CN101744832A - Chinese traditional medicine preparation for reversing multiple medicine resistance of tumor cells and preparation method thereof - Google Patents
Chinese traditional medicine preparation for reversing multiple medicine resistance of tumor cells and preparation method thereof Download PDFInfo
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- CN101744832A CN101744832A CN201010011448A CN201010011448A CN101744832A CN 101744832 A CN101744832 A CN 101744832A CN 201010011448 A CN201010011448 A CN 201010011448A CN 201010011448 A CN201010011448 A CN 201010011448A CN 101744832 A CN101744832 A CN 101744832A
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Abstract
The invention discloses a Chinese traditional medicine preparation for reversing multiple medicine resistance of tumor cells, which is prepared from the following raw material medicines in parts by weight: 1-2 parts of baicalin and 1-2 parts of jasminoidin. The preparation method comprises the following steps: firstly, preparing the baicalin and the jasminoidin, then taking and mixing the baicalin and the jasminoidin, and adding water for dissolving the mixture to prepare a mixed preparation; or taking and mixing the baicalin and the jasminoidin, adding water for dissolving the mixture, then adding starch or dextrin, carrying out heating concentration or spray drying, and then adding starch prill for preparing a granular preparation or a capsule preparation. The inventors of the application make a large amount of experimental researches, and the research results show that in the invention, the Chinese traditional medicine preparation containing 1-2 parts of baicalin and 1-2 parts of jasminoidin has obvious effect on reducing an expression rate of a medicine resistant S180 tumor cell P170 of a mouse induced by chemotherapy, and the mechanism of the Chinese traditional medicine preparation is possibly related to reduction of the P170 and over expression of TopoII. The Chinese traditional medicine preparation has the functions of inhibiting cell growth of K562/ADM medicine resistant tumor cells, reversing medicine resistance and promoting cell apoptosis.
Description
Technical field
The present invention relates to Chinese medicine preparation of a kind of reversing multiple medicine resistance of tumor cells and preparation method thereof, belong to the medicine bioengineering field.
Background technology
Tumor cell multidrug resistance is the main cause of chemotherapy failure, it also is the key factor of tumor patient death, tumor cell can produce multidrug resistance by number of ways and the chemotherapy resistance occur, these approach can brief overview be the following aspects: in the cell membrane level, memebrane protein as: P170, MRP, LRP, BCRP etc. express rising, the acceleration medicine effluxes, and makes the cell Chinese medicine accumulate minimizing; In the cytoplasm level, comprise GST, PKC, P450 reductase, multiple endochylema endoenzyme increased activity such as coenzyme II, the cell function of detoxification is strengthened, quicken the metabolism and the degraded of chemotherapeutics; In the nucleus level, quantity, activity, structure and distribution take place TOPO II change and DNA repair ability strengthen, and make the impaired minimizing of DNA; Multidrug resistance tumor cells apoptosis-related genes even also have the expression of oncogene that variation has taken place, express rising etc. as Bcl-2, Bcl-xl, the apoptosis of tumor cells of induced by chemotherapeutic agents is reduced, produce drug resistance, chemotherapeutics is produced resistance, cause the chemotherapy failure, reduce clinical chemotherapy effect.
Summary of the invention
At above-mentioned prior art, the invention provides a kind of Chinese medicine preparation that is used for reversing tumor patient multidrug resistance, improves chemotherapeutic efficacy.
The present invention is achieved by the following technical solutions:
A kind of Chinese medicine preparation of reversing multiple medicine resistance of tumor cells is made by following bulk drugs: 1～2 part of baicalin, 1～2 part of jasminoidin.
A kind of preparation method of Chinese medicine preparation of reversing multiple medicine resistance of tumor cells, step is as follows:
(1) gets Radix Scutellariae, decoct with water 2 times, each 1～3 hour, centrifugal or additive method filters (centrifugal or other conventional method), merging filtrate, concentrated filtrate is with hydrochloric acid adjust pH to 1.8～2.0,60 ℃ are incubated 30 minutes, be cooled to room temperature, placed 12 hours, filter, residue is washed till pH value 4.0 with ethanol, in residue, add 10 times of amounts (by volume) water then and stir,, add equivalent ethanol (by volume) after the dissolving and stir evenly with sodium hydroxide solution adjust pH to 7.0, placed 12 hours, filter, filtrate was with hydrochloric acid adjust pH to 1.8～2.0,80 ℃ insulation 30 minutes, be cooled to room temperature, filter, residue is washed till pH value 4.0, vacuum drying with ethanol, be ground into fine powder, promptly get the baicalin of 96.09% above purity;
(2) get Fructus Gardeniae, add the alcohol reflux twice of 5 times of amounts (5 times of amounts are meant Fructus Gardeniae weight) 80%, each 2～3 hours, filter merge extractive liquid; Reclaim ethanol and get concentrated solution; Concentrated solution is used petroleum ether and n-butanol extraction successively; N-butanol portion concentrate extractum; N-butanol portion is mixed sample, gone up silicagel column, carry out gradient elution with chloroform-methanol, when chloroform-methanol (100: 7), a large amount of powder occur, the chloroform-methanol recrystallization promptly gets white, needle-shaped crystals, is jasminoidin, and purity is more than 99%;
(3) get baicalin, the jasminoidin of above-mentioned preparation, mix, be dissolved in water, make mixture; Or: get baicalin, the jasminoidin of above-mentioned preparation, mix, be dissolved in water, add starch or dextrin then, heating concentrate or spray drying after add starch and granulate, make granule or capsule.
In the described step (1), concentrated filtrate is to 1/10 of crude drug amount volume.
In the described step (1), the concentration of hydrochloric acid is 2mol/L; The concentration of sodium hydroxide solution is 20% (mass/volume).
When taking, every day twice, each dose is: granule 1.5g, 1～2 of capsule (every 0.3～5 gram).
Tumor cell can produce multidrug resistance by number of ways and the chemotherapy resistance occur, these approach can brief overview be the following aspects: in the cell membrane level, memebrane protein as: P170, MRP, LRP, BCRP etc. express rising, quicken medicine and efflux, and make the cell Chinese medicine accumulate minimizing; In the cytoplasm level, comprise GST, PKC, P450 reductase, multiple endochylema endoenzyme increased activity such as coenzyme II, the cell function of detoxification is strengthened, quicken the metabolism and the degraded of chemotherapeutics; In the nucleus level, quantity, activity, structure and distribution take place TOPO II change and DNA repair ability strengthen, and make the impaired minimizing of DNA; Multidrug resistance tumor cells apoptosis-related genes even also have the expression of oncogene that variation has taken place expresses to raise etc. as Bcl-2, Bcl-xl, the apoptosis of tumor cells of induced by chemotherapeutic agents is reduced, and produces drug resistance.
Domestic scholars is carried out the drug-fast research of Chinese medicine reversing tumor since the nineties, and experiment in vitro confirms: Chinese medicinal components such as arteannuin, psoralen, curcumin, peimine, matrine, ligustrazine, chrysophanic acid, arsenic trioxide (As203), cinnamic aldehyde, daidzein, Genistein, dioscin, tetrandrine and Chinese medicine compound have reverse effect to the multi-drug resistance of people's drug-resistant tumor cells such as HL60/HT, K562/ADM, SGC7901/ADR, KBv200, K562/A02; Experiment confirm tetrandrine, matrine obviously reduce the expression rate of the inductive mice drug-resistant tumor of chemotherapy cell P170 in the body; Medicines such as the plain spirit of Venenum Bufonis, Realgar all can suppress the growth of HL-60/ADR cell, and reversing drug resistance promotes apoptosis, and its mechanism may be relevant with the TopoII overexpression with downward modulation P170.
The present inventor has done a large amount of experimentatioies, and result of study shows baicalin 1-2 part of the present invention, and jasminoidin 1-2 part Chinese medicine preparation has the inductive mice drug resistance of obvious reduction chemotherapy S
180The expression rate of tumor cell P170, its mechanism may be relevant with the TopoII overexpression with downward modulation P170; Suppress the growth of K562/ADM drug-resistant tumor cell, reversing drug resistance promotes apoptosis.
The specific embodiment
The present invention is further illustrated below in conjunction with the embodiment data:
Embodiment 1: the Chinese medicine preparation of reversing tumor patient multidrug resistance, raising chemotherapeutic efficacy
Needed raw material is as follows: Radix Scutellariae 500g, Fructus Gardeniae 500g.
Preparation technology is:
1) respectively Radix Scutellariae, Fructus Gardeniae are pulverized;
2) get Radix Scutellariae 500g, decoct with water 2 times, each 1.5 hours, filter, merging filtrate, filtrate are concentrated in right amount (with the ratio of crude drug amount 10: 1), with 2mol/L hydrochloric acid solution adjust pH to 1.8～2.0,60 ℃ are incubated 30 minutes, be cooled to room temperature, placed 12 hours, filter, residue is washed till pH value 4.0 with ethanol, add 10 times of water gagings and stir,, add equivalent ethanol after the dissolving and stir evenly with 20% sodium hydroxide solution adjust pH to 7.0, placed 12 hours, filter, filtrate was with 2mol/L hydrochloric acid solution adjust pH to 1.8～2.0,80 ℃ insulation 30 minutes, be cooled to room temperature, filter, residue is washed till pH value 4.0, vacuum drying with ethanol, be ground into fine powder, promptly get the baicalin (96.09-98.15) of 96.09% above purity;
3), each 2 hours, filter merge extractive liquid, with the alcohol reflux twice of Fructus Gardeniae 500g with 5 times of amounts 80%.Reclaim ethanol and get concentrated solution.Concentrated solution is used petroleum ether and n-butanol extraction successively.N-butanol portion concentrate extractum.N-butanol portion is mixed sample, gone up silicagel column, carry out gradient elution with chloroform-methanol, when chloroform-methanol (100: 7), a large amount of powder appear, the chloroform-methanol recrystallization, and (institute isolating white crystals and geniposide reference substance carry out that thin layer detects and eutectic point mensuration promptly to get 99% jasminoidin (99.70-99.73) of white, needle-shaped crystals, thin layer shows the speckle of same position, fusing point does not change, and fusing point is 167～168 ℃, determines that this compound structure is a jasminoidin);
4) baicalin 1-2 part, jasminoidin 1-2 part add appropriate amount of starch or dextrin is granulated, and makes granule or capsule.
Test data: the foundation and the mice group of mice S180 cell tumour drug resistance model
Ascitic type S
180Mice is through simulation chemotherapy PFC scheme, and cisplatin 3mg/kgip is weekly; Each 3mg/kgig of cyclophosphamide and 5-FU, once a day, continuously around, (going down to posterity once one to one during two weeks).Obtain drug resistance mice S
180Model, aseptic extraction chemotherapy are induced the drug resistance mice S in 4 weeks
180Ascites, aseptic NS is diluted to and contains cell number 1 * 10
6/ ml, every Mus 0.2mlip inoculation.Mouse inoculation drug resistance S
18024h behind the cell is divided into matched group, our granule 100mg/kg, 50mg/kg group, 14 every group at random; matched group is water 0.2ml/10gig, and our granule 100mg/kg and 50mg/kg group give 0.5%, 0.25% granule suspension 0.2ml/10gig, continuous 10 days respectively; behind last administration 24h, aseptic extraction ascites, anticoagulant heparin; the washing of PH7.4PBS liquid; the centrifugal 5min of 1500rpm, totally 3 times, back 70% ethanol is fixed; 4 ℃ of preservations are equipped with and survey.
2.2 the detection method that MDR1 expression product P170, TOPOII, LRP express
Get the fixed mice S of 70% ethanol
180Cell, by after the PH7.4 PBS liquid dilution, the centrifugal 5min of 1500rpm, once more eluting once, and to adjust cell concentration be 1 * 10
6/ ml shakes up, obtained cell suspension 500 μ l, the monoclonal antibody IgG1 working solution 20 μ l that add the fluorescein-labeled mouse-anti people of corresponding separately different sulfur hydracid, respectively get an anti-IgG1-FITC of negative control in addition, behind 37 ℃ of lucifuge reaction 20min, by the flow cytometer fluoroscopic examination.The detection coefficient of variation＜2% of instrument is measured the expression of above-mentioned 5 kinds of associated biomolecule molecules of 10,000 cells, and excitation wavelength is 488nm.
1. result of the test
3.1 to the inductive multidrug resistance ascitic type of chemotherapy mice S
180Sarcoma cell MDR1 expression product P
170The reverse effect of (P-glycoprotein, P-gp) overexpression
Get fixed each the Mus multidrug resistance S of 70% ethanol
180Tumor cell by PH7.4 PBS liquid, washs once more with the centrifugal 5min of 1500rpm, and to be made into cell concentration be 1 * 10
6/ ml suspension is by fluorescence P
170Monoclonal antibody IgG1 labelling lucifuge 30min is by each Mus S of cells were tested by flow cytometry
180Cell fluorescence intensity, statistical analysis are respectively organized mice S
180Tumor cell P
170Expression rate is as table 1.
The inductive multidrug resistance mice of table 1 pair chemotherapy S180 cell P
170Reverse effect X ± the S of overexpression
Annotate: suppression ratio (%)=(matched group-administration group) ÷ matched group * 100%.
Result of the test prompting: induced for 4 weeks with repeated transmission control group mice S after 10 days generations
180Tumor cell tumor multidrug resistance gene P
170Express there was no significant difference, the drug resistance mice S after inducing is described
180Gene expression of cells is comparatively stable.The Rhizoma Coptidis toxic materials clearing away decoction total alkali obviously reverses drug resistance mice S
180Cell drug resistant gene P
170Overexpression.Illustrate that the Rhizoma Coptidis toxic materials clearing away decoction total alkali reverses the inductive multidrug resistance cell drug resistant gene of chemotherapy P
170Expression is one of important channel of its reverse multiple drug resistance of tumor.
3.2 reverse multidrug resistance mice S
180Cell lung drug-resistant protein LRP overexpression
Each organizes mice drug resistance S
180The cell tests pre-treatment is as 3.1, and cell reacts 30min through lucifuge, and IgG1 combines with the fluorescence monoclonal, flow cytometer testing result such as table 2.
Table 2 Rhizoma Coptidis toxic materials clearing away decoction ethanol extract is to mice drug resistance S
180Cell LRP overexpression influence X ± S
Annotate: with induce after go down to posterity matched group relatively: " * * * P＜0.001; “ ﹠amp; " induce relatively back all around with chemotherapy; Tumour inhibiting rate (%)=(matched group-administration group) ÷ matched group * 100%.
Result of the test prompting: the mice drug resistance S that after chemotherapy induced for 4 weeks, went down to posterity 10 days
180Tumor cell LRP expression rate with induced for 4 weeks relatively do not have significant change, obviously reversed drug resistance S and give the Rhizoma Coptidis toxic materials clearing away decoction total alkali
180The overexpression of tumor cell LRP illustrates that alkaloid has the effect of reversing drug resistance tumor cell LRP overexpression, and this may be one of mechanism of alkaloid reverse multidrug resistance.
3.3 reversing mice S180 mdr cell DNA topoisomerase TOPOII expresses
Each organizes mice S
180The mdr cell pre-treatment is as 3.1, and room temperature lucifuge reaction 30min before the test, fluorescent monoclonal antibody IgG1 be in conjunction with 20min, measurement result such as table 3.
Table 3 couple mice S180 mdr cell TOPOII expresses influences X ± S
Annotate: compare with inducing the back matched group that goes down to posterity: " * * * " P＜0.001; “ ﹠amp; " induce all around relatively with chemotherapy; Tumour inhibiting rate (%)=(matched group-administration group) ÷ matched group * 100%.
3.4 to mice chemotherapy inducible resistance S
180Fixed each the Mus multidrug resistance S of 70% ethanol is got in apoptotic influence
180Tumor cell by PH7.4 PBS liquid, washs once more with the centrifugal 5min of 1500rpm, and to be made into cell concentration be 1 * 10
6/ ml suspension is got and is contained cell 1 * 10
6The cell suspension 500 μ l of/ml are added on PI working solution 500 μ l (concentration is 10 μ g/ml), and the lucifuge room temperature is 351nm by flow cytometer fluoremetry exciting light after placing 30min, and each organizes apoptosis rate such as table 1.
Table 1 couple chemotherapy inducing mouse drug resistance S
180Apoptotic X ± the SD that influences
Annotate: with induce after go down to posterity model control group relatively: " * * * " P＜0.001; “ ﹠amp; " and chemotherapy is induced all around, and group compares; Promotion rate (%)=(administration group-model group) ÷ model group * 100%.
Result of the test prompting: compare with inducing the back matched group that goes down to posterity, and our granule obviously improves drug resistance S
180Apoptosis rate illustrates to promote that apoptosis is one of approach of the acquired multidrug resistance of alkaloid reversing tumor.
Claims (4)
1. the Chinese medicine preparation of a reversing multiple medicine resistance of tumor cells is characterized in that, is made by following bulk drugs: 1～2 part of baicalin, 1～2 part of jasminoidin.
2. the preparation method of the Chinese medicine preparation of the described a kind of reversing multiple medicine resistance of tumor cells of claim 1, it is characterized in that: step is as follows:
(1) gets Radix Scutellariae, decoct with water 2 times, each 1～3 hour, filter merging filtrate, concentrated filtrate, with hydrochloric acid adjust pH to 1.8～2.0,60 ℃ insulation 30 minutes, be cooled to room temperature, placed 12 hours, and filtered, residue is washed till pH value 4.0 with ethanol, in residue, add 10 times of water gagings then and stir,, add equivalent ethanol after the dissolving and stir evenly with sodium hydroxide solution adjust pH to 7.0, placed 12 hours, filter, filtrate was with hydrochloric acid adjust pH to 1.8～2.0,80 ℃ insulation 30 minutes, be cooled to room temperature, filter, residue is washed till pH value 4.0, vacuum drying with ethanol, be ground into fine powder, promptly get baicalin;
(2) get Fructus Gardeniae, add the alcohol reflux twice of 5 times of amounts 80%, each 2～3 hours, filter merge extractive liquid; Reclaim ethanol and get concentrated solution; Concentrated solution is used petroleum ether and n-butanol extraction successively; N-butanol portion concentrate extractum; N-butanol portion is mixed sample, gone up silicagel column, carry out gradient elution with chloroform-methanol, when chloroform-methanol (100: 7), a large amount of powder occur, the chloroform-methanol recrystallization promptly gets white, needle-shaped crystals, is jasminoidin;
(3) get baicalin, the jasminoidin of above-mentioned preparation, mix, be dissolved in water, make mixture; Or: get baicalin, the jasminoidin of above-mentioned preparation, mix, be dissolved in water, add starch or dextrin then, heating concentrate or spray drying after add starch and granulate, make granule or capsule.
3. the preparation method of the Chinese medicine preparation of a kind of reversing multiple medicine resistance of tumor cells according to claim 1, it is characterized in that: in the described step (1), concentrated filtrate is to 1/10 of crude drug amount volume.
4. the preparation method of the Chinese medicine preparation of a kind of reversing multiple medicine resistance of tumor cells according to claim 1, it is characterized in that: in the described step (1), the concentration of hydrochloric acid is 2mol/L; The concentration of sodium hydroxide solution is 20%.
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| CN112933110A (en) * | 2021-02-03 | 2021-06-11 | 温州医科大学附属第六医院 | Marker for sensitivity of lung cancer patient to chemotherapeutic drugs and application thereof |
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| CN112933110A (en) * | 2021-02-03 | 2021-06-11 | 温州医科大学附属第六医院 | Marker for sensitivity of lung cancer patient to chemotherapeutic drugs and application thereof |
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