CN105311360B - The antineoplastic that a kind of suppression ras proto-oncogenes are overexpressed - Google Patents
The antineoplastic that a kind of suppression ras proto-oncogenes are overexpressed Download PDFInfo
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Abstract
The invention belongs to field of medicaments, be related to a kind of raw arteries and veins extract, and its preparation method and application.Research shows that about 90% pancreas, 50% colon cancer, 30% 40% adenocarcinomas of lung and 5% 40% leukaemia are as caused by ras gene overexpressions, and ras genes turn into the target for the screening decorrelation malignant tumor medicine generally acknowledged.The invention discloses a kind of preparation technology of raw arteries and veins extract, and compared with simple decocting processing, the toxicity for the life arteries and veins extract that the technique is prepared is low, and the effect for suppressing ras gene pathway excessive activations is more preferable.In addition, the invention provides the new application of raw arteries and veins extract, it can apply in suppressing ras proto-oncogenes and being overexpressed and be applied in the tumour medicine as caused by being overexpressed ras proto-oncogenes is prepared, be expected to exploitation into the antineoplastic of a new generation.
Description
Technical field
The present invention relates to a kind of raw arteries and veins extract and preparation method thereof, and in particular to raw arteries and veins extract is preparing suppression ras
Application in the antineoplastic that proto-oncogene is overexpressed and the application in suppressing ras proto-oncogenes and being overexpressed, belong to medicine
Field.
Background technology
Shengmai San is the eternal recipe of the traditional Chinese medical science, first recorded in written by Jin Dynasty doctor's Zhang Yuansu《Under medicine origin volume》" tuber of dwarf lilyturf "
In bar；Differentiation on endogenous and hospital pass the examination also on the books.It is made up of ginseng, the tuber of dwarf lilyturf, the fruit of Chinese magnoliavine, it is intended that take " the sweet benefit of ginseng
Gas, Mai Men (i.e. the tuber of dwarf lilyturf) bitter cold are purged heat the source of moisturizing, the clear respectful dry gold of acid of the fruit of Chinese magnoliavine ".
Shengmai San is used to treat that hot summer weather sweat is more earliest, the card of impairment of both QI and YIN, show as sweat is more, body is tired, body is tired, shortness of breath, pharynx
The symptoms such as dry, feeble pulse.Ancient medicine is also used for the symptoms such as gold leaf consumption wound, feeble and impalpable pulse, is used as the help of deficiency of both qi and yin patient again
Agent.This side passes through the long-term use of centuries, safely and effectively, lasting.With the development of modern medicine, the clinic of Shengmai San
Expand using continuous, its formulation is in addition to powder, also Shengmai Yin, shengmai injection；The prescription there have been developed after plus-minus changes
Join wheat drink and Shengmai San Radix Codonopsis side etc..Primarily now it is used to treat the Hemorrhagic shock in cardiovascular disease, anti-heart failure, resists myocardial ischemia
Symptom, diabetes, chronic bronchitis, all kinds of cardiogenic shocks, coronary heart disease, arrhythmia cordis etc..
New wait of soup inscription finds that Shengmai San can significantly inhibit the spontaneous metastasis of lung cancer, but testing proves this antitumor action
Not Shengmai San directly acts on cancer cell, but by improve body's immunity realize indirectly it is antitumor.Chinese patent
201010148044.0 disclose a kind of composition with antitumor action and its application in tumor is prepared,
Shengmai San and other Chinese medicine compound prescriptions are added in the antineoplastic pharmaceutical compositions with the dried venom of toads containing main ingredient by the patent, and Shengmai San is reduced
Cardiac toxic problem caused by the dried venom of toads；It is swollen in preparation treatment that Chinese patent 200510053562.9 discloses shengmai injection
Purposes in knurl toxicity-reducing medicament, hepatic and renal function can be protected in tumor-bearing mice chemotherapy process, while using Shengmai San.It is Chinese special
Profit 03142266.7 discloses shengmai injection and is preparing the purposes in treating tumour synergism medicine.Shengmai San is applied in the patent
Parenteral solution processing colon cancer, breast cancer, liver cancer and pancreas cancer cell strain, it was demonstrated that antitumor work when shengmai injection is used alone
Cut in and out with effect, Shengmai San only has relatively stable synergistic function.
About 30% human tumor is due to that activation causes RAS protein expression levels to increase to make after ras Oncogene Mutations
Into.It if RAS albumen is continuously in the state of activation, can be combined with the effect protein in downstream, pass the signal along to downstream signal member
Part, cause the abnormality proliferation of cell, lead oncogenic generation.Therefore, the screening decorrelation that ras proto-oncogenes have turned into generally acknowledged is disliked
The target of property tumour medicine.
Shengmai San only can be significantly inhibited to assess with the method for decocting suppresses ras proto-oncogene path excessive activation medicines
The excessive activation of model Caenorhabditis elegans ras paths, but there is notable toxicity simultaneously, the beautiful hidden bar of model organism can be caused
Nematode is dead.The invention provides a kind of preparation technology of raw arteries and veins extract, compared with simple decocting processing, is guaranteeing
While significantly inhibiting Caenorhabditis elegans ras path excessive activations, toxicity can be significantly reduced, the cause to Caenorhabditis elegans
Dead rate is low, and effect is more preferable.In addition, the invention provides the new application of raw arteries and veins extract, can be overexpressed suppressing ras proto-oncogenes
It is middle to apply and applied in the tumour medicine as caused by being overexpressed ras proto-oncogenes is prepared, it is swollen into resisting for a new generation to be expected to exploitation
Tumor medicine.
The content of the invention
It is an object of the invention to provide a kind of raw arteries and veins extract and its preparation technology, to provide a kind of small toxicity, inexpensively
Medicine, it is a further object of the present invention to provide a kind of new application of raw arteries and veins extract, and in particular to prepared by raw arteries and veins extract
Suppress the application in the antineoplastic that ras proto-oncogenes are overexpressed and the application in suppressing ras proto-oncogenes and being overexpressed.
The present invention is practiced by following technical proposals.
A kind of raw arteries and veins extract of the present invention, wherein raw arteries and veins extract is prepared by following method：Take ginseng, wheat
Winter and the fruit of Chinese magnoliavine, add water, soak, be heated to reflux, filter, be concentrated under reduced pressure, obtain total medicinal extract；Total medicinal extract is completely molten with distilled water
Solution, upper macroporous resin column, 24h is adsorbed, is concentrated into thick paste with elution, eluent afterwards, dried, produce.
A kind of raw arteries and veins extract of the present invention, is made up using ginseng, the tuber of dwarf lilyturf, the fruit of Chinese magnoliavine of raw material, the bulk drug
Mass ratio is 9：9：6.
A kind of raw arteries and veins extract of the present invention, macroreticular resin described in the preparation are D101 type macroreticular resins.
A kind of raw arteries and veins extract of the present invention, the described total medicinal extract of life arteries and veins extract isolate and purify elution during elution
Measure as 5 times of column volumes.
A kind of raw arteries and veins extract of the present invention, it is characterised in that the raw total medicinal extract of arteries and veins extract elution when
Eluent is ethanol solution.
A kind of raw arteries and veins extract of the present invention, it is characterised in that ethanol solution used is anhydrous during the elution
Ethanol.Now, while ensureing that raw arteries and veins extract dramatically increases Caenorhabditis elegans MT2124 WT ratios, and have
Effect composition hardly shows toxicity.
The present invention is a kind of application of raw arteries and veins extract in antineoplastic is prepared, and described tumour is by ras original cancers
Caused by being overexpressed after gene mutation.
A kind of application of the present invention for raw arteries and veins extract in suppressing ras proto-oncogenes and being overexpressed.
Raw arteries and veins extract of the present invention is preparing the application in treating antineoplastic, can add raw arteries and veins extract
Add pharmaceutically acceptable carrier such as filler, lubricant, wetting agent, promotion absorbent, absorption carrier, diluent, excipient
Deng can add sweetener, flavouring agent etc. if necessary, be prepared into injection, powder, granule, pulvis, pill, oral liquid, tablet
Deng a variety of formulations of medicine, this be it will be appreciated by those skilled in the art that.
The present invention provides a kind of preparation technology of raw arteries and veins extract and suppresses the anti-of ras proto-oncogenes overexpression preparing
The new application of tumour medicine.The application of raw arteries and veins extract disclosed by the invention has following advantage：
(1) the life arteries and veins extract effect prepared using the preparation technology in the present invention is good, can significantly reduce toxicity, make assessment
Suppressing the death rate of the model Caenorhabditis elegans of ras proto-oncogene path excessive activation medicines reduces, WT ratios rise.
(2) raw arteries and veins extract can significantly inhibit ras proto-oncogenes overexpression, can prepare treatment by ras proto-oncogenes
Applied in tumour medicine caused by overexpression.
(3) the prescription proportioning in raw arteries and veins extract between ginseng, the tuber of dwarf lilyturf and the fruit of Chinese magnoliavine can close according to Chinese prescription principle
Reason change, it still can significantly lower ras proto-oncogene excessive activations, have antitumor activity.
(4) raw arteries and veins extract only acts on the mutant RAS albumen of overexpression, and on wild type RAS albumen without influence.
The raw arteries and veins extract of prompting is applying upper safety, and it acts only on cancer cell, without acting on normal cell.From now on, Wo Menyou
Hope and apply raw arteries and veins extract in the preparation of decorrelation malignant tumor medicine, wherein, the tumour is by ras proto-oncogenes
Caused by overexpression, so as to provide more preferable treatment for such tumor patient, new way is provided for the treatment of some difficult cancers
Footpath.
(5) raw arteries and veins extract significantly lowers ras proto-oncogenes overexpression, is not caused by mutagenesis, not teratogenesis.
(6) due to ras signal paths from nematode to people it is highly conserved, in Caenorhabditis elegans let-60 gene codes protect
The RAS albumen kept, it has 83% homology compared with the RAS albumen of the mankind.If ras is mutated, in Caenorhabditis elegans
In nematode can be caused to produce more vaginal orifices, be then the malignant proliferation for causing cell in the mankind, lead oncogenic generation.Therefore, mould
Formula biology Caenorhabditis elegans (Caenorhabditis elegans) Ras/MAPK signal path excessive activation type mutant quilts
It is widely used in the antineoplastic of screening suppression ras proto-oncogene path excessive activations as test population.Therefore, it is of the invention
Employ Caenorhabditis elegans mutant strain and suppress the model of ras proto-oncogene path excessive activation medicines as assessment, and draw
Raw arteries and veins extract is overexpressed to ras proto-oncogenes obvious downward effect, has and suppresses ras proto-oncogene path excessive activations
Conclusion.
Embodiment
Specific embodiment presented below illustrates the present invention, but is not limited to these embodiments.
The preparation of embodiment all one's life arteries and veins compound
Material：Ginseng, the tuber of dwarf lilyturf, the fruit of Chinese magnoliavine are purchased from Lanzhou the Yellow River market.
(1) preparation of raw arteries and veins water decoction
Weigh ginseng 18g, tuber of dwarf lilyturf 18g, fruit of Chinese magnoliavine 12g；Decocting, total medicinal extract 29g is obtained, is dissolved in water, is settled to 100ml, most
It can be diluted afterwards by gained sample preparation into 2.5mg/ml solution for standby, during use by multiple.
(2) preparation of raw arteries and veins extract
Weigh ginseng 18g, tuber of dwarf lilyturf 18g, fruit of Chinese magnoliavine 12g；Decocting, total medicinal extract 29g is obtained, water is dissolved in, is settled to 100ml；With
D101 macroreticular resins load chromatographic column, and pretreatment is standby.Take above-mentioned medicinal extract liquid 20ml, upper D101 posts, respectively with 0%, 50%,
Ethanol solution, 5 times of column volumes rinse, finally by gained sample preparation into 2.5mg/ml solution for standby.
Embodiment two gives birth to effect of the arteries and veins water decoction to MT2124 (mutation of let-60 excessive activations)
1. principle
Ras is a kind of small molecular protein, participates in many important steps during regulation and control animal development.Ras signal paths
It is highly conserved from nematode to people, the conservative RAS albumen of let-60 gene codes in Caenorhabditis elegans, its RAS with the mankind
Albumen is compared with 83% homology.If ras is mutated, nematode can be caused to produce more vaginal orifices in Caenorhabditis elegans,
It is then the malignant proliferation for causing cell in the mankind, leads oncogenic generation.Therefore, model organism Caenorhabditis elegans
(Caenorhabditis elegans) Ras/MAPK signal paths excessive activation type mutant extensively should as test population
Suppress the antineoplastic of proto-oncogene ras path excessive activations for screening.
MT2124 is the Caenorhabditis elegans of let-60/ras excessive activations, can produce the phenotype of more vaginal orifices.Medicine is dilute
Certain concentration is released, acts on Caenorhabditis elegans MT2124, if it is possible to suppresses the more vaginal orifices generations of tested nematode population
The ratio of body, and the wild type nematode individual ratio of only one Kidney-Yin door rises, then illustrates that medicine acts on ras paths, under
The excessive activation of the path has been adjusted, there is antitumor activity.The ratio of wild type nematode is higher in colony, then drug effectiveness
It is more notable.
2. experiment material
Caenorhabditis elegans strain：Caenorhabditis elegans mutant MT2124, genotype are：let-60(n1046sd,gf)
IV, purchased from CGC (Caenorhabditis Genetics Center)；Bacterial strain：Uracil leaky mutant Escherichia coli
OP50, as the food of Caenorhabditis elegans, buy in CGC.
3. the preparation of reagent
NGM culture mediums：NaCl1.5g, K2HPO41.3g, KH2PO48.5g, peptone 1.4g, agar powder 8.5g, supplement are steamed
Distilled water is to 500mL.The 500 μ L cholesterol (5mg/mL, absolute ethyl alcohol are prepared) of filtration sterilization, high pressure steam sterilization are added after sterilizing
500 μ L1mol/L MgSO4, 500 μ L1mol/L CaCl2。
LB fluid nutrient mediums：NaCl1g, peptone 1g, yeast extract 0.5g, supplement distilled water to 100mL.1M is used after dissolving
NaOH regulation pH value is 7.0, high-temperature sterilization.
M9 buffer solutions：Na2HPO4·12H2O15.12g, KH2PO43g, NaCl5g, MgSO4·7H2O0.25g, high temperature go out
Bacterium.
S bases formula of liquid (1L)：NaCl58.5g, K2HPO410g, KH2PO46g；Supplement distilled water to 1000mL, high temperature goes out
Bacterium.
1M potassium citrates (1L)：Citric acid H2O20g, potassium citrate H2O293.5g, supplement distilled water to 1000mL,
High-temperature sterilization.
Trace(1L)：NaEDTA1.86g, FeSO4·7H2O0.69g, MnCl2·4H2O0.2g CuSO4·
5H2O0.29g, ZnSO4·7H2O0.025g, supplement distilled water to 1000mL, high-temperature sterilization.
S formula of liquid (1L)：S basal liquids 1L, 1M potassium citrate 10mL, Trace10mL, 1MCaCl23mL, 1M MgSO43mL,
5mg/mL cholesterol 1mL, prepared before use.
4. instrument
5. specific test procedure
The culture of nematode：Nematode is connected on the solid NGM plates for scribbling Escherichia coli OP50, then will be placed on 20 DEG C of training
Support and cultivated in case, synchronizing is handled when nematode grows to adult.
Nematode synchronizes：The nematode on flat board is swept away flushing liquor is sucked into centrifuge tube with M9 buffer solutions；4000rpm/
Min centrifuges 3min, removes supernatant；Add lysate (final concentration：3.2%NaClO and 1M NaOH)；Shaken in vortex instrument
7min, the polypide of nematode is set to rupture release ovum；4000rpm/min centrifuges 3min, removes supernatant, collects precipitation and obtains substantial amounts of line
Worm's ovum；M9 wash buffers twice, add 1ml M9 buffer solutions, are placed in 20 DEG C of incubators and hatch 48 hours.
Medicine acts on：This experiment is carried out on 96 orifice plates, and each processing sets five repetitions.First obtained synchronized
L1 phase nematode concentration adjust to the μ L of 80-100 bars/20.Then 140 μ L S liquid are added into every hole, 20 μ L decoctions, 20 μ L are beautiful
Hidden rhabditida liquid, 20 μ LOP50.It is put into 20 DEG C of constant-temperature shaking incubators and cultivates.After culture to adult, enter under inverted microscope
Row observation, calculates WT ratio (%)=100% × wild type nematode number/(wild type nematode number+mutant nematode number).
6. giving birth to arteries and veins water decoction suppresses ras path excessive activation biological activity determinations and its influence to MT2124 growths
Table 1 gives birth to arteries and veins water decoction and suppresses ras path excessive activation biological activity determinations and its influence to MT2124 growths
Table 1 shows that Caenorhabditis elegans ras mutant MT2124 can be acted on by giving birth to arteries and veins water decoction, significantly inhibit MT2124
More vaginal orifice phenotypes, dramatically increase Caenorhabditis elegans MT2124 WT ratios, and be in concentration-dependent relation, illustrate life
Arteries and veins water decoction has obvious downward to act on to ras proto-oncogene excessive activations, mutant is changed into wild type.It is in addition, dead by nematode
The rate of dying can be seen that Shengmai San water decoction shows toxicity to Caenorhabditis elegans, and increase with the increase toxicity of concentration.
Effect of the embodiment three lives arteries and veins extract to MT2124 (mutation of let-60 excessive activations)
1. principle, experiment material, instrument, specific test procedure are the same as embodiment two
2. giving birth to, arteries and veins extract suppresses ras path excessive activation biological activity determinations and its raw arteries and veins extract grows to MT2124
Influence
Table 2 gives birth to arteries and veins extract and suppresses ras path excessive activation biological activity determinations and its raw arteries and veins extract to MT2124 lifes
Long influence
The result of table 2 shows after being isolated and purified using D101 pillars to raw arteries and veins water decoction that D101 pillars elute effective
Composition is focused mostly in the elution section rinsed with ethanol solution, and Caenorhabditis elegans MT2124 WT ratios can be made notable
Increase (P ﹤ 0.05), rise to 40% by the 5% of blank control group, and anhydrous ethanol elution section hardly shows toxicity.This result
The raw arteries and veins extract of prompting can be applied in the antineoplastic for preparing anti-ras excessive activations.
Example IV gives birth to effect of the arteries and veins water decoction to Caenorhabditis elegans MT8189 (lin-15 mutant inactives)
Experiment material：Caenorhabditis elegans MT8189, more vaginal orifice phenotypes, genotype:Lin-15b (n765) X, purchased from CGC
(Caenorhabditis Genetics Center)；Escherichia coli OP50, uracil leaky mutant, as beautiful hidden bar line
The food of worm, purchased from CGC.
Specific experiment step：With embodiment two.
It is located at ras upstream in lin-15 (n765) X, suppresses the expression of ras genes, MT8189 nematode strains under normal circumstances
Gene inactivation causes nematode to be in more vaginal orifice phenotypes in system.
The present embodiment gives birth to arteries and veins water decoction
100%, illustrate that raw arteries and veins water decoction can not suppress lin-15 (n765) X more vaginal orifice phenotypes, according to the upper principle of heredity, raw arteries and veins
Since more vaginal orifice phenotypes caused by the reversible let60/ras proto-oncogene excessive activations downstream of water decoction, then also suppress
MT8189 more vaginal orifice phenotypes, but expected results are not observed in this experiment, i.e., raw arteries and veins water decoction can not suppress on let60/ras
Swim gene lin-15 (n765) X more vaginal orifice phenotypes.This is probably because what is included in lin-15 (n765) x-ray worm is wild type
Let60/ras is copied, and raw arteries and veins water decoction only works to the let60/ras of overexpression, unwise to wild type let60/ras
Sense, i.e., raw arteries and veins water decoction act only on the RAS albumen of excessive activation, and insensitive to wild type RAS albumen, this illustrates the life arteries and veins water
What decoction only acted on ras excessive activations causes tumour cell, without acting on normal cell.
In addition, raw arteries and veins extract can not similarly suppress let60/ras upstream genes lin-15 (n765) X more vaginal orifice tables
Type.
Embodiment five gives birth to effect of the arteries and veins water decoction to Caenorhabditis elegans CB1275 (lin-1 mutant inactives)
Experiment material：Caenorhabditis elegans genotype：Lin-1 (e1275) IV, purchased from CGC (Caenorhabditis
Genetics Center)；Escherichia coli OP50, uracil leaky mutant, as the food of Caenorhabditis elegans, it is purchased from
CGC。
Specific experiment step：With embodiment two.
Lin-1 is a gene in ras downstreams, and it can be the negative of vaginal orifice development by the nuclear factor of MAPK phosphorylations to be
Regulatory factor, the signal path of the vaginal orifice development in regulation let-60 downstreams.After lin-1 mutant inactives, nematode is more vaginal orifice phenotypes.
Because lin-1 is nuclear factor, the process of vaginal orifice cell development is adjusted.If medicine is to more vaginal orifice tables of the gene Inactivating mutations
Type has significant inhibitory action, then illustrates that medicine can directly suppress the development of vaginal orifice precursor, prevent its form more vaginal orifices and
In non-specific CDCC.
It is 100% to Caenorhabditis elegans CB1275 (lin-1 mutant inactives) mutation rate that the present embodiment, which gives birth to arteries and veins water decoction,
Illustrate that more vaginal orifice phenotypes of CB1275 strain nematodes are not affected by the influence of raw arteries and veins extract, this shows that medicine acts on ras signals
Path can suppress the false vaginal orifice of ras excessive activations formation, it is impossible to suppress the false vaginal orifice phenotype of lin-1 mutant inactives formation, explanation
Raw arteries and veins water decoction acts on ras without acting on lin-1, it was demonstrated that medicine has been lowered ras excessive activation rather than directly suppressed
What the propagation of cell worked, i.e., it is that ras pathways rely on that raw arteries and veins water decoction, which works, rather than extensive cell toxicant is made
With.
In addition, raw arteries and veins extract can not equally suppress more vaginal orifice phenotypes of CB1275 strain nematodes.
Shown by above example, Shengmai San only by decocting, can significantly significantly inhibit Caenorhabditis elegans ras and dash forward
Variant MT2124 more vaginal orifice phenotypes, dramatically increase Caenorhabditis elegans MT2124 WT ratios, and are in concentration dependant
Relation, illustrate that raw arteries and veins water decoction has obvious downward to act on to ras proto-oncogene excessive activations, mutant is changed into wild type.But
It is only to use decocting, raw arteries and veins water decoction can increase Caenorhabditis elegans toxicity with the increase of concentration.The invention provides one kind
The preparation technology of arteries and veins extract is given birth to, after being isolated and purified using D101 pillars to raw arteries and veins water decoction, gives birth to the effective of arteries and veins extract
Composition focuses mostly in anhydrous ethanol elution section, dramatically increases Caenorhabditis elegans MT2124 WT ratios, and effectively into
Divide and hardly show toxicity., can be in suppressing ras proto-oncogenes and being overexpressed present invention also offers the new application of raw arteries and veins extract
Suppress to apply in the antineoplastic that is overexpressed after ras Oncogene Mutations using and preparing, be expected to exploitation into a new generation
Antineoplastic.
Claims (3)
1. a kind of raw arteries and veins extract, it is characterised in that raw arteries and veins extract is prepared by following method：It is 9 in mass ratio：9：6 take
Ginseng, the tuber of dwarf lilyturf and the fruit of Chinese magnoliavine, add water, soak, be heated to reflux, filter, be concentrated under reduced pressure, obtain total medicinal extract；
Total medicinal extract is completely dissolved with distilled water, upper D101 types macroporous resin column, adsorbs 24h, it is anhydrous with 5 times of column volumes afterwards
Ethanol elution, eluent are concentrated into thick paste, dry, produce.
2. application of the raw arteries and veins extract in antineoplastic is prepared as claimed in claim 1, described tumour is former by ras
Caused by being overexpressed after oncogene mutation.
3. raw arteries and veins extract is preparing the application in suppressing ras proto-oncogenes overexpression medicine as claimed in claim 1.
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