CN101062026A - Function of pseudolarix acetic acid as medicine for inducing tumor cells to die - Google Patents
Function of pseudolarix acetic acid as medicine for inducing tumor cells to die Download PDFInfo
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- CN101062026A CN101062026A CN 200610009948 CN200610009948A CN101062026A CN 101062026 A CN101062026 A CN 101062026A CN 200610009948 CN200610009948 CN 200610009948 CN 200610009948 A CN200610009948 A CN 200610009948A CN 101062026 A CN101062026 A CN 101062026A
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Abstract
The invention discloses an application with hibiscus peel acetic acid to evoke tumor cell to die, which is characterized by the following: extracting effective element of hibiscus peel acetic acid from hibiscus peel; using hibiscus peel acetic acid to antimycotics haemostasis, nerve dermitis and so on aspects; unknowing making mechanism of hibiscus peel; restricting development of hibiscus peel . This invention can resolve the above problems.
Description
Technical field:
The present invention relates to a kind of novel application of compound, be specifically related to of the application of a kind of Pseudolarix acid B as the medicine of inducing apoptosis of tumour cell.
Background technology:
The Chinese medicine Cortex Pseudolaricis is dry root bark or the near root bark of pinaceae plant golden larch (Pseudolarix Kaempferi Gorden).See the supplementary Amplifications of the Compendium of Materia Medica that Qing Dynasty's ZHAO Xue-Min is shown the earliest, warm in nature, acrid in the mouth, poisonous.Return lung, spleen channel.Have parasite killing, itching relieving effect.Tcm clinical practice is used for killing parasites for relieving itching, tinea manus and pedis, neurodermatitis, cancerous protuberance etc.
Pseudolarix acid B is a kind of effective ingredient that extracts from Cortex Pseudolaricis, and its chemical constitution is a diterpenoid acid.In application and research in the past, Pseudolarix acid B is mainly used in aspects such as anti-true hemostasis and neurodermatitis.The experiment report is arranged, and Pseudolarix acid B can suppress the propagation of hepatoma cell line, but mechanism of action is indeterminate.Though Cortex Pseudolaricis has been used for the treatment of tumor, and certain curative effect is arranged, because the mechanism of action of its active component is still indeterminate, has limited it and used as the exploitation of cancer therapy drug and with the compatibility of other drug.
The Pseudolarix acid B chemical structural formula
Summary of the invention:
The purpose of this invention is to provide of the application of a kind of Pseudolarix acid B that from the golden larch root bark, extracts as the medicine of inducing apoptosis of tumour cell.
Above-mentioned purpose realizes by following technical scheme:
A kind of Pseudolarix acid B that extracts from the golden larch root bark is as the application of the medicine of inducing apoptosis of tumour cell.
The above-mentioned Pseudolarix acid B that from the golden larch root bark, extracts as the medicine of inducing apoptosis of tumour cell directly as the application of cancer therapy drug.
The above-mentioned Pseudolarix acid B that extracts from the golden larch root bark is done the application of the lead compound of cancer therapy drug as the medicine of inducing apoptosis of tumour cell.
This technical scheme has following beneficial effect:
1. the present invention, the Pseudolarix acid B that extracts from the golden larch root bark can become PTS to use with the other drug compatibility.
2. the present invention, the Pseudolarix acid B that extracts from the golden larch root bark also can directly be developed to cancer therapy drug.
3. the present invention, it is little, rapid-action that the Pseudolarix acid B that extracts from the golden larch root bark has dosage as cancer therapy drug, the outstanding effect characteristics.
4. the Pseudolarix acid B that extracts from the golden larch root bark of the present invention is as cancer therapy drug, and the generation that it can suppress tumor cell impels the apoptosis of tumor cell.
Description of drawings:
Accompanying drawing 1 is for observing normal HeLa cell and adding the sketch map of HeLa cell that concentration is the Pseudolarix acid B of 5 μ M at inverted microscope;
Accompanying drawing 2 is for observing normal HeLa cell and adding the sketch map of HeLa cell that concentration is the Pseudolarix acid B of 5 μ M under fluorescence microscope;
Accompanying drawing 3 is for observing normal A375-S2 cell and adding the sketch map of A375-S2 cell that concentration is the Pseudolarix acid B of 5 μ M at inverted microscope;
Accompanying drawing 4 is for observing normal A375-S2 cell and adding the sketch map of A375-S2 cell that concentration is the Pseudolarix acid B of 5 μ M under fluorescence microscope;
Accompanying drawing 5 is induced HeLa cell sketch map behind the HeLa cell generation apoptosis for Pseudolarix acid B;
Accompanying drawing 6 is induced A375-S2 cell sketch map behind the A375-S2 cell generation apoptosis for Pseudolarix acid B.
The specific embodiment of the present invention:
Embodiment 1:
The present invention adopts 2 kinds of cell strains to carry out cell culture, by the dyeing of morphological observation method, nuclear fluorescence observe and dna fragmentation fractional analysis experiment confirm Pseudolarix acid B induce HeLa cell and A375-S2 cell death mechanism to be cell death inducing.Pseudolarix acid B can be used as the lead compound of cancer therapy drug, also can directly be developed to PTS or become PTS with the other drug compatibility.
It is little, rapid-action that Pseudolarix acid B has dosage as cancer therapy drug, the outstanding effect characteristics.
Experiment one, morphological observation
One, principle
This experiment confirms the inhibition growth of tumour cell of Pseudolarix acid B and the feature of inducing apoptosis of tumour cell from morphocytology.Effectively anticancer component has specific biological effect to the tumor cell of In vitro culture, can make to carry outer tumor cell and produce morphologic variation, and the generation of inhibition tumor cell impels the apoptosis of tumor cell.Under optical microscope, apoptotic cell shows, karyopyknosis, and chromosome breakage, part is along the dense pleomorphism high density granular district of gathering into of nuclear membrane; The nuclear membrane shrinkage, the disperse of parcel chromosome is in cytoplasm after the disintegrate; The comprehensive shrinkage of cell, cell membrane shrinkage evagination; But the 26S Proteasome Structure and Function of cell membrane still is kept perfectly, and content does not have and overflows; At last, cell membrane parcel organelle or nuclear fragmentation form the apoptotic body littler than cell.When apoptosis took place the attached cell of In vitro culture, cell diminished round, loses adhesion and comes off from adhering to.After fluorescent dye Hoechst 33258 dyeing, have granule in the cell and form.
Experiment material
(1) material and instrument
1, human cervical carcinoma HeLa and Humanmachine tumour A375-S2 cell; 2, RPMI-1640 culture fluid, 10% hyclone, 2mmolL
-1Glutamine; 3,0.25% trypsin; 4, CO
2Incubator, 5%CO
2, 37 ℃; 5, inverted microscope; 6, fluorescence microscope
(2) experimental technique
1, inverted microscope is observed
With every bottle 1 * 10
5The density of individual cell is inoculated in the 50mL Tissue Culture Flask, and adding concentration behind the cultivation 24h is the Pseudolarix acid B of 0 and 5 μ M, and respectively at 0,24h observes with inverted microscope.
2, fluorescence staining
With every bottle 1 * 10
5The density of individual cell is inoculated in the 50mL Tissue Culture Flask, and adding concentration behind the cultivation 24h is the Pseudolarix acid B of 0 and 5 μ M, cultivates 24h, collecting cell, PBS are washed 1 time, the centrifugal 5min of 1000 * g, with 1mL 3.7% paraformaldehyde re-suspended cell, room temperature is 1h fixedly, the centrifugal 5min of 1000 * g, abandon supernatant, wash 1 time, cell is resuspended among the 100 μ L PBS with PBS, obtained cell suspension 10 μ L, add 2 μ L Hoechst 33258 (1mM), 37 ℃ of dyeing 15min, fluorescence microscope is observed down.
Three, result
Shown in Fig. 1-4, optical microscope is observed down 5 μ M Pseudolarix acid Bs and is acted on cell rounding behind the cell 24h, and foaming produces apoptotic body.Fluorescence microscope is observed the whole indigo plants of normal cell down and is dyed, and cell wall is complete, and behind the Pseudolarix acid B 5 μ M function cells 24h, visible tangible granular fluorophor is assembled, and promptly apoptotic body is the representative configuration of cell generation apoptosis.
Testing two microplate reader detects
One, principle
Developer thiophene seat blue (MTT) can be reduced to the bluish violet crystal of slightly solubility and be deposited in the cell by the succinate dehydrogenase in the living cells mitochondrion, and dead cell does not have this phenomenon.Bluish violet crystal in dimethyl sulfoxide (DMSO) the energy dissolved cell is measured its absorbance value with enzyme-linked immunosorbent assay instrument at 490nm wavelength place, the quantity of reflection living cells that can be indirect.In certain cell quantity scope, the amount that the MTT crystal forms is directly proportional with cell number.This method is highly sensitive, good reproducibility, simple to operate, economic, economical, easily automatization, "dead" pollution.
Two, experiment material
(1) material and instrument
1, the RPMI-1640 culture fluid (Hyclone, Logan, UT, USA); 2, MTT; 3, DMSO; 4, enzyme-linked immunosorbent assay instrument (Tecan, Salzburg, Austria)
(2) experimental technique
With every hole 5 * 10
4Cell density be inoculated in 96 orifice plates, every hole 100 μ L add 0,0.16,0.8,4 respectively after the overnight incubation, the Pseudolarix acid B of 20,100,500 μ M, in 12,24,36, every hole adds 5mg/ml MTT 15 μ L behind the 48h, after continuing to cultivate 4h, gives up supernatant.Every hole adds DMSO 150 μ L, and vibration 10min measures the OD value with the enzyme linked immunological instrument at 490nm.
The result
| Cell lines | HeLa | A375 |
| Time(h) IC 50(μM) | 24 4.8 | 24 3.6 |
Test three dna fragmentation electrophoresis experiments
One, principle
Endonuclease enzyme activation during apoptosis in the nuclear, the endonuclease that has activated is cut into breach with the DNA chain in the nucleosome bonding pad, form the oligonucleotide fragment of some 180～200bp or its multiple, on agarose gel electrophoresis, be " stepped " collection of illustrative plates (DNA ladder).This is used as judges one of objective indicator that has or not the apoptosis generation.
Two, experiment material
(1) material and instrument
1, RPMI-1640 culture fluid; 2, ethidium bromide; 3, agarose; 4, RNase; 5, proteinase K
(2) experimental technique
Collect 1 * 10
6Individual above cell, the centrifugal 10min of 1000 * g washes 2 times with PBS, add 100 μ L cell pyrolysis liquids (10mM Tris-HCl pH 7.4,10mM EDTA pH 8.0,0.5%TritonX-100) 4 ℃ of cracking 10min, the centrifugal 20min of 25000 * g collects supernatant.Add 40ng/LRNase, hatch 1h for 37 ℃, add 40ng/L proteinase K again, hatch 1h for 37 ℃, add 20 μ L5M NaCl and 120 μ L isopropyl alcohols ,-20 ℃ are spent the night.The centrifugal 15min of 25000 * g removes supernatant, and the centrifugal 5min of 25000 * g removes supernatant once more, with Tris-EDTA (pH7.5) buffer dissolution precipitation, in the bromophenol blue that adds 2 μ L.With 2% agarose gel 100V electrophoresis 40min, 0.5mg/L ethidium bromide staining 15min, distillation washing 30min takes pictures under the uviol lamp.
Three, result
Shown in Fig. 5-6, behind 2.5,5, the 10 μ M Pseudolarix acid B effect 36h, can induce HeLa, A375-S2 cell that tangible dna fragmentationization takes place, i.e. inducing cell generation apoptosis.
Claims (3)
1. a Pseudolarix acid B that extracts from the golden larch root bark is as the application of the medicine of inducing apoptosis of tumour cell.
According to the described Pseudolarix acid B that from the golden larch root bark, extracts of claim 1 as the medicine of inducing apoptosis of tumour cell directly as the application of cancer therapy drug.
3. do the application of the lead compound of cancer therapy drug as the medicine of inducing apoptosis of tumour cell according to claim 1 or the 2 described Pseudolarix acid Bs that from the golden larch root bark, extract.
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| Application Number | Priority Date | Filing Date | Title |
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| CN 200610009948 CN101062026A (en) | 2006-04-24 | 2006-04-24 | Function of pseudolarix acetic acid as medicine for inducing tumor cells to die |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106138034A (en) * | 2016-06-28 | 2016-11-23 | 中国人民武装警察部队后勤学院 | Corter pseudolaricis acetic acid or derivatives thereof prevents and treats the application in ulcerative colitis medicine in preparation |
| CN108524496A (en) * | 2018-03-09 | 2018-09-14 | 天津国际生物医药联合研究院 | Pseudolarix acid B is being prepared for treating the application in pulmonary fibrosis disease drug |
| CN113230249A (en) * | 2021-05-07 | 2021-08-10 | 广州中医药大学(广州中医药研究院) | Application of pseudolaric acid B in serving as or preparing Hedgehog signal path inhibitor |
| CN114668757A (en) * | 2022-03-29 | 2022-06-28 | 葛鹏飞 | Application of pseudolaric acid B in preparing medicine for preventing and/or treating malignant tumor |
| CN115252602A (en) * | 2022-05-17 | 2022-11-01 | 吉林大学 | Application of pseudolaric acid B in pharmacodynamic research of antitumor drugs |
-
2006
- 2006-04-24 CN CN 200610009948 patent/CN101062026A/en active Pending
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106138034A (en) * | 2016-06-28 | 2016-11-23 | 中国人民武装警察部队后勤学院 | Corter pseudolaricis acetic acid or derivatives thereof prevents and treats the application in ulcerative colitis medicine in preparation |
| CN108524496A (en) * | 2018-03-09 | 2018-09-14 | 天津国际生物医药联合研究院 | Pseudolarix acid B is being prepared for treating the application in pulmonary fibrosis disease drug |
| CN113230249A (en) * | 2021-05-07 | 2021-08-10 | 广州中医药大学(广州中医药研究院) | Application of pseudolaric acid B in serving as or preparing Hedgehog signal path inhibitor |
| CN114668757A (en) * | 2022-03-29 | 2022-06-28 | 葛鹏飞 | Application of pseudolaric acid B in preparing medicine for preventing and/or treating malignant tumor |
| CN114668757B (en) * | 2022-03-29 | 2023-08-08 | 葛鹏飞 | Application of pseudolaric acid B in preparing medicine for preventing and/or treating malignant tumor |
| CN115252602A (en) * | 2022-05-17 | 2022-11-01 | 吉林大学 | Application of pseudolaric acid B in pharmacodynamic research of antitumor drugs |
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Open date: 20071031 |