, so that the probe can emit very weak fluorescent signals, in the presence of GSH, the GSTs can catalyze the combination of the GSH and electron-deficient recognition sites in the probe, so that the fluorescent groups are released, and the fluorescent signals quenched by the probe can be recovered through the GSTs. Preferably, the fluorescent group of the probe in step (2) is provided by resorufin and the quenching group is provided by 2, 4-dinitrobenzenesulfonyl chloride. The inventor finds that in the preferred embodiment, the probe has stronger specificity, selectivity and sensitivity on the activity of GSTs, the cytotoxicity of the probe is low, the synthesis method is simpler and the cost is reduced.

According to the invention, the preparation method of the probe comprises the following steps: under the anaerobic condition, mixing a fluorescent agent with triethylamine in an organic solvent II, and then mixing with a quencher III; wherein the fluorescent agent is resorufin, and the quencher is 2, 4-dinitrobenzenesulfonyl chloride. Wherein, the anaerobic condition can be realized by introducing nitrogen or argon. The inventors have found that in this preferred embodiment, it is advantageous to increase the yield of the probe.

The organic solvent used in the preparation of the probe is not particularly limited, and the probe can be used for the synthesis reaction of the fluorescent agent, the triethylamine and the quencher. Preferably, the organic solvent II is at least one selected from the group consisting of tetrahydrofuran, N-dimethylformamide, N-dimethylacetamide, and dimethylsulfoxide.

In the present invention, the conditions for mixing II and III in preparing a probe are not particularly limited, and generally, the conditions for mixing include temperature, stirring speed, and stirring time. Preferably, the mixture II satisfies at least the following conditions: stirring at 15-25 deg.C for 25-35 min; the blend III satisfies at least the following conditions: the temperature is 15-25 ℃, and the stirring time is 10-15 h.

According to the present invention, the method for preparing the probe further comprises: and removing the organic solvent II from the mixed solution obtained by mixing the III, and purifying by silica gel chromatography to obtain the probe. Illustratively, the silica gel chromatography purification process uses a petroleum ether-ethyl acetate mixture as an eluent to improve the purity of the purified probe.

According to the invention, in the step (2), the concentration of the probe is 3-8mg/L, the concentration of the glutathione is 180-220 μ M and the concentration of the extract to be screened is 15-25 μ M based on the glutathione S-transferase with the concentration of 10 mg/L. The inventor finds that the swertia extract which has an inhibiting effect on GSTs can be screened more sensitively and accurately in the preferred embodiment.

According to the invention, the process of mixing I comprises: mixing the extract to be screened with the glutathione S-transferase I-1, and then mixing with the probe and the glutathione I-2; the mixture I-1 at least satisfies the following conditions: the temperature is 0-40 deg.C, and the time is 10-20 min; the mixture I-2 at least satisfies the following conditions: the temperature is 35-40 deg.C, and the time is 15-25 min. The inventor finds that under the preferred embodiment, the substance to be screened and the glutathione S-transferase are mixed I-1 to uniformly mix the extract to be screened and the GSTs, so that when the substance to be screened and the GSTs are mixed I-2, if the extract to be screened has an inhibiting effect on the GSTs, the fluorescence change of the probe has higher sensitivity.

at least one of (1).

According to the present invention, the pharmaceutical composition may be a pharmaceutical composition for inhibiting glutathione S-transferase activity, or a pharmaceutical composition for anti-tumor. The pharmaceutical composition can also contain a pharmaceutically acceptable carrier, and the dosage form of the pharmaceutical composition can be any one dosage form such as tablets, capsules, pills, suspensions and the like.

The present invention will be described in detail below by way of examples.

In the following examples, UV-1800 UV spectrophotometer was used for UV-visible absorption spectroscopy, F-2500 spectrophotometer was used for fluorescence measurement, Agilent 1100 was used for High Performance Liquid Chromatography (HPLC), Bruker Ascend 600MHz spectrometer (Bruker Biospin GmbH, Germany) equipped with 5 mm CPP BBO was used for nuclear magnetic resonance spectroscopy at 25 ℃, Olinbas FY1200 (objective 60 fold) was used for confocal fluorescence imaging, and K2 cell-measuring instrument (Nexcelom, USA) was used for apoptosis analysis.

In the following examples, GSTs, GSH, cysteine (Cys) and homocysteine (Hcy) were purchased from Sigma Aldrich, usa, GSTs monoclonal antibody was purchased from Abbkine Scientific co, usa, Ltd, trypsin, Bovine Serum Albumin (BSA), cytochrome c (cytc), Lysozyme (Lysozyme), vitamin c (vc) was purchased from Solarbio T, BCA protein detection kit was purchased from beyond, Ethenoic Acid (EA) was purchased from shanghai-source biotechnology limited, resorufin was purchased from tokyo chemical, 2, 4-dinitrobenzenesulfonyl chloride was purchased from china energy chemical group, and acetylcholinesterase (AchE) and butylcholinesterase (BchE) were extracted and purified from brain and blood, respectively; other raw materials and solvents were all commercial HPLC grade reagents; all solutions were made up in distilled water and stored at 4 ℃.

The room temperature is 25. + -. 5 ℃ unless otherwise specified.

Examples

The preparation process of the probe RP comprises the following steps:

(1-1) introducing N₂Under the condition of (1), 0.28mmol of 2, 4-dinitrobenzenesulfonyl chloride is dissolved in 10mL of tetrahydrofuran to obtain a quencher solution;

(1-2) introducing N₂Adding 0.28mmol of triethylamine into 5mL of tetrahydrofuran, mixing with 0.28mmol of resorufin powder II, stirring at 20 ℃ for 30min, mixing with the quencher solution obtained in the step (1-1) III, and stirring at 20 ℃ for 12h to obtain a mixed solution;

(1-3) after evaporating the mixture obtained in the step (1-2) to remove tetrahydrofuran, performing silica gel chromatography purification by using a petroleum ether-ethyl acetate mixture (the volume ratio of petroleum ether to ethyl acetate is 2:1) as an eluent to obtain a probe RP (yellow solid, 8.3mg, yield 27.7%).

The preparation process of the swertia pseudochinensis extract to be screened comprises the following steps:

(2-1) extracting swertia davidi (dry whole plant) with 95% ethanol under reflux for 3 times (2 h/time), mixing extractive solutions, rotary evaporating to recover solvent, and vacuum drying to obtain total extract;

(2-2) adding 2L of water into the total extract obtained in the step (2-1), stirring and ultrasonically dispersing the extract in the water to obtain a suspension, sequentially and respectively extracting with different solvents of petroleum ether, dichloromethane, ethyl acetate and n-butyl alcohol, combining organic solvent layers, and respectively recovering under reduced pressure to obtain extracts of 4 extraction parts: petroleum ether extract, dichloromethane extract, ethyl acetate extract and n-butanol extract;

(2-3) subjecting the petroleum ether layer extract obtained in the step (2-2) to 200-mesh and 300-mesh silica gel column chromatography, and performing silica gel column chromatography by using petroleum ether: gradient elution is carried out on ethyl acetate (1:0-0:1) by a solvent system to obtain 7 fractions (FrA1, FrA2, … and FrA7), the fraction FrA3 is repeatedly washed by different solvents to obtain an extract C1 to be screened, the fraction FrA4 is repeatedly washed and recrystallized by different solvents to obtain an extract C2 to be screened, and the fraction FrA6 is subjected to petroleum ether: repeatedly performing silica gel column chromatography with acetone solvent system to obtain extracts C3, C4, C5 and C7; fraction FrA7 was purified from petroleum ether: isocratic elution is carried out on a solvent system of acetone (9:1) on a silica gel column to obtain an extract C8 to be screened;

subjecting the ethyl acetate layer extract obtained in the step (2-2) to 200-mesh and 300-mesh silica gel column chromatography, and performing silica gel column chromatography by using petroleum ether: a solvent system gradient of acetone (1:0-0:1) followed by dichloromethane: gradient elution with methanol (1:0-0:1) solvent system afforded 9 fractions (FrB1, FrB2, …, FrB9), fraction FrB7 was purified by dichloromethane: repeatedly performing silica gel column chromatography in methanol solvent system to obtain compound extract C9, C10, fraction FrB9, and separating with dichloromethane: silica gel column chromatography was repeated in an isocratic solvent system of methanol (98: 2) to obtain extract C6 to be screened.

The specific structural formula of the extract C1-C10 to be screened is shown in Table 1.

TABLE 1

The screening process of the swertia:

11 parts of 100. mu.L of PBS buffer containing GSTs and having pH7.4 were added to the substance to be screened (C1-C10) and a blank as a control so that the concentrations of C1-C10 were 20. mu. M, GSTs, respectively, and then, after interacting for 15min at room temperature, mixed with probe RP and GSH so that the concentration of probe RP was 5. mu.g/mL and the concentration of GSH was 200. mu.M, incubated at 37 ℃ for 20min, and the color of each reaction solution was observed and the fluorescence intensity was measured.

As shown in fig. 1, the visual change of the color of the reaction solution indicates that, at the same concentration (20 μ M), the substances to be screened (C1-C10) have different effects on the activity of GSTs, wherein, 3 compounds (C1, C2 and C7) have stronger inhibitory action on the activity of GSTs, and the reaction solution of the three compounds is nearly colorless, i.e., the three compounds can inhibit the activity of GSTs, and cannot trigger the binding of GSH with the electron-deficient recognition sites in the probe RP, and the reaction of releasing the fluorescent signal cannot proceed, so that the fluorescent group of the probe RP cannot be released, and red fluorescence cannot be observed. C1, C2 and C7 screened by this screening process are inhibitors of GSTs.

Test example

1. Relationship of C1, C2 and C7 concentrations to inhibition of GSTs

The fluorescence intensity was measured by taking 3 groups of 100. mu.L of PBS buffer containing GSTs and having pH7.4, namely, group C1, group C2 and group C7 so that the concentration of GSTs in the corresponding group was 10. mu.g/mL and the concentration of C1, C2 or C3 was 0. mu.M, 5. mu.M, 10. mu.M, 15. mu.M and 20. mu.M, and then, after interacting with each other at room temperature for 15min, mixing with probe RP and GSH so that the concentration of probe RP was 5. mu.g/mL and the concentration of GSH was 200. mu.M, incubating for 20min under 37 ℃ adjustment. As shown in FIGS. 2, 3 and 4, the inhibitory effects of C1, C2 and C7 on GSTs are concentration-dependent, IC₅₀9.626 μ M, 9.626 μ M and 9.410 μ M, respectively.

2. Molecular docking of C1, C2, C7 with GSTs

Molecular docking studies were performed using molecular docking software (moe.2015), first, C1, C2, C7 were transformed into 3D mode by Chem3D (PerkinElmer), then the energy of the compound was protonated and minimized, thus obtaining stable 3D structure and saved in ". moe file" format; a new database is created in the MOE, the saved ". MOE file" is imported into the database, and the GSTs amino acid sequences are downloaded from the NCBI database (PDB code: 5x79), the crystal structures of the GSTs are transformed and their active binding sites are virtualized to interface with the compounds.

The results of molecular docking show that C1 binds to amino acid residues Ala 407 and Thr 317 through hydrogen bonds, C2 binds to Thr 232 and Ser 229, and C7 binds to Glu 78, Ser 114, Ser 116 and Phe 57, and the model explains the possible interactions of C1, C2 and C7 with GSTs and the regulation mechanism of GSTs activity.

3. Fluorescent monitoring of GSTs Activity in HepG2 cells under C7 Regulation

MTT assay of probe RP: HepG2 cells (5X 10)³Individual cells/well) in 100. mu.L DMEM medium (10% fetal bovine serum, 1% streptomycin, 37 ℃ C., 5% CO)₂) Culturing in a 96-well plate for 48h, and washing with PBS for 3 times; the medium (1% fetal bovine serum) containing the probe RP prepared in preparation examples in different concentrations was added to each well, and the concentrations of the probe RP were 0. mu.g/mL, 1. mu.g/mL, 2.5. mu.g/mL, 5. mu.g/mL, 10. mu.g/mL, 20. mu.g/mL, and 25. mu.g/mL, respectively, and further incubated for 24 hours; after the original DMEM is removed, 100 mu L of fresh DMEM medium is added into each well, 1% MTT solution (5mg/mL) is added into each well, the mixture is incubated for 4 hours, finally 100 mu L of DMSO is added into each well, the mixture is kept in the dark and is strongly shaken for 15min, and then the cell viability is determined. As shown in FIG. 5, probe RP was less cytotoxic to HepG2 cells.

Fluorescent monitoring of GSTs activity in HepG2 cells: HepG2 cells in DMEM (10% fetal bovine serum, 1% streptomycin, 37 ℃, 5% CO)₂) After culturing for 48h, treating by different methods (the cells of the control group are not treated by any medicine, 500 mu M EA is added into the cells of the EA group for 1h of incubation, and 10 mu M C7 is added into the cells of the C7 group for 24h of incubation), and respectively obtaining cell lysate extracts, wherein EA is an effective inhibitor of all GST isozymes, and the EA group is used as a positive control;

cell lysate extracts were prepared as follows: collecting cells, treating with trypsin and centrifuging for 5min, adding 1mL cold PBS to clean cells, lysing with 0.5mL frozen cell lysate on ice for 30min, performing pulse ultrasound on ice for 3 times, 5s each time, centrifuging at 4 deg.C at 12000rpm for 20min, and collecting supernatant; the concentration of cell supernatant (562nm) was quantitatively determined using BCA kit, expression of GSTs in cell extracts was detected using Western felt, proteins (20. mu.g) were separated using SDS-PAGE (1.5h), transferred to PVDF membrane (Millipore, USA) for 1.5h, 5% skim milk was membrane-sealed at room temperature for 2h, and the expression level of GSTs protein was detected, as shown in FIG. 8;

cell lysate extracts (1 μ L) of the control group, EA group, and C7 group were mixed with probe RP (final concentration 5 μ g/mL) respectively, added to 100 μ L of PBS buffer (ph7.4), incubated at 37 ℃ for 20min, and then subjected to fluorescence intensity measurement (Ex/Em ═ 572/585nm) using PBS buffer (ph7.4) containing probe RP (final concentration 5 μ g/mL) as a blank control, and the results are shown in fig. 6 and fig. 7.

Fluorescence imaging monitoring of GSTs activity in HepG2 cells: HepG2 cells were plated in 12-well plates with 1000. mu.L of DMEM (10% fetal bovine serum, 1% streptomycin, 37 ℃ C., 5% CO)₂) After 48h incubation and PBS wash, the cells were incubated with C7 (10. mu.M) in DMEM medium containing 1% fetal bovine serum for 24h and 48h, respectively, and pre-treated with ethacrylic acid (EA, 500. mu.M) for 60min before fluorography, and then incubated with Hoechst 33342 (150. mu.L, 30min) and probe RP (10. mu.g/mL, 20min), respectively, followed by confocal laser scanning to observe fluorescence, as shown in FIGS. 9 and 10.

As shown in fig. 9 and 10, HepG2 cells exhibited intense red fluorescence after incubation with probe RP, which simultaneously monitored the activity of GST in the cytoplasm and nucleus, as evidenced by the superposition of red fluorescence from RP and blue fluorescence from Hoechst 33342; in contrast, EA-treated cells showed ultra-weak fluorescence due to strong inhibition of GSTs by EA, and the fluorescence signal of C7-treated cells was also significantly decreased, indicating that C7 had an inhibitory effect on intracellular GSTs activity. As the incubation time was extended to 48h, the fluorescence of the C7 treated cells was significantly reduced and EA treated cells had little apparent fluorescence. As shown in FIGS. 6-8, fluorescence measurements and western blot results showed that the expression of GSTs was lowest in EA-treated cells, and that C7 immediately followed the EA, which was consistent with the fluorescence imaging measurements described above.

4. Apoptosis assay

HepG2 cells (5X 10)³Individual cells/well) in 100. mu.L DMEM medium (10% fetal bovine serum, 1% streptomycin, 37 ℃ C., 5% CO)₂) Is/are as followsCulturing in a 96-well plate for 48h, and washing with PBS for 3 times; adding culture medium (1% fetal bovine serum) containing different concentrations of C7 into wells of a group C7, adding culture medium (1% fetal bovine serum) containing different concentrations of Cisplatin into wells of a group Cisplatin (Cisplatin), adding culture medium (1% fetal bovine serum) containing different concentrations of C7 and Cisplatin into wells of a group Cisplatin + C7, wherein the concentrations of C7 or Cisplatin in the groups C7 and Cisplatin are respectively 0. mu.M, 1. mu.M, 3. mu.M, 5. mu.M, 7. mu.M, 10. mu.M and 15. mu.M, and the concentrations of Cisplatin in the groups Cisplatin + C7 are respectively 0. mu.M, 1. mu.M, 3. mu.M, 5. mu.M, 7. mu.M, 10. mu.M and 15. mu.M, the concentration of C7 is 5. mu.M, and further 24 h; after removing the original DMEM, 100. mu.L of fresh DMEM medium was added to each well, 1% MTT solution (5mg/mL) was added thereto, and the mixture was incubated for 4 hours, and finally 100. mu.L of DMSO was added to each well, which was vigorously shaken in the dark for 15min, and then the cell viability was measured, and the results are shown in FIG. 11 and FIG. 12.

HepG2 cells were cultured in 1000. mu.L DMEM medium (10% fetal bovine serum, 1% streptomycin, 37 ℃ C., 5% CO) in 6-well plates₂) After 48h incubation, cells were cultured in DMEM medium containing 1% fetal bovine serum, and C7 (10. mu.M), Cisplatin (Cisplatin, 10. mu.M), Cisplatin and C7(Cisplatin + C7, 10. mu.M + 5. mu.M), and a blank were added as controls, incubated for 48h, washed with PBS, treated with trypsin (without EDTA) for 4min, and finally collected, stained with Annexin-FITC and PI, and apoptosis was detected, as shown in FIG. 13.

As can be seen in FIG. 11, the cell viability was concentration dependent for C7, cisplatin, and C7, with the strongest cell killing effect for cisplatin combined with C7. FIG. 12 shows the IC of C7, cisplatin, and C7 in combination₅₀Difference in value, IC of C7₅₀The highest value was 23.97. + -. 0.023. mu.M, IC for cisplatin₅₀The value was 9.24. + -. 0.009. mu.M, whereas the IC was obtained after cisplatin in combination with C7₅₀The value is obviously reduced to 3.54 +/-0.018 mu M, and the effect of synergistically killing tumor cells by reducing the activity of GSTs through C7 when the cisplatin is combined with C7 is shown. The MTT experimental result is shown in FIG. 13, the apoptosis induced by the combination of cisplatin and C7 is changed from 58.22% to 80.89%, and the C7 alone does not show the capacity of obviously inducing apoptosis, which indicates that the C7 causes the sensitization of cisplatin on HepG2 cells by inhibiting the activity of GSTs。

The preferred embodiments of the present invention have been described above in detail, but the present invention is not limited thereto. Within the scope of the technical idea of the invention, many simple modifications can be made to the technical solution of the invention, including combinations of various technical features in any other suitable way, and these simple modifications and combinations should also be regarded as the disclosure of the invention, and all fall within the scope of the invention.

Claims

1. The application of the swertia extract in preparing the medicament for inhibiting the activity of glutathione S-transferase is characterized in that the swertia extract is selected from the following components:

at least one of (1).

2. The use as claimed in claim 1, wherein the screening method of the swertia extract comprises the following steps:

3. The use according to claim 2, wherein the extraction in step (1) comprises: extracting swertia davidii with an organic solvent I under reflux to obtain an extracting solution, and drying the extracting solution to obtain a total extract;

4. The use as claimed in claim 3, wherein the silica gel column separation is 200-300 mesh silica gel.

5. Use according to claim 2, wherein in step (2) the fluorescent group of the probe is provided by resorufin and the quenching group is provided by 2, 4-dinitrobenzenesulfonyl chloride.

6. The use according to claim 5, wherein the method of preparing the probe comprises the steps of: under the anaerobic condition, mixing a fluorescent agent with triethylamine in an organic solvent II, and then mixing with a quencher III;

wherein the fluorescent agent is resorufin, and the quencher is 2, 4-dinitrobenzenesulfonyl chloride.

7. The use according to claim 6, wherein the organic solvent II is selected from at least one of tetrahydrofuran, N-dimethylformamide, N-dimethylacetamide and dimethylsulfoxide;

8. The screening method according to claim 6 or 7, wherein the preparation method of the probe further comprises: and removing the organic solvent II from the mixed solution obtained by mixing the III, and purifying by silica gel chromatography to obtain the probe.

9. The use as claimed in claim 2, wherein in step (2), the concentration of the probe is 3-8mg/L, the concentration of the glutathione is 180-220 μ M, and the concentration of the extract to be screened is 15-25 μ M, based on the glutathione S-transferase with the concentration of 10 mg/L;

10. A pharmaceutical composition is characterized by comprising an extract of swertia pseudochinensis, or a pharmaceutically acceptable salt, hydrate or solvate of the extract of swertia pseudochinensis, wherein the extract of swertia pseudochinensis is selected from the group consisting of:

at least one of (1).