CN1689574A - Application of alisol B23-mono acetic ester and alisma rhizome extract in reversal of cancer cell multi-drug tolerance - Google Patents
Application of alisol B23-mono acetic ester and alisma rhizome extract in reversal of cancer cell multi-drug tolerance Download PDFInfo
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- CN1689574A CN1689574A CN 200410038420 CN200410038420A CN1689574A CN 1689574 A CN1689574 A CN 1689574A CN 200410038420 CN200410038420 CN 200410038420 CN 200410038420 A CN200410038420 A CN 200410038420A CN 1689574 A CN1689574 A CN 1689574A
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- extract
- rhizoma alismatis
- alisol
- cell
- monoacetate
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Abstract
The present invention relates to the application of alisol B23-monoacetate and other alisma extract in reversing the resistance of cancer cell on drugs. The present invention also relates to the application of medicine preparation with alisol B23-monoacetate and other alisma extract. Alisol B23-monoacetate and other alisma extract is applied in treating drug resistance caused by excessive expression of P-glucoprotein to raise the sensitivity of drug resisting cell on various kinds of anticancer medicine, to generate synergist with anticancer medicine and to reduce toxic side effect.
Description
Technical field
The present invention relates to the application in the inverse cancer cell multidrug resistance of alisol B 23-monoacetate and Rhizoma Alismatis extract.Rhizoma Alismatis of the present invention is meant Alismataceae plant Rhizoma Alismatis (Alisma plantagoOrientale (Sam) Juzep) and rhizome medicine Rhizoma Alismatis (Rhizoma Alismatis) thereof.
Background technology
The cancer patient of accepting long-term chemotherapy tends to occur the phenomenon of multiple medicines patience (MDR) in treatment: the growth of still can surviving under the effect of certain single cancer therapy drug of part cancerous cell, and show subsequently and can tolerate a large amount of structures and the incoherent cancer therapy drug of function simultaneously, thereby weakened chemotherapeutical curative effect greatly.
The molecule mechanism that causes multiple medicines patience is a lot, some of them are determined by demonstration: comprise cell surface transport protein (P-glycoprotein, MRP and LRP) overexpression and activation, the overactivity of glutathion detoxification system enzyme, the downward modulation or the gene mutation of DNA topological isomer enzyme gene expression, the overexpression of the sudden change of caused by tumor suppressor p 53 and Bcl-2 gene.
Wherein, be found the earliest by the multiple medicines patience that overexpression caused of P-glycoprotein (P-gp), and also be a kind of molecule mechanism that is studied the most thorough.P-gp is a kind of transport protein that is positioned on the cell membrane, it can rely on energy that the ATP hydrolysis produces with intracellular chemical transport to the extracellular.Scientist is verified, and in the cancerous cell of P-gp overexpression, because the translocation of P-gp, the accumulation of various hydrophobic anticancer drugs is greatly diminished, thereby can't reach the desired valid density of treatment.
At present, scientist finds that some existing clinical medicines or unification compound can treat this multiple medicines patience that is caused by P-gp, as: isoptin (Verapamil) (a kind of coronary artery expansion medicine), reserpine (a kind of depressor), cephalosporin (cephalosorin), Gramicidin (a kind of antibiotic), cyclosporin A and derivant thereof (mostly being immunosuppressant greatly), and some lipophilic compounds.These medicines or chemical compound all are the aromatic micromolecule of hydrophobicity usually, and they can emulatively be combined on the P-gp, thereby suppress the transportation of P-gp to cancer therapy drug.Yet the P-gp inhibitor that these are developed often shows serious toxic and side effects clinically, thereby is not widely used.Therefore, seek the research and development focus that low toxicity and single-minded effective P-gp inhibitor become scientist.
On the other hand, in China, traditional Chinese herbal medicine is used for the existing very long history of treatment of cancer, and Chinese herbal medicine also often is used in combination with the antitumor Western medicine to improve curative effect.Based on this, we imagine in these medical herbs, may contain the natural drug that can reverse the multiple medicines patience that is caused by P-glycoprotein overexpression, and because Chinese medicine often than the gentle low toxicity of Western medicine, becomes clinical effective inversion agent so the inversion agent of being come out by tcm development may replace original medicine.Under this imagination, obtained having the Rhizoma Alismatis extract that reverses active alisol B 23-monoacetate and contain this material through overtesting, it has overcome the limitation (obvious toxic and side effects is arranged) of existing inversion agent, and the mechanism of action is clear and definite.
Summary of the invention
The object of the present invention is to provide alisol B 23-monoacetate (being called for short MG-02) and comprised the new purposes of the Rhizoma Alismatis extract (abbreviation RAE) of alisol B 23-monoacetate, i.e. the application of inverse cancer cell multidrug resistance.Alisol B 23-monoacetate is clear and definite with the reverse effect of the Rhizoma Alismatis extract that comprises alisol B 23-monoacetate, can reverse the multidrug resistance that is caused by the P-gp overexpression, and improve cancerous cell the sensitivity of cancer therapy drug and the drug effect of increase cancer therapy drug.Simultaneously, the invention still further relates to alisol B 23-monoacetate and the application of the Rhizoma Alismatis extract that comprises alisol B 23-monoacetate in preparation inverse cancer cell multidrug resistance medicine.
Rhizoma Alismatis extract of the present invention is to obtain by one of following method, but the Rhizoma Alismatis extract with purposes of the present invention has more than to be limited to from these methods and obtains:
(1) Rhizoma Alismatis dry product coarse powder is doubly measured methanol or alcohol at normal temperature, heating or reflux, extract, with 8-20, extracts 1-5 time.Extracting solution normal pressure or removal of solvent under reduced pressure are fully extracted with chloroform after adding suitable quantity of water, and chloroform extracted solution removes through normal pressure or concentrating under reduced pressure and desolvates, and promptly obtains Rhizoma Alismatis extract;
(2) Rhizoma Alismatis dry product coarse powder is doubly measured chloroform room temperature, heating or reflux, extract, with 8-20, extracts 1-5 time.Normal pressure or removal of solvent under reduced pressure promptly get Rhizoma Alismatis extract;
(3) Rhizoma Alismatis dry product coarse powder, with supercritical carbon dioxide fluid (including 2.5%-10% ethanol), at 50 ℃ with down to room temperature, extract in supercritical carbon dioxide extraction still internal recycle, the extract solvent removed by evaporation at reduced pressure, add chloroform and fully extract, normal pressure or concentrating under reduced pressure remove and desolvate, and promptly get Rhizoma Alismatis extract.
In addition, Rhizoma Alismatis extract also can obtain by other method: the solvent with middle polarity adds extractions such as acetone, ethyl acetate, perhaps use extractions such as aqueous methanol, aquiferous ethanol, aqueous acetone, mainly contained the mixture of alisol B 23-monoacetate again by extraction or column chromatography mode.
The medicine of inverse cancer cell multidrug resistance of the present invention comprises oral formulations or ejection preparation.Wherein oral formulations comprises tablet, capsule, granule, oral liquid or pill.Above-mentioned preparation is prepared routinely.When the preparation peroral dosage form, add required various conventional adjuvant in alisol B 23-monoacetate or the Rhizoma Alismatis extract, as disintegrating agent, lubricant, binding agents etc. are prepared into any peroral dosage form commonly used with the oral formulations method of routine.For example, alisol B 23-monoacetate or Rhizoma Alismatis extract and suitable excipient such as starch or derivatives thereof, lactose, cellulose derivative etc. can be mixed, make capsule in incapsulating.Perhaps when adding above-mentioned adjuvant, add binding agent such as carboxymethyl cellulose, Radix Acaciae senegalis and water and obtain granule; Adding Mel etc. is made pill.Also alisol B 23-monoacetate or Rhizoma Alismatis extract can be added binding agents such as carboxymethyl cellulose, Radix Acaciae senegalis, add microcrystalline Cellulose, modified starch etc. and make filler and disintegrating agent, the machine pressing for preparing tablet by routine becomes tablet.When preparation is used for the injectable formulation of the outer approach of intestinal, alisol B 23-monoacetate or Rhizoma Alismatis extract can be mixed with the required additives and the solvent for injection that suits, carry out sterilization treatment then and make.For example, alisol B 23-monoacetate or Rhizoma Alismatis extract are dissolved in the sterile distilled water liquid or physiological saline solution liquid with solubilizing agent and/or cosolvent, also can use stabilizing agent and/or buffer, carry out the preparation of ejection preparation.
Outstanding progress of the present invention and remarkable result are that alisol B 23-monoacetate is little with the toxic and side effects of the Rhizoma Alismatis extract that contains alisol B 23-monoacetate, have the effect of clear and definite inverse cancer cell multidrug resistance, and have the effect that improves the cancer therapy drug drug effect.
Description of drawings
Mutual relation in Fig. 1 Rhizoma Alismatis extract, alisol B 23-monoacetate and isoptin and each the cancer therapy drug drug combination
Fig. 2 Rhizoma Alismatis extract and alisol B 23-monoacetate are to the restorative influence of the cytotoxicity of vincristine in the multiple medicines patience cancerous cell
Fig. 3 Rhizoma Alismatis extract, alisol B 23-monoacetate and isoptin are to the influence of amycin accumulation in the multiple medicines patience cancerous cell
Fig. 4 Rhizoma Alismatis extract and alisol B 23-monoacetate are to the influence of rhodamine-123 transportation in the multiple medicines patience cell
Fig. 5 Rhizoma Alismatis is carried thing the influence that P-gp expresses is detected
The specific embodiment
Rhizoma Alismatis activity extract of the present invention, and the extract pharmacologically active obtains by method shown in the following embodiment.Involved method is the technological means that those skilled in the art can grasp and use.But following examples must not be interpreted as the restriction to claim of the present invention of going up in all senses.
Embodiment 1: the preparation of Rhizoma Alismatis extract (method one)
Jiangxi Rhizoma Alismatis dry product coarse powder 1-kg, with 95% alcohol reflux 3 times, each 2 hours, the ethanol consumption was 10L, 8L, 8L.Filter, merge three times extracting solution, ethanol is removed in decompression, and residue adds 500mL water suspendible, fully extracts 3 times with chloroform, each consumption 400mL, and the combined chloroform extracting solution, concentrating under reduced pressure removes and desolvates, and promptly obtains Rhizoma Alismatis extract 30g, yield 3%.
Embodiment 2: the preparation of Rhizoma Alismatis activity extract (method two)
Jiangxi Rhizoma Alismatis dry product coarse powder 1kg uses chloroform reflux, extract, 3 times, and each 2 hours, the chloroform consumption was 10L, 8L, 8L.Put coldly, filter, merge three times extracting solution, removal of solvent under reduced pressure promptly obtains Rhizoma Alismatis extract 25g, yield 2.5%.
Embodiment 3: the preparation of Rhizoma Alismatis activity extract (method three)
Jiangxi Rhizoma Alismatis dry product coarse powder 1kg, with supercritical carbon dioxide fluid (including 5% ethanol) as entrainer, under 40 ℃, the condition of pressure 10Mpa, extraction circulates in the supercritical carbon dioxide extraction still, extract after 2 hours, at room temperature, adjust the extraction-container temperature, pressure and discharge carbon dioxide.Gained extractum is the supercritical carbon dioxide extraction extract of Rhizoma Alismatis.Get this extract and add chloroform and fully extract (3 times, each consumption be 500ml), the combined chloroform extracting solution, concentrating under reduced pressure removes and desolvates, and promptly gets Rhizoma Alismatis extract 34g, yield 3.4%.
Embodiment 4: the isolation identification that has the effective ingredient of the multiple medicines patience that reverse caused by the P-gp overexpression in the Rhizoma Alismatis activity extract
The separation of effective site chemical constituent
Get embodiment 1 gained chloroform extract (30g) and use dissolve with methanol, be scattered in proper silica gel, dry method dress silica gel (200-300 order) post.With petroleum ether-ethyl acetate (10: 2,10: 5,2: 10) eluting successively, obtain 1,2,3 three fractions.After measured, fraction 1 shows biological activity.With fraction 1 once more through silica gel column chromatography, use benzene-ethyl acetate (20: 1---4: 1) eluting successively, every 50mL is a fraction, monitor with silica gel thin-layer chromatography (TLC), the fraction that merges same composition is also tested each eluting position, gets an active fractions from eluate in 4: 1, and the gained active fractions is through silica gel column chromatography repeatedly, benzene-ethyl acetate (4: 1) eluting gets single-activity composition I.I repeatedly behind the recrystallization, obtains white crystal 130mg through normal hexane-benzene.Analyze through HPLC, purity is greater than 98%.
I MG-02, white crystal, through MS,
1H,
13C-NMR, its data are identical with alisol B 23-monoacetate.Warp
1H NMR,
13C NMR and MS determine that molecular formula is C
32H
50O
5FABMSm/z：537(M+Na)，515(M+H)。
1H-NMRδ(CDCL3)ppm：4.61(1H，td，J＝8.8，2.4Hz，23-H)，3.81(1H，td，J＝10.8，5.6Hz，11-aH)，2.74(1H，d，J＝8.8Hz，24-H)，2.55(1H，dd，J＝13.2，5.6Hz，11-eH)，2.16(1H，ddd)，2.08(3H，s，CH
3CO)，1.72(1H，d，J＝10.8Hz，9-aH)，1.34，1.32，1.15，1.08，1.06，1.05，0.98(3Heach，s，CH
3)，1.03(3H，d，J＝6.8Hz，21-CH
3)。
13C-NMR δ (CDCL3) ppm sees attached list one.
Table one
| ??No. | ????1 | Alisol B 23-monoacetate | ????No. | ?1 | Alisol B 23-monoacetate |
| ????1 ????2 ????3 ????4 ????5 ????6 ????7 ????8 ????9 ????10 ????11 ????12 ????13 ????14 ????15 ????16 | ????30.9 ????36.7 ????220.1 ????46.9 ????48.4 ????20.0 ????34.1 ????40.7 ????49.9 ????36.9 ????70.2 ????34.5 ????138.1 ????57.0 ????30.6 ????29.1 | ????30.9 ????36.7 ????220.1 ????46.9 ????48.4 ????20.0 ????34.1 ????40.7 ????49.9 ????36.9 ????70.1 ????34.4 ????138.1 ????57.0 ????30.6 ????29.1 | ????17 ????18 ????19 ????20 ????21 ????22 ????23 ????24 ????25 ????26 ????27 ????28 ????29 ????30 ????Ac | ?134.2 ?23.1 ?25.6 ?27.8 ?20.0 ?33.7 ?71.4 ?65.0 ?58.4 ?19.4 ?24.6 ?29.5 ?20.0 ?23.8 ?169.9 ?21.1 | ????134.1 ????23.1 ????25.6 ????27.8 ????20.0 ????33.7 ????71.4 ????65.0 ????58.4 ????19.3 ????24.6 ????29.5 ????20.0 ????23.8 ????169.9 ????21.1 |
Structure is as follows:
Embodiment 5: Rhizoma Alismatis extract and alisol B 23-monoacetate are to the reverse effect of the multiple medicines patience that caused by the P-gp overexpression
(1) the cancer cell in vitro bulk-growth suppresses to detect
1. material and method
1.1 medicine: testing drug is respectively Rhizoma Alismatis extract, alisol B 23-monoacetate, actinomycin D, puromycin, paclitaxel, vincristine, amycin and isoptin.
1.2 sensitivity and multiple medicines patience cancerous cell: testing used cell line is that human body hepatoma cell line HepG2, leukaemia are that K562 and multiple medicines patience subcellular fraction thereof are HepG2-DR and K562-DR, all from the Dr.Judy Chan of Hong Kong Chinese University laboratory, these two kinds of multiple medicines patience cell lines are all carried out selectivity with amycin to sensitivity blast cell system and are progressively cultivated and set up (promptly improve constantly the concentration of amycin in the cultivation of sensitivity cancerous cell, thereby the mdr cell that retains survival playing the purpose of screening).The condition of culture of all cells is as follows: at 37 ℃, and C0
2In the environment of content 5%, grow in and contain in 10% hyclone and the antibiotic RPMI-1640 culture fluid of 100U/mL.For keeping the phenotypic characteristic of multiple medicines patience, 1.2 μ M and 0.1 μ M amycin have been added in the culture fluid of HepG2-DR and K562-DR respectively.
1.3 medicinal application: cell to be detected is dispersed into the individual cells suspension, by 5 * 10
3The cells/well branch is planted in 96 orifice plates, use the pastille culture fluid after 24 hours instead, per 3 holes are a detectable concentration, and medicine and cytosis were measured drug cell toxicity with sulfur rhodamine B (SRB) method after 72 hours, and experiment is established culture fluid blank group-I in addition and reached cell matched group-II that not dosing is handled.
1.4SRB method is measured the half cytostatics amount concentration (IC of medicine cell growth
50): medicine and cytosis be after 72 hours, (comprises and cultivate the white matched group in the night sky) that 50% trichloroacetic acid that adds pre-cooling was 4 ℃ of following fixed cells one hour in all holes.Discard fixative and also float, dry with the distillation washing, the SRB dyeing with 0.4% 10 minutes, the dyeing liquor that the acetic acid flush away of reuse 1% is unnecessary is also air-dry.At last, to be dissolved in the Tris buffer solution (PH10.5) of 10mM with the bonded SRB of cell, measure the absorbance A value with ultraviolet spectrophotometer 515nm wavelength, with the color intensity of measuring SRB under the condition, this moment, color intensity and the cell number of SRB were proportionate, and can this estimate comparison as cell quantity.Cell survival rate (%)=(experimental port A value/control wells-IIA value) * 100%, IC
50It for cell survival rate 50% o'clock drug level.
2. result
2.1 antineoplastic agent, Rhizoma Alismatis extract and alisol B 23-monoacetate are for the cytotoxicity of HepG2 and HepG2-DR cell: as shown in table 1, HepG2 is to actinomycin D, puromycin, paclitaxel, vincristine and amycin all demonstrate high susceptibility, and Rhizoma Alismatis extract and alisol B 23-monoacetate are all lower to the cytotoxicity of drug sensitive cell HepG2 and mdr cell HepG2-DR: no matter be the Rhizoma Alismatis extract or the IC of alisol B 23-monoacetate
50All be higher than the anticarcinogen 100-100 that this institute adopts, 000 times (table 1).The used multiple medicines patience cell line HepG2-DR of this test selects to cultivate out under the effect of amycin, but this multiple medicines patience cell is except that showing intensive adriamycin-resistant, to actinomycin D, and puromycin, paclitaxel, vincristine also show intensive drug resistance.This cell can be assessed by the tolerance factor the drug-resistant intensity of different pharmaceutical and (tolerate the factor and equal the IC of drug effect in HepG2-DR
50Value and the I that acts on HepG2
C50Ratio (table 1)).The tolerance factor of Rhizoma Alismatis extract and alisol B 23-monoacetate is very little, shows that promptly they are very little for the effect difference of two kinds of cell lines.
Table 1: Rhizoma Alismatis extract, alisol B 23-monoacetate and cancer therapy drug are to hepatoma carcinoma cell
The growth inhibitory effect of HepG2, multiple medicines patience cell HepG2-DR
| ?????????????????IC 50 * | |||
| HepG2 | ?HepG2-DR | The tolerance factor ** | |
| Rhizoma Alismatis extract (μ g/mL) | 35.79±1.43 | ?46.80±3.62 | ????1.3 |
| Alisol B 23-monoacetate (μ M) | 18.69±3.56 | ?19.44±3.86 | ????1.1 |
| Actinomycin D (μ M) | 0.002±0.0004 | ?2.19±0.50 | ????1095 |
| Puromycin (μ M) | 0.35±0.01 | ?311±92.6 | ????888 |
| Paclitaxel P (μ M) | 0.003±0.0003 | ?4.30±0.23 | ????1433 |
| Vincristine (μ M) | 0.0004±0.0002 | ?0.46±0.03 | ????1150 |
| Amycin (μ M) | 0.055±0.004 | ?41.6±2.30 | ????756 |
(2) assessment of drug combination in the multiple medicines patience cell
1. material and method
1.1 medicine: testing drug is respectively Rhizoma Alismatis extract, alisol B 23-monoacetate, actinomycin D, puromycin, paclitaxel, vincristine, amycin and isoptin (positive control).
1.2 sensitivity and multiple medicines patience cancerous cell: as described in (one) 1.2.
1.3 medicinal application: cancer therapy drug is mixed by 1: 1 with Rhizoma Alismatis extract, alisol B 23-monoacetate or isoptin.On the other hand, cell to be detected is dispersed into the individual cells suspension, by 5 * 10
3The cells/well branch is planted in 96 orifice plates, use the pastille culture fluid after 24 hours instead, per 3 holes are a detectable concentration, and medicine and cytosis were measured drug cell toxicity with sulfur rhodamine B (SRB) method after 72 hours, and experiment is established culture fluid blank group-I in addition and reached cell matched group-II that not dosing is handled.
1.4 cell growth measurement: as described in (one) 1.4.
1.5 drug combination assessment: detect the effect of drug combination and compare with the effect of independent medication by the SRB method.The gained comparative result, i.e. interaction between medicine can be represented with association index (CI): CI is greater than 4 expression antagonisms, and CI is less than 0.8 expression synergism.If the CI value between 0.8 and 4, is then represented adduction, referring to Fig. 1, wherein MG-02 is an alisol B 23-monoacetate.
2. result: we studies show that no matter Rhizoma Alismatis extract still is an alisol B 23-monoacetate and can both improve the sensitivity of multiple medicines patience HepG2-DR cell for antineoplastic agent.As shown in table 2, under the effect of Rhizoma Alismatis extract or alisol B 23-monoacetate, the HepG2-DR cell is improved largely to the sensitivity of anticarcinogen.By IC
50Value as seen, the two has all almost completely recovered the toxic action of puromycin to HepG2-DR, while HepG2-DR cell has also improved 30 times for the sensitivity of amycin, and the sensitivity of actinomycin D and paclitaxel has been improved 10 times respectively.In addition, it is comparatively similar with the ability that alisol B 23-monoacetate reverses multiple medicines patience that we also observe Rhizoma Alismatis extract, and compare with isoptin, all shown stronger reverse effect.
By analysis to medication combined index (CI), Rhizoma Alismatis extract or alisol B 23-monoacetate with the synergism (Fig. 1) that all demonstrates in the use in various degree uniting of different anticarcinogens, wherein actinomycin D and Rhizoma Alismatis extract or alisol B 23-monoacetate, the drug combination of paclitaxel and Rhizoma Alismatis extract or alisol B 23-monoacetate have all shown the most significant synergism.Simultaneously, Rhizoma Alismatis extract still is that alisol B 23-monoacetate all can not strengthen the sensitivity of HepG2 cell to anticarcinogen, and Rhizoma Alismatis extract or alisol B 23-monoacetate are united use with cancer therapy drug and all shown tangible adjection in the HepG2 cell.This explanation, Rhizoma Alismatis extract and alisol B 23-monoacetate are relevant to the active function of the raising of cancerous cell susceptibility degree and P-gp.
Table 2. Rhizoma Alismatis extract, alisol B 23-monoacetate and isoptin are to the influence to cancer therapy drug sensitivity of HepG2 and HepG2-DR cell
| ????IC 50-HepG2-DR | ????IC 50-HepG2 | Tolerance factor * | |
| Actinomycin D, (μ M)+Alisma extract+alisol B 23-monoacetate+isoptin puromycin, (μ M)+Alisma extract+alisol B 23-monoacetate+isoptin taxol, (μ M)+Alisma extract+alisol B 23-monoacetate+isoptin vincristine, (μ M)+Alisma extract+alisol B 23-monoacetate+isoptin adriamycin, (μ M)+Alisma extract+alisol B 23-monoacetate+isoptin | ????2.19±0.50 ????0.21±0.03 ????0.31±0.18 ????0.58±0.06 ????311±92.6 ????1.18±0.40 ????1.93±0.74 ????6.45±1.63 ????4.30±0.23 ????0.43±0.15 ????0.60±0.09 ????0.75±0.05 ????0.46±0.03 ????0.12±0.02 ????0.18±0.02 ????0.16±0.01 ????41.6±2.30 ????1.42±0.38 ????1.18±0.25 ????1.40±0.04 | ????0.002±0.0004 ????0.002±0.0002 ????0.002±0.0003 ????0.002±0.0008 ????0.35±0.01 ????0.196±0.013 ????0.242±0.104 ????0.290±0.061 ????0.003±0.0003 ????0.002±0.001 ????0.003±0.002 ????0.002±0.001 ????0.0004±0.0002 ????0.0003±0.0001 ????0.0003±0.0001 ????0.0003±0.0002 ????0.055±0.004 ????0.049±0.019 ????0.053±0.019 ????0.046±0.018 | ????1095 ????105 ????155 ????290 ????888 ????6 ????8 ????22 ????1433 ????215 ????200 ????375 ????1150 ????400 ????600 ????533 ????756 ????29 ????22 ????30 |
(3) Rhizoma Alismatis extract and alisol B 23-monoacetate are induced G to vincristine in multiple medicines patience cell
2The restorative influence of/M stagnation
1. material and method
1.1 medicine: testing drug is respectively Rhizoma Alismatis extract, alisol B 23-monoacetate, vincristine, and isoptin (positive control).
1.2 instrument: (CA), the gained data then adopt MacintoshCellQuest software to analyze to the FACSCAN cells were tested by flow cytometry for Becton DickinsonImmunocytometry Systems, San Jose.
1.3 sensitivity and multiple medicines patience cancerous cell: as described in (one) 1.2.
1.4 medicinal application: HepG2 or HepG2-DR cell are respectively by the mixture of the Rhizoma Alismatis extract (2.5 μ g/mL or 25 μ g/mL) of vincristine (300ng/mL) and vincristine and variable concentrations, or with the mixture process of alisol B 23-monoacetate (1 μ M or, 10 μ M) 24 hours, and to adopt untreated cell be matched group.
1.5 sample treatment and analysis: the cell after the processing is with after the PBS washed twice of pre-cooling, and the ethanol with 70% fixedly spends the night in-20 ℃.The cell that is fixed was handled 30 minutes down at 37 ℃ with 200 μ g/mL ribonuclease after washing with PBS.Add propidium iodide solution (final concentration is 40 μ g/mL) at last, it is combined with DNA.Handle after 10 minutes, cell is analyzed with the FACSCAN flow cytometer immediately.
2. result: referring to Fig. 2, Rhizoma Alismatis extract and alisol B 23-monoacetate are to the restorative influence of the cytotoxicity of vincristine in the multiple medicines patience cancerous cell, wherein, Fig. 2 (A) is the influence of Rhizoma Alismatis extract in the HepG2 cell, Fig. 2 (B) is the influence of Rhizoma Alismatis extract in the HepG2-DR cell, Fig. 2 (C) is the influence of alisol B 23-monoacetate in the HepG2 cell, and Fig. 2 (D) is the influence of alisol B 23-monoacetate in the HepG2-DR cell.Vincristine shows that for the cytotoxicity of cancerous cell the mitotic spindle of inhibition forms and thereby the inducing cell cycle arrest makes cell enter procedural apoptosis in the G2/M-stage.Handled 24 hours through vincristine, can cause the HepG2 cell obviously to be stagnated in G
2/ M-the stage (Fig. 2 A, C).But the cell cycle to multiple medicines patience HepG2-DR cell does not have similar effect under the same conditions.After vincristine and Rhizoma Alismatis extract or alisol B 23-monoacetate Combined Treatment, the HepG2-DR cell is stuck in G
2The ratio in/M stage increases along with the increase of Rhizoma Alismatis extract or alisol B 23-monoacetate dosage.This result shows that Rhizoma Alismatis crude extract and unification compound all can reverse the phenotype drug-resistant effect of multiple medicines patience cell to vincristine.
Embodiment 6 Rhizoma Alismatis are proposed the detection to the functional translocation of P-gp of thing and alisol B 23-monoacetate
(1) Rhizoma Alismatis is carried thing and alisol B 23-monoacetate the effect of amycin accumulation in the multiple medicines patience cell is detected
1, material and method
1.1 medicine: testing drug is respectively Rhizoma Alismatis extract, alisol B 23-monoacetate, amycin and isoptin (positive control).
1.2 cell: as described in embodiment 5 () 1.2.
1.3 instrument: as described in embodiment 5 (three) 1.2.
1.4 amycin accumulation test: 1 * 10
6Individual cell was cultivated 1 hour in 37 ℃ in the culture fluid of Rhizoma Alismatis extract that contains 10 μ M amycin and variable concentrations or alisol B 23-monoacetate.The amycin content of cultivating in the cell of back can pass through cells were tested by flow cytometry, and the gained data are analyzed with Macintosh CellQuest software.In the experiment, isoptin is used as positive control as known P-gp inhibitor.
2. result: HepG2-DR and K562-DR cell line are attributed to the overexpression of P-gp to the tolerance effect of chemotheraping preparation, compare with its sensitivity blast cell system, the antitumor drug content that is accumulated in these cells weakens its effect owing to reaching required valid density.Referring to Fig. 3, Rhizoma Alismatis extract, alisol B 23-monoacetate and isoptin are to the influence of amycin accumulation in the multiple medicines patience cancerous cell.Rhizoma Alismatis extract, alisol B 23-monoacetate all are the IC by separately
50Multiple add i.e. more separately reverse effect under same toxicity condition.Each organizes the content of amycin by representing with the ratio of matched group (sample that only has amycin to handle).In this research, cell was cultivated 1 hour in together with the culture fluid of Rhizoma Alismatis extract, alisol B 23-monoacetate or the isoptin of variable concentrations in containing 10 μ M amycin, subsequently the fluorescence intensity by amycin in the cells were tested by flow cytometry cell.HepG2-DR (Fig. 3 A) and K562-DR cell (Fig. 3 B) are under the effect of Rhizoma Alismatis extract or alisol B 23-monoacetate, intracellular amycin content obviously improves, in addition, be that Rhizoma Alismatis extract or alisol B 23-monoacetate all can accumulate more amycin than isoptin, this shows inversion agent Rhizoma Alismatis extract or the alisol B 23-monoacetate reverse effect stronger than isoptin that have to the multiple medicines patience cell of P-gp overexpression.
(2) Rhizoma Alismatis is proposed the detection to rhodamine-123 transportation influence in the multiple medicines patience cell of thing and alisol B 23-monoacetate
1, material and method
1.1 medicine: testing drug is respectively Rhizoma Alismatis extract, alisol B 23-monoacetate, rhodamine-123 and isoptin (positive control).
1.2 cell: as described in embodiment 5 () 1.2.
1.3 instrument: as described in embodiment 5 (three) 1.2.
1.4 rhodamine-123 transportation test: containing 1 * 10
6Add Rh-123 in the cell suspension of individual cell, make final concentration 5ug/mL, cultivate 1 hour to absorb Rh-123 at 37 ℃ subsequently, the PBS washed cell of reuse pre-cooling 2 times is to remove the unnecessary Rh-123 in extracellular, cell after the washing is resuspended in the fresh medium, add RAE or alisol B 23-monoacetate and continue and cultivated 1 hour at 37 ℃, this moment, Rh-123 can discharge in cell by the effect of P-gP.The fluorescence intensity of the interior Rh-123 of cell can be passed through the FACSCAN cells were tested by flow cytometry, and (CA), the gained data then adopt Macintosh CellQuest software to analyze for BectonDickinson Immunocytometry Systems, San Jose.
2. result: adopt flow cytometer can measure the influence of Rhizoma Alismatis extract or alisol B 23-monoacetate to rhodamine-123 in the P-gp overexpression cell (a kind of fluorogenic substrate of P-gp) transportation.Referring to Fig. 4, Rhizoma Alismatis extract and alisol B 23-monoacetate are to the influence of rhodamine-123 transportation in the multiple medicines patience cell, cell in the culture fluid that contains rhodamine-123 37 ℃ cultivated one hour, thick line is represented is the fluorescence intensity of rhodamine-123 in the cell behind this hour.Extracellular subsequently rhodamine-123 is removed, and adds or does not add Rhizoma Alismatis extract (25 μ g/mL), alisol B 23-monoacetate (1-40 μ M) or isoptin (10 μ M) and continue to cultivate one hour.Fine rule is represented after back one hour residual rhodamine-123 fluorescence intensity in the cell, after dotted line is represented back one hour under Rhizoma Alismatis extract, alisol B 23-monoacetate or isoptin effect the fluorescence intensity of residual rhodamine-123 in the cell.From Fig. 4 as seen, absorbed the HepG2 and the K562 cell of rhodamine-123, dyestuff was removed 1 hour from cell culture fluid after, the fluorescence intensity in the cell still remained unchanged substantially.Then significantly reduction of the content of rhodamine-123 in HepG2-DR and the K562-DR cell under the same case, we have further confirmed the transportation active function of P-glycoprotein with this.Experimental result shows, Rhizoma Alismatis extract still is the loss of fluorescent material in the mdr cell (Fig. 4) of all obviously having slowed down of alisol B 23-monoacetate, and the reverse effect of alisol B 23-monoacetate is proportional with used dosage under study for action.Wherein, Fig. 4 A is rhodamine in the sensitivity cell-123 test, and Fig. 4 B is that Rhizoma Alismatis extract is in multiple medicines patience cell.Loaded: the fluorescence intensity of rhodamine-123 in the cell after first hour; Residual: residual rhodamine-123 fluorescence intensity in the cell after second hour; Isoptin: after second hour under the isoptin effect fluorescence intensity of residual rhodamine-123 in the cell; RAE: after second hour under the Rhizoma Alismatis extract effect fluorescence intensity of residual rhodamine-123 in the cell, Fig. 4 C: the effect of alisol B 23-monoacetate in multiple medicines patience cell.DMSO is the solvent control group.
Embodiment 7 Rhizoma Alismatis are carried thing the influence that P-gp expresses are detected
1, material and method
1.1 medicine: testing drug is a Rhizoma Alismatis extract.
1.2 cell: as described in embodiment 5 () 1.2.
1.3 the immunity that P-gp expresses in cell seal stain technology for detection analysis: handle various cells 0,4,8 and 12 hours with Rhizoma Alismatis extract.Cell after processing centrifuging and taking after placing 30 minutes in the buffer (50mMTrisPH7.4,100mMNaCl, 2mMEDTA, 1% sodium deoxycholate, 0.1%SDS, 1%triton X-100,2mM PMSF, 1% aprotinin) of pre-cooling gets total protein.With Bu Laidefu detection method (Bradford assay) test sample protein concentration.In this test, 40 μ g total proteins are transferred on the nitrocellulose membrane by electromigration after the 8%SDS/PAGE electrophoretic separation.With 5% skim milk/0.1%Tween-20/TBS (10mM Tris pH7.5,100mM NaCl) seals this film, at room temperature add anti--P-gp antibody response 1 hour subsequently, the secondary antibody reaction 1 hour that at room temperature adds the horseradish peroxidase labelling is again measured by ECL by the albumen of antibody labeling at last and is developed.
2. result: experimental result as shown in Figure 5, Rhizoma Alismatis is carried thing the influence that P-gp expresses is detected.HepG2-DR and K562-DR cell and its blast cell have relatively all been expressed high-caliber P-gp (Fig. 5 A, Fig. 5 B).After the Rhizoma Alismatis extract of 50ug/mL is handled 12 hours, in still being the K562-DR cell, HepG2-DR all do not have to detect the change (Fig. 5 A, Fig. 5 B) of observable P-gp protein level.
Claims (6)
1. the application of alisol B 23-monoacetate in preparation inverse cancer cell multidrug resistance medicine.
2. the application of Rhizoma Alismatis extract in preparation inverse cancer cell multidrug resistance medicine.
3. application according to claim 2, described Rhizoma Alismatis extract are to obtain by one of following method:
(1) Rhizoma Alismatis dry product coarse powder is doubly measured methanol or alcohol at normal temperature, heating or reflux, extract, with 8-20, extracts 1-5 time.Extracting solution normal pressure or removal of solvent under reduced pressure are fully extracted with chloroform after adding suitable quantity of water, and chloroform extracted solution removes through normal pressure or concentrating under reduced pressure and desolvates, and promptly obtains Rhizoma Alismatis extract;
(2) Rhizoma Alismatis dry product coarse powder is doubly measured chloroform room temperature, heating or reflux, extract, with 8-20, extracts 1-5 time.Normal pressure or removal of solvent under reduced pressure promptly get Rhizoma Alismatis extract;
(3) Rhizoma Alismatis dry product coarse powder, with containing the alcoholic acid supercritical carbon dioxide fluid of 2.5%-10%, at 50 ℃ with down to room temperature, extract in supercritical carbon dioxide extraction still internal recycle, the extract solvent removed by evaporation at reduced pressure, add chloroform and fully extract, normal pressure or concentrating under reduced pressure remove and desolvate, and promptly get Rhizoma Alismatis extract.
4. application according to claim 1 is characterized in that, described medicine is oral formulations or ejection preparation.
5. application according to claim 2 is characterized in that, described medicine is oral formulations or ejection preparation.
6. according to claim 4 or 5 described application, it is characterized in that described oral formulations comprises tablet, capsule, granule, oral liquid or pill.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101411711A (en) * | 2008-11-24 | 2009-04-22 | 上海现代药物制剂工程研究中心有限公司 | Composition containing alisol A and alisol A 24-acetic ester and use |
| CN101717425B (en) * | 2008-10-09 | 2011-09-21 | 中国科学院昆明植物研究所 | 11,23,24-Tris-oxygen-butyryl alisol A ,as well as medicine composite and application thereof |
| CN102645364A (en) * | 2012-05-03 | 2012-08-22 | 沈阳药科大学 | Renal toxicity causing toxic effect portion of rhizoma alismatis and method for preparing toxic effect portion |
| CN104666322A (en) * | 2015-02-06 | 2015-06-03 | 天津中医药大学 | Novel use of 24-acetyl alisol F |
-
2004
- 2004-04-26 CN CNB2004100384205A patent/CN100553638C/en not_active Expired - Fee Related
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101717425B (en) * | 2008-10-09 | 2011-09-21 | 中国科学院昆明植物研究所 | 11,23,24-Tris-oxygen-butyryl alisol A ,as well as medicine composite and application thereof |
| CN101411711A (en) * | 2008-11-24 | 2009-04-22 | 上海现代药物制剂工程研究中心有限公司 | Composition containing alisol A and alisol A 24-acetic ester and use |
| CN101411711B (en) * | 2008-11-24 | 2013-06-19 | 上海现代药物制剂工程研究中心有限公司 | Composition containing alisol A and alisol A 24-acetic ester and use |
| CN102645364A (en) * | 2012-05-03 | 2012-08-22 | 沈阳药科大学 | Renal toxicity causing toxic effect portion of rhizoma alismatis and method for preparing toxic effect portion |
| CN104666322A (en) * | 2015-02-06 | 2015-06-03 | 天津中医药大学 | Novel use of 24-acetyl alisol F |
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