CN110575461A - New application of liquorice - Google Patents
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Abstract
The invention discloses the induction action of active ingredients of liquiritin, liquiritigenin, isoliquiritin, isoliquiritigenin, chalcone A and glycyrrhetinic acid in two main categories of flavonoids and pentacyclic triterpenes in liquorice on efflux transporter P-gp. Mainly through researching mRNA expression, protein expression and efflux transport function of P-gp in cells after administration, the active ingredient in liquorice has induction effect on P-gp. Provides a basis for reasonable application of medicine compatibility and clinical pharmacological research, provides a basis for further understanding of the detoxification mechanism of the liquorice, and has important significance for safe and effective clinical medication.
Description
Technical Field
The invention belongs to the technical field of medicines, relates to a new application of liquorice, and particularly relates to a new application of liquorice in inducing P-gp so as to reduce foreign substances from entering cells.
Background
The liquorice is used as a traditional classical Chinese medicine and has the pharmacological effects of tonifying spleen and qi, eliminating phlegm and stopping cough, relieving spasm and pain, clearing away heat and toxic materials, harmonizing the drugs and the like. Licorice root can be used singly or in combination. When used in combination, the Chinese herbal medicines are mostly used as the raw materials and have the functions of detoxification and synergism. P-gp is used as the classical category of efflux transporters, the induction effect of liquorice on the efflux transporters is researched, the detoxification mechanism of liquorice is better understood, and the traditional Chinese medicine has the characteristics of complex diversity and narrow therapeutic window of some toxic traditional Chinese medicines.
Disclosure of Invention
The invention aims to disclose a new application of liquorice in inducing P-gp.
The invention utilizes two major active ingredients of flavone and pentacyclic triterpene in liquorice, namely liquiritin, liquiritigenin, isoliquiritin, isoliquiritigenin, licochalcone A and glycyrrhetinic acid, to respectively act on LS-180 and Caco-2 cells, and shows that the active ingredients in the liquorice have induction effect on P-gp in RT-PCR, WesternBlot, transport test and function verification test.
Drawings
FIG. 1: influence of main active ingredients in licorice on expression of LS-180 cell P-gp mRNA;
FIG. 2: influence of main active ingredients in licorice on P-gp protein;
FIG. 3: influence of main active ingredients in liquorice on LS-180 cell P-gp efflux rhodamine 123;
FIG. 4: influence of main active ingredients in liquorice on P-gp efflux rhodamine 123 of Caco-2 cells;
FIG. 5: the effect of Caco-2 cells on P-gp efflux rhodamine 123 as a function of efflux time after administration.
Detailed Description
The object of the invention is achieved by the following examples:
Example 1: RT-PCR
1. Materials and methods
(1) Instrument and reagent
the instrument comprises the following steps: heraccell 150i carbon dioxide incubator: product of Thermo Scientific corporation, usa.
Model AB54-S analytical balance: the product of the mertler-tollido instruments (shanghai) ltd.
Model 3K15 high speed refrigerated centrifuge: product of Sigma, Germany.
Sorvall Legend Micro 17R high speed refrigerated centrifuge: product of Thermo Scientific corporation, usa.
Eppendorf Mastercycler ep Realplex2qPCR instrument: product of Eppendorf company, germany.
24-well polycarbonate membrane transfer plate, cell culture dish, centrifuge tube, pipette, Corning Costar Co
Reagent: liquiritin reference (lot No. 17030801), liquiritigenin reference (lot No. 17022104), isoliquiritigenin reference (lot No. 17031204), isoliquiritigenin reference (lot No. 17012503), chalcone a reference (lot No. 17032501), chinese drug biological product assay; beta-glycyrrhetinic acid control (batch No. 1129A022), Solarbio corporation; rifampin control (batch No. 11246), bosentan (batch No. HYA0013), saquinavir control (batch No. 05907). DMSO, analytical grade, Kaixin chemical industries, Inc. of Tianjin. RPIM1640 medium, Gibco Laboratories, Inc.; DMEM medium was from Hyclone. Trypsin-EDTA, cell cryopreservation, Gibco Inc. Fetal Bovine Serum (FBS), bio-engineering ltd, national sea, langzhou. Quercetin, batch number: q8010; penicillin streptomycin mixed solution (100 ×), batch number: 20170907, respectively; phospatebuffered Saline (PBS) (1 ×), lot number: 8112372, respectively; DEPC water, batch number: 20170727, available from Solarbio. Ultrapure water, HBSS, self-made in the laboratory. TRNzol Reagent: manufactured by tiangen biochemical technology (beijing) ltd (tiangenbiotech (beijing) co. ltd), lot No.: q5607. Reverse transcription kit Transcriptor First StrandcDNA Synthesis kit, produced by Roche, lot number: 21457720. real-time quantification kit Fast StartUniversal SYBR Green Master (Rox), produced by Roche corporation, lot number: 24759100. PCR primers: synthesized by Shenzhen Huada Gene science and technology Limited (Huada) (BGI (Shenzhen, China)), the concentration of the primers is 10 mu M after the primers are dissolved by double distilled water, and the primer sequences are shown in Table 1.
TABLE 1 efflux transporter RT-PCR primer sequences
(2) Cell culture
After LS-180 cells were recovered at 2X 105Perml density was inoculated into 100mm by 20mm cell culture dishes, 10mL RPMI-1640 medium containing 10% FBS, 1% diabody (penicillin 10000U/mL and streptomycin 10000. mu.g/mL) was added. Cells were incubated at 37 ℃ with 5% CO2The culture is carried out in the incubator, the liquid is changed every other day, when the cells grow to 80-90%, 0.25% pancreatin is used for digestion and passage, and LS-180 cells in logarithmic phase are selected for experiment.
(3) Experiment grouping
Blank group, positive drug group (rifampicin 10. mu.M, bosentan 10. mu.M, saquinavir 10. mu.M, quercetin 10. mu.M) and administration group (liquiritin 10. mu.M, liquiritigenin 10. mu.M, isoliquiritigenin 10. mu.M, licochalcone A1. mu.M and glycyrrhetinic acid 10. mu.M).
(4) Experimental methods
LS-180 at cell density 2 x105Inoculating into Transwell polycarbonate membrane 24-well plate, changing culture medium every other day, continuously changing blank and medicated culture medium for 3 days, 7 days, after 10 days, removing culture medium by suction, extracting total RNA by Trizol method, and reverse transcribing cell total RNA into cDNA according to the operation instruction of reverse transcription kit. The cDNA was subjected to real-time fluorescent quantitative PCR according to the instructions of Fast StartUniversal SYBR Green Master (Rox) of Roche. The PCR reaction conditions are as follows: 95 ℃ for 10min, 95 ℃ for 15s, 60 ℃ for 1min, 40 cycle, beta-actin is the reference gene, 3 times of parallel experiments are carried out by setting the same gene of the same template with 3 times of holes.
(5) Data processing and statistics
All measurements were entered using Microsoft excel to calculate mean, standard deviation and t-test for significance from the blank group for each group.
2. Test results
Influence of major active ingredients in licorice on expression of LS-180 cell P-gp mRNA: the active ingredients in licorice induced the expression of P-gp mRNA to different extents, see Table 2, FIG. 1.
TABLE 2 influence of active ingredients in Glycyrrhiza on the expression of P-gp mRNA
Note: p <0.05, P <0.01 compared to blank group.
Example 2: western Blot
3. Materials and methods
(1) Instrument and reagent
The instrument comprises the following steps: an incubator: product of Thermo Scientific corporation, usa.
Model AB54-S analytical balance: the product of the mertler-tollido instruments (shanghai) ltd.
Model 3K15 high speed refrigerated centrifuge: product of Sigma, Germany.
Sorvall Legend Micro 17R high speed refrigerated centrifuge: product of Thermo Scientific corporation, usa.
Power supply of basic electrophoresis apparatus (Weetake Limited ELITE 300PLUS E5W0541)
Protein imprinting instrument (Weitaike Limited company YRdmies SW07D0567)
Desk type constant temperature oscillator (China Shanghai essence macro experimental facilities Co., Ltd. THZ-312)
Gel imaging analysis System (U.S. Li-COR Odyssey two-color infrared laser imaging System)
24-well polycarbonate membrane transfer plate, cell culture dish, centrifuge tube, pipette, Corning Costar Co
Reagent: liquiritin reference (lot No. 17030801), liquiritigenin reference (lot No. 17022104), isoliquiritigenin reference (lot No. 17031204), isoliquiritigenin reference (lot No. 17012503), chalcone a reference (lot No. 17032501), chinese drug biological product assay; beta-glycyrrhetinic acid control (batch No. 1129A022), Solarbio corporation; rifampicin control (batch No. 11246), bosentan (batch No. HYA 0013). DMSO, analytical grade, Kaixin chemical industries, Inc., Tianjin; RPIM1640 medium, Gibco Laboratories, Inc.; DMEM medium was from Hyclone. Trypsin-EDTA, cell cryopreservation, Gibco; fetal Bovine Serum (FBS), bio-engineering ltd, national sea, langzhou; penicillin streptomycin mixed solution (100 ×), batch number: 20170907, respectively; phospate Buffered Saline (PBS) (1 ×), lot number: 8112372, respectively; DEPC water, batch number: 20170727, available from Solarbio. Ultrapure water, HBSS, self-made in the laboratory. ABCB1(P-gp), batch number: a11747; ABCG2(BCRP), lot number: a6159 and ABCC2(MRP2), lot number: a8405 antibody was purchased from abclonatechology.gapdh antibody, lot No.: ab181602 was purchased from Abcam. Anti-Rabbit secondary antibody, batch number: a23920 is purchased from Abkkine.
(2) Cell culture
After LS-180 cells were recovered at 2X 105Perml density was inoculated into 100mm by 20mm cell culture dishes, 10mL RPMI-1640 medium containing 10% FBS, 1% diabody (penicillin 10000U/mL and streptomycin 10000. mu.g/mL) was added. Cells were incubated at 37 ℃ with 5% CO2The culture is carried out in the incubator, the liquid is changed every other day, when the cells grow to 80-90%, 0.25% pancreatin is used for digestion and passage, and LS-180 cells in logarithmic phase are selected for experiment.
(3) Experiment grouping
Blank group, positive drug group (rifampicin 10. mu.M, bosentan 10. mu.M) and administration group (liquiritin 10. mu.M, liquiritigenin 10. mu.M, isoliquiritigenin 10. mu.M, licochalcone A1. mu.M and glycyrrhetinic acid 10. mu.M).
(4) Experimental methods
LS-180 at cell density 2 x105Inoculating into Transwell polycarbonate membrane 6-well plate, changing culture medium every other day, continuously changing blank and medicated culture medium for 7 days, removing culture medium by suction, cracking cell with lysis solution RIPA, extracting total protein, operating according to kit method, drawing standard curve and calculating protein amount in each sample. SDS-PAGE protein electrophoresis, each sample is taken with a volume of 20 mug protein, electrophoresis is carried out, a model is transferred, a confining liquid containing 5% skimmed milk powder is used, shaking table oscillation is carried out for 1.5h, primary antibody is incubated overnight at 4 ℃, TBST is washed for 3 times, horseradish enzyme labeled goat anti-mouse IgG secondary antibody is incubated for 2h at room temperature, TBST is washed for 3 times, ECL reagent kit is used for showing clear bands on PVDF membrane, and gel imager is used for imaging.
(6) Data processing and statistics
Results were grey scale analyzed using Gel-Pro Analyzer 4 software.
2. Results of the experiment
Influence of the main active ingredients in licorice on P-gp protein: the main active ingredients in licorice significantly increased the expression of P-gp protein, see FIG. 2, Table 3.
TABLE 3P-gpwestern blot protein Gray values
C is control
P-1 is rifampicin
P-2 is bosentan
S-1-6 is liquitin, liquitinigenin, isoliquitinin, isoliquitinigenin, liquitinigenin, Licochalcone
Example 3: efflux function experiment-flow cytometer and Varioskan Flash multifunctional reading instrument for detecting LS-180 cell P-gp efflux function
1. Materials and methods
(1) Instrument and reagent
The instrument comprises the following steps: incubator, Sorvall Legend Micro 17R high speed refrigerated centrifuge, Varioskan Flash multifunctional reader: product of Thermo Scientific corporation, usa.
Model AB54-S analytical balance: the product of the mertler-tollido instruments (shanghai) ltd.
Model 3K15 high speed refrigerated centrifuge: product of Sigma, Germany.
BD FACSAria II flow cytometer: BD company, usa.
24-well polycarbonate membrane transfer plate, cell culture dish, centrifuge tube, pipette, Corning Costar Co
Reagent: liquiritin reference (lot No. 17030801), liquiritigenin reference (lot No. 17022104), isoliquiritigenin reference (lot No. 17031204), isoliquiritigenin reference (lot No. 17012503), chalcone a reference (lot No. 17032501), chinese drug biological product assay; beta-glycyrrhetinic acid control (batch No. 1129A022), Solarbio corporation; rifampin control (batch No. 11246), bosentan (batch No. HYA 0013); rhodamine 123, batch 1015A053, available from Sigma; DMSO, analytical grade, Kaixin chemical industries, Inc., Tianjin; RPIM1640 medium, Gibco Laboratories, Inc.; DMEM medium was from Hyclone. Trypsin-EDTA, cell cryopreservation, Gibco; fetal Bovine Serum (FBS), bio-engineering ltd, national sea, langzhou; penicillin streptomycin mixed solution (100 ×), batch number: 20170907, respectively; phospate Bufferedline (PBS) (1 ×), lot number: 8112372, respectively; DEPC water, batch number: 20170727, available from Solarbio. Ultrapure water, HBSS, self-made in the laboratory.
(2) Cell culture
After LS-180 cells were recovered at 2X 105Perml density was inoculated into 100mm by 20mm cell culture dishes, 10mL RPMI-1640 medium containing 10% FBS, 1% diabody (penicillin 10000U/mL and streptomycin 10000. mu.g/mL) was added. Cells were incubated at 37 ℃ with 5% CO2The culture is carried out in the incubator, the liquid is changed every other day, when the cells grow to 80-90%, 0.25% pancreatin is used for digestion and passage, and LS-180 cells in logarithmic phase are selected for experiment.
(3) Experiment grouping
Flow cytometry: blank group, positive drug group (rifampicin 10 μ M, bosentan 10 μ M) and administration group (liquiritin 10 μ M, liquiritigenin 10 μ M, isoliquiritin 10 μ M, isoliquiritigenin 10 μ M, licochalcone A1 μ M and glycyrrhetinic acid 10 μ M), positive inhibitor group verapamil 25 μ M.
Varioskan Flash multifunctional reader: blank group, positive drug group (Rifampicin 10 μ M, bosentan 10 μ M) and administration group (liquiritin, liquiritigenin, isoliquiritigenin-5 μ M, 12.5 μ M, 25 μ M, 50 μ M; licochalcone A, glycyrrhetinic acid-2.5 μ M, 5 μ M, 10 μ M, 20 μ M), positive inhibitor group verapamil 25 μ M.
(4) Experimental methods
Flow cytometer
LS-180 at cell density 2 x105Inoculating into 12-well plate of Transwell polycarbonate membrane, changing culture medium every other day, continuously changing blank and medicated culture medium for 7 days, removing culture medium by suction, adding HBSS solution preheated at 37 deg.C, balancing at 37 deg.C for 20min, removing HBSS by suction, adding 1.5mL preheated HBSS solution containing 0.5 μ M rhodamine 123, incubating at 37 deg.C for 15min and 30min respectively, removing HBSS by suction, washing with cold PBS for 3 times, adding blank HBSS solution (positive inhibitor group and HBSS solution containing 25 μ M verapamil), incubating for 15min, suspending 0.125% pancreatin 500 μ l cold PBS, and measuring drug concentration in cells in flow cytometer with excitation wavelength of 485nm and emission wavelength of 525 nm. The relative fluorescence intensity of rhodamine 123 ═ A was calculated according to the following formulaExperimental group/ABlank control group*100％。
Varioskan Flash multifunctional reading instrument
LS-180 at cell density 2 x105Inoculating into Transwell polycarbonate membrane 96 pore plate, changing culture medium every other day, continuously changing blank and medicated culture medium for 7 days, removing culture medium by suction, adding preheated HBSS solution at 37 deg.C, balancing at 37 deg.C for 20min, removing HBSS by suction, adding preheated HBSS solution containing 0.1 μ M rhodamine 123 1.5mL, incubating at 37 deg.C for 15min respectively, removing HBSS by suction, washing with cold PBS for 3 times, adding blank HBSS solution (positive inhibitor group and HBSS solution containing 25 μ M verapamil), incubating for 15min, removing HBSS solution by suction, adding 200 μ l lysis solution containing 0.1% triton-X100, measuring in Varioskan Flash multifunctional reader with excitation wavelength of 485nm and emission wavelength of 525nmThe concentration of the drug in the cells is determined. The relative fluorescence intensity of rhodamine 123 ═ A was calculated according to the following formulaExperimental group/ABlank control group100%. 3 multiple wells are arranged on the same gene of the same template, and 3 times of parallel experiments are carried out.
(5) Data processing and statistics
All measurements were entered using Microsoft excel to calculate mean, standard deviation and t-test for significance from the blank group for each group.
2. Test results
Influence of main active ingredients in liquorice on LS-180 cell P-gp efflux rhodamine 123:
(1) Flow cytometer
The active ingredients in the liquorice reduce the concentration of rhodamine 123 in LS-180 cells to different degrees, which indicates that the discharge of P-gp to the rhodamine 123 is increased, and the effect of P-gp is induced. See table 4.
TABLE 4 ratio of rhodamine 123 content in dosed cells to blank
(2) varioskan Flash multifunctional reading instrument
The active ingredients in licorice reduce the concentration of rhodamine 123 in LS-180 cells to different degrees, and the content of rhodamine 123 in cells is reduced along with the increase of the concentration of liquiritin and liquiritigenin. In conclusion, the active ingredients in the liquorice indicate that the efflux of P-gp to rhodamine 123 is increased, and the P-gp effect is induced. See tables 5-7, FIG. 3.
TABLE 5 Effect of active ingredients of Glycyrrhiza on Glycyrrhiza glycosides and Liquiritins on LS-180 intracellular rhodamine 123 content
TABLE 6 influence of active ingredients of Glycyrrhiza on Isoliquiritin and isoliquiritigenin on the content of LS-180 intracellular rhodamine 123
TABLE 7 influence of Glycyrrhiza uralensis active ingredients on Glycyrrhetinic acid and chalcone A on LS-180 intracellular rhodamine 123 content
Note: p <0.05, P <0.01 compared to blank group.
Example 4: caco-2 transport assay
1. Materials and methods
(1) Instrument and reagent
The instrument comprises the following steps: an incubator: product of Thermo Scientific corporation, usa.
Model AB54-S analytical balance: the product of the mertler-tollido instruments (shanghai) ltd.
Model 3K15 high speed refrigerated centrifuge: product of Sigma, Germany.
Sorvall Legend Micro 17R high speed refrigerated centrifuge: product of Thermo Scientific corporation, usa.
24-well polycarbonate membrane transfer plate, cell culture dish, centrifuge tube, pipette, Corning Costar Co
Reagent: liquiritin reference (lot No. 17030801), liquiritigenin reference (lot No. 17022104), isoliquiritigenin reference (lot No. 17031204), isoliquiritigenin reference (lot No. 17012503), chalcone a reference (lot No. 17032501), chinese drug biological product assay; beta-glycyrrhetinic acid control (batch No. 1129A022), Solarbio corporation; verapamil control (lot 18578), MCE; rhodamine 123, Sigma company; DMSO, analytical grade, Kaixin chemical industries, Inc., Tianjin; RPIM1640 medium, Gibco Laboratories, Inc.; DMEM medium was from Hyclone. Trypsin-EDTA, cell cryopreservation, Gibco; fetal Bovine Serum (FBS), bio-engineering ltd, national sea, langzhou; penicillin streptomycin mixed solution (100 ×), batch number: 20170907, respectively; phospate Buffered Saline (PBS) (1 ×), lot number: 8112372, respectively; DEPC water, batch number: 20170727, available from Solarbio. Ultrapure water, HBSS, self-made in the laboratory.
(2) Cell culture
After the Caco-2 cells are recovered, the recovery rate is 2 multiplied by 105Perml density was inoculated in 100mm by 20mm cell culture dishes, 10mL DMEM medium containing 10% FBS, 1% diabody (penicillin 10000U/mL and streptomycin 10000. mu.g/mL) was added. Cells were incubated at 37 ℃ with 5% CO2The culture is carried out in the incubator, the culture solution is changed every other day, when the cells grow to 80-90%, 0.5% pancreatin is used for digestion and passage, and Caco-2 cells in logarithmic phase are selected for experiment.
(3) Experiment grouping
Blank group, administration group (liquiritin 10. mu.M, liquiritigenin 10. mu.M, isoliquiritigenin 10. mu.M, licochalcone A1. mu.M and glycyrrhetinic acid 10. mu.M), and positive inhibitor group (verapamil 25. mu.M).
(4) experimental methods
Caco-2 at cell density 2 x105The cells were seeded in a Transwell polycarbonate membrane 12-well plate, and 0.5mL of cell suspension was added per well at the top end (AP) of the cell layer and 1.5mL of fresh medium per well at the bottom end (BL) of the cell layer, and cultured continuously for 21 days to obtain a fully differentiated cell monolayer. Continuously replacing blank and medicated culture medium for 3 days, removing culture medium by suction, removing culture medium at AP and BL ends of Transwell chamber, adding HBSS solution preheated at 37 deg.C, and balancing at 37 deg.C for 20 min; absorbing HBSS, adding 0.5mL blank HBSS solution at the AP end, and adding 1.5mL preheated HBSS solution containing 20 μ M rhodamine 123 at the BL end (adding HBSS solution containing 25 μ M verapamil and 20 μ M rhodamine 123 to the positive inhibitor group); incubating by a constant temperature oscillator at 37 ℃, sucking the transport solution at the AP end for 5min, 15min and 30min respectively, filling the transport solution at the AP end with a blank HBSS solution after sucking, and measuring the concentration of the drug in the transport solution. 3 multiple wells are arranged on the same gene of the same template, and 3 times of parallel experiments are carried out.
(5) Data processing and statistics
All measurements were entered using Microsoft excel to calculate mean, standard deviation and t-test for significance from the blank group for each group.
2. Test results
The main active ingredients in the liquorice have influence on P-gp efflux rhodamine 123 of Caco-2 cells and the influence of the change of the Caco-2 cells along with efflux time on the P-gp efflux rhodamine 123 after administration: the active ingredients in the liquorice enhance the discharge of rhodamine 123 to different degrees, which shows that the medicine has an induction effect on P-gp, and the content of rhodamine 123 on the top layer of a transwell is continuously increased along with the increase of discharge time. Tables 8-9, FIGS. 4-5.
TABLE 8 Effect of Glycyrrhiza active ingredients on Caco-2 cell-mediated rhodamine 123 transport (basal side)
TABLE 9 Effect of Glycyrrhiza active ingredients on Caco-2 cell-mediated rhodamine 123 transport (apical side)
Note: p <0.05, P <0.01 compared to blank group.
Claims (3)
1. Use of Glycyrrhrizae radix and its main active ingredient in preparing antidote is provided.
2. The use as claimed in claim 1, wherein the main active ingredient of licorice is selected from the group consisting of liquiritin, liquiritigenin, isoliquiritin, isoliquiritigenin, chalcone A and glycyrrhetinic acid.
3. The use of claim 1, wherein the use is licorice induction of P-gp to reduce entry of foreign material into a cell.
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| CN201810593611.XA CN110575461A (en) | 2018-06-11 | 2018-06-11 | New application of liquorice |
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| CN201810593611.XA CN110575461A (en) | 2018-06-11 | 2018-06-11 | New application of liquorice |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1810796A (en) * | 2005-01-24 | 2006-08-02 | 车庆明 | Prepn process of licoflavone from licorice and licorice slag |
| CN107929241A (en) * | 2017-11-08 | 2018-04-20 | 武汉大学 | A kind of licochalcone A liposome and its preparation method and application |
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2018
- 2018-06-11 CN CN201810593611.XA patent/CN110575461A/en active Pending
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