CN1823987A - Application of coptic toxin resolving decoction in preparation of medicine for predventing and treating tumour chemotherapy tolerance - Google Patents
Application of coptic toxin resolving decoction in preparation of medicine for predventing and treating tumour chemotherapy tolerance Download PDFInfo
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Abstract
A Chinese medicine Detoxicating coptis decoction for suppressing the resistance of tumor cells to chemicotherapeutic medicines to improve the curative effect of chemicotherapeutic medicines is prepared from coptis root, scutellaria root, phellodendron bark and capejasemine fruit.
Description
(1) technical field under
The present invention relates to of the application of a kind of Rhizoma Coptidis toxic materials clearing away decoction, belong to the new purposes of Rhizoma Coptidis toxic materials clearing away decoction at preparation prophylactic treatment drug resistance of tumor chemotherapy medicine.
(2) background technology
Rhizoma Coptidis toxic materials clearing away decoction is former to be stated from " medical secrets of official ", mainly is made up of Rhizoma Coptidis, Radix Scutellariae, Cortex Phellodendri and Fructus Gardeniae four Chinese medicine, is the heat-clearing and toxic substances removing side of representative.
(3) summary of the invention
The object of the present invention is to provide a kind of medicine that utilizes Rhizoma Coptidis toxic materials clearing away decoction to prepare the prophylaxis of tumours chemotherapy resistance.
The present invention realizes by following measure: Rhizoma Coptidis toxic materials clearing away decoction is in the application of preparation prophylaxis of tumours chemotherapy resistance medicine, and the medicinal raw material of Rhizoma Coptidis toxic materials clearing away decoction is Rhizoma Coptidis 3-9 gram, Radix Scutellariae 3-9 gram Cortex Phellodendri 3-9 gram Fructus Gardeniae 6-12 gram.
The preparation method of Rhizoma Coptidis toxic materials clearing away decoction can adopt common decocting liquid, and is oral after filtering; Also can adopt alcohol extracting method to extract, extracting solution is made common dosage form after concentrating.
Modern pharmacology studies confirm that, Rhizoma Coptidis toxic materials clearing away decoction contains berberine (berberine), coptisine (coptisine), 13-methyl-.psi.-coptisine. (worenine), jateorhizine (jatrorrhizine), phellodendrine (phellodedrine), palmatine (palmatine), Dauricine multiple alkaloids such as (menisperine), and Chinese traditional medicine biology alkali is the Chinese medicinal ingredients that a class has extensive biological effect.
Can prove that by experiment Rhizoma Coptidis toxic materials clearing away decoction prevention administration can obviously increase the inductive mice S of simulation clinical chemotherapy
180Apoptosis rate, the expression rate of increase apoptosis factor Fas reduces adhesion factor CD54 expression rate.Rhizoma Coptidis toxic materials clearing away decoction prevention administration can obviously reduce the inductive mice S of simulation clinical chemotherapy
180The expression product P of cell drug resistant gene MDR
170, lung drug-resistant protein LRP expression, suppress the active generation that obviously suppresses tumor cell multidrug resistance of cell topoisomerase II (TOPOII).The mechanism of action of the acquired multidrug resistance of Rhizoma Coptidis toxic materials clearing away decoction reversing tumor suppresses cell DNA TOPOII activity, reduces the drug resistance of cell to chemotherapeutics; Help the absorption of chemotherapeutics, thus reversing multiple medicine resistance of tumor cells.
(4) specific embodiment
Embodiment 1:
Get Rhizoma Coptidis 9 grams, Radix Scutellariae 6 grams, Cortex Phellodendri 6 restrains, and Fructus Gardeniae 9 grams merge, and decoct with water twice, merge decoction liquor, are divided into two parts, and are oral, sooner or later respectively once.
Embodiment 2:
Get 4 kilograms of Rhizoma Coptidis, 8 kilograms of Radix Scutellariaes, 8 kilograms of Cortex Phellodendris, 6 kilograms of Fructus Gardeniaes are ground into fine powder after the merging, add the ethanol of 8 times of weight 75%, extract twice, merge extractive liquid, filters decompression recycling ethanol, be condensed into dry extract, pulverize the back an amount of starch of adding and make tablet, the 0.5g/ sheet amounts to 10000.
Usage and consumption: oral, three times on the one, each 2.
Embodiment 3:
Rhizoma Coptidis 6g, Cortex Phellodendri 6g, Radix Scutellariae 6g,, Fructus Gardeniae 6g pulverizes 70% alcohol reflux, and 2 times, each 1.5h revolves and steams 40 ℃ of evaporations 5.7g that gets dry extract, and grinds and is made into 1% and 0.5% suspension for fine powder.
Test case 1: Rhizoma Coptidis toxic materials clearing away decoction is to multidrug resistance mice S
180The influence that apoptosis of tumor cells, antiapoptotic factors Fas and CD54 express
1. test material
1.1 test drug: Rhizoma Coptidis toxic materials clearing away decoction is assisted preparation by my plant chamber professor Yang Shubin of institute, and is substantially the same manner as Example 3; Cisplatin for inj: Shandong Qilu Pharmaceutical Factory product, lot number: 01111611; Cyclophosphamide: Hualian Pharmaceutical Co., Ltd., Shanghai's product, lot number: 030703; 5-fluorouracil: Tianjin gold credit aminoacid company limited product, lot number: the accurate word H12020959 of traditional Chinese medicines.
1.2 experimental animal: Kunming mouse is provided the quality certification number: Shandong kinoplaszm word: 200210023 by Shandong University's Experimental Animal Center; Mice S
180Ascitic type tumor kind Mus: provide by pharmacological room of medicine institute of Shandong Medical University.
2. test method
2.1 mice group and administration
18-22g is divided into 4 groups at random, 14 every group, is respectively normal control S
180Group, model group, Rhizoma Coptidis toxic materials clearing away decoction 100mg/kg and 50mg/kg group, 3 S of aseptic extraction
180Mouse ascites is diluted to by aseptic NS and contains oncocyte 1 * 10
6/ ml shakes up, and every mouse peritoneal inoculation 0.2ml behind the inoculation 24h (except that the normal control group), all begins to give cisplatin 3mg/kgip, and is weekly; Each 3mg/kgig of cyclophosphamide and 5-FU, once a day.Inoculate the 15th day, the aseptic extraction ascites of the mice of survival goes down to posterity one to one, behind the 24h that goes down to posterity, gives above-mentioned chemotherapeutics again.In the inductive while of chemotherapy, model group and normal control mice S
180Group gives cold water 0.2ml/10gig, and Rhizoma Coptidis toxic materials clearing away decoction 2/kg and 50mg/kg group give 0.5%, 0.25% Rhizoma Coptidis toxic materials clearing away decoction suspension 0.2ml/10gig respectively, be total to around the administration, behind the drug withdrawal 24h, each mouse ascites 2ml of aseptic extraction, go in the anticoagulant heparin test tube, the centrifugal 5min of 1500rpm removes supernatant, by the rinsing of PH7.4PBS liquid, centrifugal eluting 3 times, with 70% ethanol, 1.0ml fixes, 4 ℃ of preservations, to be measured.
2.2 apoptosis is measured
The fixed mice S of 70% ethanol
180Before the cell, PI dyeing, by centrifugal again eluting after the dilution of PBS liquid once, and transfer to and contain tumor cell 1 * 10
6/ ml, shake up, get cell suspension 500 μ l, the PI working solution 500 μ l that add 10 μ g/ml are behind 37 ℃ of lucifuge reaction 30min, by the FACScan flow cytometer, exciting light 351nm fluorescent scattering detects, 10,000 cells of every specimen, test data is by Macintosh650 computer utility ModiFit software (U.S. company BD provides), analytical data.
2.3Fas and CD
54The detection of expressing
Get the fixed mice S of 70% ethanol
180Cell, by after the PH7.4PBS 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 30min, by the flow cytometer fluoroscopic examination.The detection coefficient of variation＜2% of instrument is measured the expression of above-mentioned 2 kinds of associated biomolecule molecules of 10,000 cells, and excitation wavelength is 488nm.
3 result of the tests
3.1 the prevention administration is to chemotherapy inducible resistance mice S
180Apoptotic influence
Get the fixed S that respectively organizes of 70% ethanol
180Cell behind PH7.4PBS liquid eluting, and is adjusted cell concentration to 1 * 10
6Behind/the ml, obtained cell suspension PI dyeing, behind 37 ℃ of lucifuge reaction 30min, by the flow cytometer fluoroscopic examination, exciting light is 488nm, records and respectively organizes mice S
180Apoptosis rate is as table 1
Table 1 prevention administration is to multidrug resistance mice S
180Apoptotic X ± the SD that influences
| Group | N | Apoptosis rate | Increment rate (%) |
| Normal S180 groups of cells model cell group Rhizoma Coptidis toxic materials clearing away decoction 100mg/kg | 11 10 10 | 2.07±1.13** 4.47±2.17 14.82±6.32*** | - 115.94& 231.54 |
| Rhizoma Coptidis toxic materials clearing away decoction 50mg/kg | 10 | 11.87±5.36*** | 165.55 |
Annotate: compare with model group: "
*" P＜0.01, "
* * *" P＜0.001; “ ﹠amp; " and chemotherapy is induced all around, and group compares; Increment rate (%)=(alkaloid group-model group) ÷ model group * 100%.
Result of the test prompting: Rhizoma Coptidis toxic materials clearing away decoction increases obviously that chemotherapy is induced and the mice S that causes
180The apoptosis of cell (P＜0.001) illustrates that the acquired multidrug resistance of its intervention chemotherapy is relevant with its promotion mdr cell apoptosis, and increasing the drug-resistant tumor apoptosis may intervene one of important mechanisms of tumor cell multidrug resistance by Rhizoma Coptidis toxic materials clearing away decoction.
3.2 the prevention administration is induced multidrug resistance mice S to chemotherapy
180Apoptosis of tumor cells factor Fas (CD
95) influence expressed
The PBS liquid fixed mice S of 70% ethanol of eluting once more learns from else's experience
180Cell, adjusting cell concentration by PBS is 1 * 10
6/ ml, by Fas fluorescent monoclonal antibody IgG1 in conjunction with 20min, by flow cytometer fluoroscopic examination, excitation wavelength 488nm, result such as table 2
Table 2 prevention administration is to drug resistant gene mice S
180Cell Fas (CD
95) express influence X ± SD
| Group | N | The Fas expression rate | Increment rate (%) |
| Normal S180 groups of cells model cell group Rhizoma Coptidis toxic materials clearing away decoction 100mg/kg | 11 10 10 | 7.21±3.88 11.15±7.34 58.18±22.76*** | - 54.65& 421.74 |
| Rhizoma Coptidis toxic materials clearing away decoction 50mg/kg | 10 | 49.67±23.12*** | 345.47 |
Annotate: compare with model group: "
* *" P＜0.001; “ ﹠amp; " and chemotherapy is induced all around, and group compares; Increment rate (%)=(alkaloid group-model group) ÷ model group * 100%.
The result of the test prompting: Rhizoma Coptidis toxic materials clearing away decoction is to the inductive multidrug resistance mice of chemotherapy S
180Cell Fas expression rate has tangible potentiation, illustrates that Chinese traditional medicine biology alkali increases drug resistance S
180Apoptosis is with its increase apoptogene Fas (CD
95) express and be correlated with.
3.3 the prevention administration is induced multidrug resistance mice S to chemotherapy
180Tumor cell adhesion molecule CD
54The influence of expressing
Cell is handled and is detected all with 3.1, and fluoroscopic examination is respectively organized mice drug resistance S
180Cell adhesion factor CD
54Express, as table 3.
Table 3 prevention administration is to drug resistant gene mice S
180Adhesion factor CD54 expresses influences X ± SD
| Group | N | The CD54 expression rate | Suppression ratio (%) |
| Normal S180 groups of cells model cell group Rhizoma Coptidis toxic materials clearing away decoction 100mg/kg Rhizoma Coptidis toxic materials clearing away decoction 50mg/kg | 11 10 10 10 | 89.44±37.48 79.75±37.44 7.89±2.76*** 20.65±5.78 | - 10.83& 90.11 12.67 |
Annotate: compare with model group: "
* *" P＜0.001.“ ﹠amp; " and chemotherapy is induced all around, and group compares increment rate (%)=(alkaloid group-model group) ÷ model group * 100%.
Result of the test prompting: induce 4 weeks and repeated transmission generation 10 days S
180The CD of cell
54Expression rate basically identical, Rhizoma Coptidis toxic materials clearing away decoction obviously suppress drug resistance S
180Cell adhesion factor CD
54Expression rate, reduced the iuntercellular adhesion, suppress the transfer of sticking of tumor cell, thereby improved chemotherapy effect.Illustrating and reduce the effect of drug-resistant tumor cell adhesion, may also be one of mechanism of alkaloid reverse multiple drug resistance of tumor.
Discuss:
Tumor cell multidrug resistance is the one of the main reasons that causes the chemotherapy failure, and is closely related with tumor patient death, intervenes and reverse multiple drug resistance of tumor, improves chemotherapeutic efficacy, for oncotherapy positive meaning arranged.
During the generation of the acquired multidrug resistance MDR of chemotherapy of tumors, apoptosis of tumor cells gene Fas expresses and reduces, reduces the apoptosis of tumor cells rate; Cell adhesion factor CD54 expresses enhancing, promotes that tumor cell sticks transfer, and tumor cell reduces the sensitivity of chemotherapeutics
[4～6]Stop chemotherapeutics to act on tumor cell, and produce drug resistance.We are application simulation clinical tumor patient clinical chemotherapy inducing mouse S
180Cellular expression produces drug resistance, observe the influence of Rhizoma Coptidis toxic materials clearing away decoction vivo medicine-feeding to drug resistance of tumor cell apoptosis rate and associated biomolecule active substance apoptosis factor Fas, CD54 expression, result's Rhizoma Coptidis toxic materials clearing away decoction prevention administration as can be known can obviously increase the inductive mice S of simulation clinical chemotherapy
180Apoptosis rate, the expression rate of increase apoptosis factor Fas reduces adhesion factor CD54 expression rate.Illustrate that Rhizoma Coptidis toxic materials clearing away decoction prevention administration can suppress the generation of tumor cell multidrug resistance by above-mentioned approach, this has great importance for the clinical generation that prevents and reduce the chemotherapy multidrug resistance of application Chinese medicine, raising clinical chemotherapy curative effect.
Test case 2: the coptis detoxifcation decoction extract is to acquired multidrug resistance mice S
180Tumor cell P
170, the influence expressed of LRP, TOPOII
1. test material
1.1 test drug: the Rhizoma Coptidis toxic materials clearing away decoction ethanol extract is assisted preparation by my plant chamber professor Yang Shubin of institute, and is substantially the same manner as Example 3; Cisplatin for inj: Shandong Qilu Pharmaceutical Factory product, lot number: 01111611; Cyclophosphamide: Hualian Pharmaceutical Co., Ltd., Shanghai's product, lot number: 030703; 5-fluorouracil: Tianjin gold credit aminoacid company limited product, lot number: the accurate word H12020959 of traditional Chinese medicines.
1.2 experimental animal: Kunming mouse is provided the quality certification number: Shandong kinoplaszm word: 200210023 by Shandong University's Experimental Animal Center; Mice S
180Ascitic type tumor kind Mus: provide by pharmacological room of medicine institute of Shandong Medical University.
1.3 test reagent: iodate pyrimidine PI:C27H34N4I2, numbering [25535-16-4], U.S. Sigarm company product, lot number: NO.247-081-0; MDR expression product P
170The monoclonal antibody IgC1 of (P-glycoprotein, P-gp), TOPOII, LRP, different sulfur hydracid fluorescein (FITC) labelling and immune negative control product IgC1-FITC are provided by Beijing Yue Tai biotech firm by U.S. pharmingen company product.
2. test method
2.1 grouping and administration
18-22g is divided into 4 groups at random, 14 every group, is respectively normal control S180 group, model group, Rhizoma Coptidis toxic materials clearing away decoction 100mg/kg and 50mg/kg group, 3 S of aseptic extraction
180Mouse ascites is diluted to by aseptic NS and contains oncocyte 1 * 10
6/ ml shakes up, and every mouse peritoneal inoculation 0.2ml behind the inoculation 24h (except that the normal control group), all begins to give cisplatin 3mg/kgip, and is weekly; Each 3mg/kgig of cyclophosphamide and 5-FU, once a day
[1]Inoculate the 15th day, the aseptic extraction ascites of the mice of survival goes down to posterity one to one, behind the 24h that goes down to posterity, gives above-mentioned chemotherapeutics again.In the inductive while of chemotherapy, model group and normal control mice S
180Group gives cold water 0.2ml/10gig, and Rhizoma Coptidis toxic materials clearing away decoction 100mg/kg and 50mg/kg group give 0.5%, 0.25% Rhizoma Coptidis toxic materials clearing away decoction suspension 0.2ml/10gig respectively, be total to around the administration, behind the drug withdrawal 24h, each mouse ascites 2ml of aseptic extraction, go in the anticoagulant heparin test tube, the centrifugal 5min of 1500rpm removes supernatant, by the rinsing of PH7.4PBS liquid, centrifugal eluting 3 times, with 70% ethanol, 1.0ml fixes, 4 ℃ of preservations, to be measured.
2.2P
170, the detection expressed of TOPOII, LRP
Get the fixed mice S of 70% ethanol
180Cell, by after the PH7.4PBS 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 30min, 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.
3. result of the test
3.1 to the inductive multidrug resistance ascitic type of chemotherapy mice S
180Sarcoma cell MDR expression product P
170The influence of (P-glycoprotein, P-gp)
When giving induced by chemotherapeutic agents, give the Rhizoma Coptidis toxic materials clearing away decoction ethanol extract, continuously around, record and respectively organize mice S
180Ascites cells P
170, expression rate such as table 4.
Table 4 prevention administration is to acquired multidrug resistance mice S
180Cell membrane sugar mucin P
170That expresses influences X ± SD
| Group | N | Expression rate | Suppression ratio (%) |
| Normal S 180Groups of cells model cell group Rhizoma Coptidis toxic materials clearing away decoction ethanol extract high dose | 11 10 10 | 3.61±1.75*** 13.13±5.33 6.12±3.32*** | - 53.39 |
| Rhizoma Coptidis toxic materials clearing away decoction ethanol extract low dosage | 10 | 8.35±2.36** | 36.41 |
Annotate: compare with model control group: " * * " P＜0.01, " * * * " P＜0.001.
The result of the test prompting: after the simulation clinical chemotherapy induced for 4 weeks, ascitic type mice S
180Its P of cell
170Expressing obviously increases; The Rhizoma Coptidis toxic materials clearing away decoction ethanol extract significantly suppresses the S after chemotherapy is induced
180Cell P
170Expression, illustrate its prevention administration can obviously reduce the drug resistance of tumor cell expression of gene, intervene the generation of acquired multidrug resistance.
3.2 the prevention administration is to the acquired multidrug resistance S of mice
180The influence that cell multidrug-associated protein LRP expresses
Respectively organize mice S after 70% ethanol is fixing
180Cell after immunofluorescence monoclonal antibody body IgG1 combination, detects its fluorescence intensity by flow cytometer, statistics mice drug resistance S
180Tumor cell lung drug resistance LRP expression rate such as table 5
Table 5 prevention administration is to the acquired multidrug resistance S of mice
180Cell multidrug-associated protein LRP expresses influences X ± SD
| Group | N | The LRP expression rate | Suppression ratio (%) |
| Normal S180 groups of cells model cell group Rhizoma Coptidis toxic materials clearing away decoction ethanol extract high dose | 11 10 11 | 4.99±1.75*** 62.73±15.24 5.80±3.90*** | - 90.75 |
| Rhizoma Coptidis toxic materials clearing away decoction ethanol extract low dosage | 10 | 11.97±5.10*** | 80.92 |
Annotate: compare with model group: " * * * " P＜0.001.Suppression ratio is: (model group-alkaloid group) ÷ model group * 100% (expression rate).
Result of the test prompting: the mice S after the simulation clinical chemotherapy is induced
180Cellular expression drug resistance associated protein lung drug-resistant protein (Lung resistant protein.LRP) obviously increases, and the Rhizoma Coptidis toxic materials clearing away decoction ethanol extract can effectively reduce chemotherapy inducing mouse S
180The expression rate of cell LRP.Illustrate that suppressing LRP may be one of approach of Chinese traditional medicine biology alkali intervention tumor MDR.
3.3 the prevention administration is induced multidrug resistance mice S to chemotherapy
180Cell DNA topoisomerase II (DNA, TopoisomeraseII, TOPOII) active influence
The fixed S of 70% ethanol
180Cell by being made into cell suspension behind the PH7.4PBS liquid eluting, after the combination of fluorescence monoclonal resistance, is excited by 488nm, measures fluorescence intensity in the cell, detects and respectively organizes cell TOPOII activity, as table 6
Table 6 prevention administration is to mice drug resistance S
180Active X ± the SD that influences of cell TOPOII
| Group | N | The topOII activity | Suppression ratio (%) |
| Normal S180 groups of cells model cell group Rhizoma Coptidis toxic materials clearing away decoction ethanol extract high dose | 11 10 10 | 5.46±1.95*** 26.66±10.02 15.95±8.31** | - 40.17 |
| Rhizoma Coptidis toxic materials clearing away decoction ethanol extract low dosage | 10 | 20.31±2.10* | 23.82 |
Annotate: with model control group S
180Cell compares: " * " P＜0.05, " * * " P＜0.01, " * * * " P＜0.001.Suppression ratio is: (model group-alkaloid group) ÷ model group * 100% (expression rate).
Result of the test prompting: simulate clinical chemotherapy and induce back mice S
180Cell TOPOII is active obviously to be strengthened, and the Rhizoma Coptidis toxic materials clearing away decoction ethanol extract obviously suppresses chemotherapy and induces back S
180The TOPOII activity of cell illustrates that suppressing the TOPOII activity is one of approach of Chinese traditional medicine biology alkali intervention tumor cell MDR.
Discuss:
Tumor cell multidrug resistance is the one of the main reasons that causes the chemotherapy failure, and is closely related with tumor patient death, intervenes and reverse multiple drug resistance of tumor, improves chemotherapeutic efficacy, for oncotherapy positive meaning arranged.
The generation of the acquired multidrug resistance MDR of chemotherapy of tumors can be seen tumor cell multidrug resistance gene, drug-resistant protein overexpression, performances such as related enzyme activity increase such as tumor cell DNA topoisomerase TOPOII.During the overexpression of drug resistant gene and drug-resistant protein, chemotherapeutics can be discharged cell and stoped medicine to enter nucleus through transmembrane transport, make chemotherapeutics can not bring into play cytotoxic effect; The TOPOII increased activity strengthens the DNA repair ability, the cytotoxicity of antagonism chemotherapeutics, and reproducibility glutathione enzymatic activity increases, and combines with chemotherapeutics, stops chemotherapeutics to act on tumor cell, and produces drug resistance.
We are application simulation clinical tumor patient clinical chemotherapy inducing mouse S
180Cellular expression produces drug resistance, observes Rhizoma Coptidis toxic materials clearing away decoction ethanol extract vivo medicine-feeding to drug resistance of tumor cell associated biomolecule active substance P
170, the influence expressed of LRP, TOPOII, result's Rhizoma Coptidis toxic materials clearing away decoction ethanol extract prevention administration as can be known can obviously reduce the inductive mice S of simulation clinical chemotherapy
180The expression product P of cell drug resistant gene MDR
170, lung drug-resistant protein LRP expression, suppress the activity of cell topoisomerase II (TOPOII).Illustrate that Rhizoma Coptidis toxic materials clearing away decoction ethanol extract prevention administration can obviously suppress the generation of tumor cell multidrug resistance, it intervenes mechanism may be that the influence of related enzyme activity realizes by the regulation and control to drug resistant gene and drug-resistant protein expression.
Test case 3: the coptis detoxifcation decoction extract reverses mice S
180Cell multidrug resistance correlation molecule P
170, the research expressed of LRP, TOPOII
1. test material
1.1 test drug: Rhizoma Coptidis toxic materials clearing away decoction is assisted preparation by my plant chamber professor Yang Shubin of institute, and is substantially the same manner as Example 3; Cisplatin for inj: Shandong Qilu Pharmaceutical Factory product, lot number: 01111611; Cyclophosphamide: Hualian Pharmaceutical Co., Ltd., Shanghai's product, lot number: 030703; 5-fluorouracil: Tianjin gold credit aminoacid company limited product, lot number: the accurate word H12020959 of traditional Chinese medicines.
1.2 experimental animal: Kunming mouse is provided the quality certification number: Shandong kinoplaszm word: 200210023 by Shandong University's Experimental Animal Center; Mice S
180Ascitic type tumor kind Mus: provide by pharmacological room of medicine institute of Shandong Medical University.
1.3 test reagent: iodate pyrimidine PI:C27H34N4I2, numbering [25535-16-4], U.S. Sigarm company product, lot number: NO.247-081-0; MDR1 expression product P
170, the monoclonal antibody IgC1 of TOPOII, LRP, different sulfur hydracid fluorescein (FITC) labelling and immune negative control product IgC1-FITC be by U.S. pharmingen company product, provide by Beijing Yue Tai biotech firm.
2. test method
2.1 mice S
180The foundation and the mice group of cell tumour drug resistance model
S
180Mice (ascitic type) 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, and get the drug resistance mice S that each chemotherapy induced for 4 weeks
180Ascites 2ml, behind the anticoagulant heparin, PBS liquid eluting three times, 70% ethanol is fixed, 4 ℃ of preservations.Mouse inoculation drug resistance S
18024h behind the cell is divided into matched group, Rhizoma Coptidis toxic materials clearing away decoction 100mg/kg, 50mg/kg group, 14 every group at random, matched group is water 0.2ml/10gig, and Rhizoma Coptidis toxic materials clearing away decoction 100mg/kg and 50mg/kg group give 0.5%, 0.25% Rhizoma Coptidis toxic materials clearing away decoction 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.
2.2MDR1 expression product P
170, the detection method expressed of TOPOII, LRP
Get the fixed mice S of 70% ethanol
180Cell, by after the PH7.4PBS 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.
3. result of the test
3.1 to the inductive multidrug resistance ascitic type of chemotherapy mice 8
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 PH7.4PBS 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 7.
Table 7 coptis detoxifcation decoction extract is to the inductive multidrug resistance mice of chemotherapy S180 cell P
170Reverse effect X ± the SD of overexpression
| Group | n | Expression rate | Tumour inhibiting rate (%) |
| Around inducing, chemotherapy organizes matched group Rhizoma Coptidis toxic materials clearing away decoction 100mg/kg | 9 11 10 | 24.01±7.48 28.12±9.79 5.52±2.61 *** | - 17.12& 80.37 |
| Rhizoma Coptidis toxic materials clearing away decoction 50mg/kg | 10 | 9.16±3.67 *** | 67.43 |
Annotate: with induce after go down to posterity matched group relatively: "
* *" P＜0.001; “ ﹠amp; " and chemotherapy is induced all around, and group compares; Tumour inhibiting rate (%)=(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.Rhizoma Coptidis toxic materials clearing away decoction obviously reverses drug resistance mice S
180Cell drug resistant gene P
170Overexpression.Illustrate that Rhizoma Coptidis toxic materials clearing away decoction 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 reversing, the coptis detoxifcation decoction extract obtains 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 8.
Table 8 coptis detoxifcation decoction extract reversing drug resistance S
180Cell LRP overexpression influence X ± SD
| Group | n | The LRP expression rate | Suppression ratio (%) |
| Around inducing, chemotherapy organizes matched group Rhizoma Coptidis toxic materials clearing away decoction 100mg/kg | 9 11 10 | 70.21±21.75 67.65±25.42 6.86±3.39 *** | - -3.65& 89.86 |
| Rhizoma Coptidis toxic materials clearing away decoction 50mg/kg | 10 | 10.76±6.28 *** | 84.09 |
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 Rhizoma Coptidis toxic materials clearing away decoction
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 alkaloid reverses mice S180 mdr cell DNA topoisomerase TOPOII and 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 9.
Table 9 coptis detoxifcation decoction extract reverses the X ± SD that influences of mice S180 mdr cell TOPOII expression
| Group | N | The topOII expression rate | Suppression ratio (%) |
| Around inducing, chemotherapy organizes matched group Rhizoma Coptidis toxic materials clearing away decoction 100mg/kg | 9 11 10 | 22.57±12.38 24.45±14.35 6.18±3.38 *** | - 8.33& 74.72 |
| Rhizoma Coptidis toxic materials clearing away decoction 50mg/kg | 10 | 8.58±4.10 *** | 64.91 |
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%.
Result of the test prompting: the acquired drug-resistance mice S after the remarkable reverse of Rhizoma Coptidis toxic materials clearing away decoction is induced by chemotherapy
180The overexpression of cell TOPOII, and the matched group that went down to posterity after inducing 10 days with induce back its expression rate no significant difference of 4 when week, illustrate that Rhizoma Coptidis toxic materials clearing away decoction is by reversing the overexpression of TOPOII, the reverse of intervention chemotherapy multidrug resistance.
4. discuss
Suppress the expression of tumor multi-medicine drug-resistant gene, drug-resistant protein and tumor cell TOPOII, promote that the multidrug resistance tumor cells apoptosis is the effective way of the acquired tumor multi-medicine drug-resistant of reverse (MDR) of generally acknowledging.Chinese traditional medicine biology alkali is that a class has multiple bioactive Chinese medicinal ingredients, and effect such as it is antibiotic, arrhythmia, calcium antagonism and blood sugar lowering has been accepted extensively by the world of medicine, and tetrandrine is external to K
562Chemical sproof influence etc. cell line also has report in recent years
[8]We have induced mice S with the success of simulation clinical chemotherapy PFC scheme
180The overexpression of cell multidrug resistance correlation factor at the somatic cell model, observe the common alkaloid of Chinese medicine to mice drug resistance S with the modal oral administration of Chinese medicine
180The reverse of the expression of cell drug resistance correlation factor and apoptotic influence, the experimental result prompting: Rhizoma Coptidis toxic materials clearing away decoction obviously reduces mice drug resistance S
180The expression of the expression product P170 of cell multidrug resistance gene MDR1, lung drug-resistant protein LRP and cell DNA topoisomerase TOPOII.The mechanism of action of the acquired multidrug resistance of Rhizoma Coptidis toxic materials clearing away decoction reversing tumor is described, (MDR1, MDR1 are the main expressing genes of tumor drug resistance, and it is mainly by mucin P with its reduction multidrug resistance gene
170Being also referred to as P-gp constitutes) thereby and drug resistance associated protein LRP express inhibition chemotherapeutics toxicity; Suppress cell DNA TOPOII activity, reduce the drug resistance of cell chemotherapeutics; Help the absorption of chemotherapeutics, thus reversing multiple medicine resistance of tumor cells.
Test case 4: Rhizoma Coptidis toxic materials clearing away decoction reverses mice drug resistance S
180Sarcoma chemotherapy resistance;
1. test material
1.1 test drug: Rhizoma Coptidis toxic materials clearing away decoction is assisted preparation by my plant chamber professor Yang Shubin of institute, and is substantially the same manner as Example 3; Cisplatin for inj: Shandong Qilu Pharmaceutical Factory product, lot number: 01111611; Cyclophosphamide: Hualian Pharmaceutical Co., Ltd., Shanghai's product, lot number: 030703; 5-fluorouracil: Tianjin gold credit aminoacid company limited product, lot number: the accurate word H12020959 of traditional Chinese medicines.
1.2 experimental animal: Kunming mouse is provided the quality certification number: Shandong kinoplaszm word: 200210023 by Shandong University's Experimental Animal Center; Mice S
180Ascitic type tumor kind Mus: provide by pharmacological room of medicine institute of Shandong Medical University.
2. test method
2 mice S
180The foundation of cell tumour drug resistance model and Rhizoma Coptidis toxic materials clearing away decoction reverse the effect of chemotherapy resistance
S
180Mice (ascitic type) 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 is inoculated in left oxter, be divided into matched group at random, Rhizoma Coptidis toxic materials clearing away decoction 100mg/kg adds cyclophosphamide 20mg/kg and cyclophosphamide 20mg/kg, every group 14, matched group is water 0.2ml/10gig, successive administration 10 days, behind last administration 24h, peel off tumor, scales/electronic balance weighing, result such as table 10:
Table 10 coptis detoxifcation decoction extract reverses mice drug resistance S
180Sarcoma chemotherapy resistance X ± SD
| Group | N | The heavy mg of tumor | Suppression ratio (%) |
| Matched group cyclophosphamide 20mg/kg Rhizoma Coptidis toxic materials clearing away decoction 100mg/kg adds cyclophosphamide 20mg/kg | 14 14 14 | 1.57±0.38 1..15±0.35 ** 0.96.±3.78 *** | - 26.75 42.03 |
The result of the test prompting: the coptis detoxifcation decoction extract obviously improves cyclophosphamide 20mg/kg to drug resistance mice S
180The tumour inhibiting rate of sarcoma reverses mice drug resistance S
180Sarcoma chemotherapy resistance.
3. discuss
The coptis detoxifcation decoction extract obviously improves cyclophosphamide 20mg/kg to drug resistance mice S
180The tumour inhibiting rate of sarcoma illustrates that Rhizoma Coptidis toxic materials clearing away decoction has the mice of reverse drug resistance S
180The effect of sarcoma chemotherapy resistance.The main generation because of tumor multi-medicine drug-resistant of chemotherapy of tumors resistance, the tumor-inhibiting action of reduction chemotherapeutics, coptis detoxifcation decoction extract obviously improve the tumor that presses down of cyclophosphamide and make rate, have shown that it reverses the effect of chemotherapy resistance.
Claims (1)
1. Rhizoma Coptidis toxic materials clearing away decoction is in the application of preparation prophylaxis of tumours chemotherapy resistance medicine, and the medicinal raw material of Rhizoma Coptidis toxic materials clearing away decoction is Rhizoma Coptidis 3-9 gram, Radix Scutellariae 3-9 gram Cortex Phellodendri 3-9 gram Fructus Gardeniae 6-12 gram.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102309591A (en) * | 2011-09-20 | 2012-01-11 | 湖南农业大学 | Formula and preparation method of compound coptis injection |
| CN102764324A (en) * | 2012-07-27 | 2012-11-07 | 金忠和 | Medicine composition for treating neoplastic diseases and improving immunity |
| CN114767756A (en) * | 2022-04-25 | 2022-07-22 | 复旦大学附属华山医院 | Application of coptis chinensis detoxification decoction in preparation of sensitizer for tumor immune checkpoint blockade therapy |
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| CN1437874A (en) * | 2002-02-10 | 2003-08-27 | 罗广生 | Apparatus for making bean-curd with filling by once-moulding and producing method thereof |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102309591A (en) * | 2011-09-20 | 2012-01-11 | 湖南农业大学 | Formula and preparation method of compound coptis injection |
| CN102764324A (en) * | 2012-07-27 | 2012-11-07 | 金忠和 | Medicine composition for treating neoplastic diseases and improving immunity |
| CN114767756A (en) * | 2022-04-25 | 2022-07-22 | 复旦大学附属华山医院 | Application of coptis chinensis detoxification decoction in preparation of sensitizer for tumor immune checkpoint blockade therapy |
| CN114767756B (en) * | 2022-04-25 | 2023-04-07 | 复旦大学附属华山医院 | Application of coptis chinensis detoxification decoction in preparation of sensitizer for tumor immune checkpoint blockade therapy |
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