CN104706937A - Application of pulse-activating powder in inhibition of ras proto-oncogene overexpression - Google Patents
Application of pulse-activating powder in inhibition of ras proto-oncogene overexpression Download PDFInfo
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Abstract
The invention belongs to the traditional Chinese medicine field, relates to an application of a pulse-activating powder in inhibition of ras proto-oncogene overexpression, and specifically, relates to the application in preparation of anti-tumor drugs for inhibiting ras proto-oncogene overexpression. Researches indicate that about 90% of pancreatic cancer, 50% of colon cancer, 30%-40% of lung adenocarcinoma and 5%-40% of leukemia are caused by ras gene overexpression, and a Ras protein becomes a universally accepted target for screening drugs resistant to related malignant tumors. Animal experiments indicate that the pulse-activating powder can specifically down-regulate overexpression caused after ras proto-oncogene mutation, but does not act on wild-type ras proto-oncogenes, so that the pulse-activating powder has low toxicity and no teratogenic effect. The invention provides the new use of the pulse-activating powder, and the pulse-activating powder can be applied in preparing the drugs for tumors caused by the ras proto-oncogene overexpression, and is expected to be developed into a new-generation anti-tumor drug.
Description
Technical field
The present invention relates to SHENGMAI SAN suppressing the application in ras proto-oncogene process LAN, being specifically related to the application in the antitumor drug of process LAN after preparation suppresses ras Oncogene Mutation, belonging to the field of Chinese medicines.
Background technology
SHENGMAI SAN is the eternal recipe of the traditional Chinese medical science, and head is loaded in Jin Dynasty doctor's Zhang Yuansu and shows in " under medicine origin volume " " Radix Ophiopogonis " bar; Differentiation on endogenous and hospital pass the examination also on the books.Be made up of Radix Ginseng, Radix Ophiopogonis, Fructus Schisandrae Chinensis, be intended to get " the sweet QI invigorating of Radix Ginseng, Mai Men (i.e. Radix Ophiopogonis) bitter cold is purged heat the source of moisturizing, the clear respectful dry gold of acid of Fructus Schisandrae Chinensis ".
SHENGMAI SAN is used for the treatment of summer-heat hyperhidrosis the earliest, the card of impairment of both QI and YIN, shows as hyperhidrosis, body is tired, body is tired, breathes hard, the symptom such as dry pharynx, deficient pulse.Ancient medicine is also for gold leaf consumption wound, and the symptoms such as faint pulse being about to disappear, are used as again the tonifying recipes of deficiency of both QI and YIN patient.This side is through the life-time service of centuries, safe and effective, lasting.Along with the development of modern medicine, the clinical practice of SHENGMAI SAN constantly expands, and its dosage form, except powder, also has SHENGMAI YIN, SHENGMAI ZHUSHEYE; This prescription there have been developed Rhizoma Zingiberis Recens drink and SHENGMAI SAN Radix Codonopsis side etc. after plus-minus change.Symptom, diabetes, chronic bronchitis, all kinds of cardiogenic shock, coronary heart disease, the arrhythmia etc. such as be mainly used in now treating shock, anti-heart failure in cardiovascular diseases, resist myocardial ischemia.
The discovery SHENGMAI SAN such as soup inscription is new significantly can suppress the spontaneous metastasis of pulmonary carcinoma, but experiment proves that this antitumor action is not that SHENGMAI SAN directly acts on cancerous cell, but indirectly realize antitumor by improving body's immunity.Chinese patent 201010148044.0 disclose a kind of there is antitumor action compositions and application in preparation tumor, SHENGMAI SAN and other Chinese medicine compound are added to in the antineoplastic pharmaceutical compositions containing principal agent Venenum Bufonis by this patent, and SHENGMAI SAN decreases the cardiac toxicity problem that Venenum Bufonis brings; Chinese patent 200510053562.9 discloses the purposes of SHENGMAI ZHUSHEYE in preparation treatment tumor attenuation medicine, in tumor-bearing mice chemotherapy process, applies SHENGMAI SAN simultaneously and can protect hepatic and renal function.Chinese patent 03142266.7 discloses the purposes of SHENGMAI ZHUSHEYE in preparation treatment tumor synergism medicine.Apply SHENGMAI SAN injection process colon cancer, breast carcinoma, hepatocarcinoma and pancreas cancer cell strain in this patent, prove that antitumor action effect when SHENGMAI ZHUSHEYE is applied separately cuts in and out, the SHENGMAI SAN synergistic function that only tool is more stable.Relate to the antitumor application of SHENGMAI SAN in foregoing invention patent, be all confined to SHENGMAI SAN as the adjuvant drug application in tumor therapeutic procedure.
The human tumor of about 30% causes because activation after ras Oncogene Mutation causes Ras protein expression level to increase.If Ras albumen continues to be in the state of activation, can be combined with the effect protein in downstream, pass the signal along to downstream signal element, cause the abnormality proliferation of cell, lead oncogenic generation.Therefore, Ras proto-oncogene has become the target of generally acknowledged screening decorrelation malignant tumor medicine.
Research shows single medicinal material Radix Ginseng, the Fructus Schisandrae Chinensis of composition SHENGMAI SAN and all has antitumor action Radix Ophiopogonis, but they are formulated into into after SHENGMAI SAN, people have but only found in antitumor application aspect the synergistic function that its tool is more stable to it, therefore can only using the adjuvant drug of this prescription as treatment tumor.The invention discloses SHENGMAI SAN and have significant inhibitory action to process LAN caused after Ras Oncogene Mutation, and do not act on wild type ras proto-oncogene, therefore toxicity is low, and without teratogenesis.The present invention has broken away from the constraint of traditional view, proves that SHENGMAI SAN prescription has and significantly lowers ras proto-oncogene process LAN activity, can suppress apply in the medicine of the decorrelation malignant tumor of ras proto-oncogene process LAN in preparation, and be not only secondary purpose.
Summary of the invention
The object of this invention is to provide the novelty teabag of Chinese medicine compound SHENGMAI SAN in pharmacy, relate to SHENGMAI SAN and suppress the application in ras proto-oncogene process LAN, be specifically related to the application suppressed in preparation in the antitumor drug of ras proto-oncogene process LAN, to providing a kind of anti-tumor activity high, toxicity is little, cheap antitumor drug.
The formula of Chinese medicine compound SHENGMAI SAN of the present invention is:
Formula 1: the mass ratio of Radix Ginseng, Radix Ophiopogonis, Fructus Schisandrae Chinensis is 9:9:6.
Formula 2: the mass ratio of Radix Ginseng, Radix Ophiopogonis, Fructus Schisandrae Chinensis is 9:18:9.
Formula 3: the mass ratio of Radix Ginseng, Radix Ophiopogonis, Fructus Schisandrae Chinensis is 9:5.4:5.4.
SHENGMAI SAN prescription of the present invention, its prescription ratio rationally can be changed according to Chinese prescription principle, and does not change the purposes of this prescription in the antitumor drug of preparation suppression ras proto-oncogene process LAN, and this it will be appreciated by those skilled in the art that.
The application of Chinese medicine compound SHENGMAI SAN of the present invention in preparation treatment antitumor drug, Chinese medicine compound SHENGMAI SAN can be added pharmaceutically acceptable carrier as filler, lubricant, wetting agent, penetration enhancement agent, absorption carrier, diluent, excipient etc., sweeting agent, flavouring agent etc. can be added if desired, be prepared into the multiple dosage form of the medicines such as injection, powder, granule, powder, pill, oral liquid, tablet, this it will be appreciated by those skilled in the art that.
The invention provides a kind of SHENGMAI SAN prescription suppresses the antitumor drug of ras proto-oncogene process LAN novelty teabag in preparation.The application of SHENGMAI SAN prescription disclosed by the invention has following advantage:
(1) SHENGMAI SAN prescription significantly can suppress ras proto-oncogene process LAN, can treat in the tumour medicine caused by ras proto-oncogene process LAN apply in preparation.
(2) Radix Ginseng in SHENGMAI SAN prescription, prescription proportioning between Radix Ophiopogonis and Fructus Schisandrae Chinensis rationally can be changed according to Chinese prescription principle, and it still significantly can lower ras proto-oncogene excessive activation, has anti-tumor activity.
(3) SHENGMAI SAN prescription only acts on the mutant Ras albumen of process LAN, and on wild-type Ras protein without impact.Prompting SHENGMAI SAN safety in application, it only acts on cancerous cell, and can not act on normal cell.From now on, we are expected to SHENGMAI SAN to be applied in the preparation of decorrelation malignant tumor medicine, and wherein, described tumor is caused by the process LAN of ras proto-oncogene, thus providing better treatment for this type of tumor patient, the treatment for some difficult cancers provides new approach.
(4) ras proto-oncogene process LAN significantly lowered by SHENGMAI SAN prescription, is not that mutagenesis causes, not teratogenesis.
(5) because ras signal path is from nematicide to people's high conservative, the Ras albumen that let-60 gene code is conservative in Caenorhabditis elegans, it has the homology of 83% compared with the Ras albumen of the mankind.If ras suddenlys change, nematicide can be caused in Caenorhabditis elegans to produce many vaginal orifices, be then the malignant proliferation causing cell in the mankind, lead oncogenic generation.Therefore, model organism Caenorhabditis elegans (
caenorhabditis elegans) Ras/MAPK signal path excessive activation type mutant is widely used in as test population screening the antitumor drug suppressing ras proto-oncogene path excessive activation.Therefore, this experiment have employed Caenorhabditis elegans mutant suppresses ras proto-oncogene path excessive activation medicine model as assessment, and has shown that SHENGMAI SAN has obvious downward effect to ras proto-oncogene process LAN, has the conclusion suppressing ras proto-oncogene path excessive activation.
Below provide specific embodiment to suppress to apply in the antitumor drug of ras proto-oncogene process LAN in preparation to realize SHENGMAI SAN prescription of the present invention, but be not limited to these embodiments.
detailed description of the invention
the preparation of embodiment one SHENGMAI SAN prescription
Material: Radix Ginseng, Radix Ophiopogonis, Fructus Schisandrae Chinensis are purchased from medicine city, the Yellow River, Lanzhou.
The SHENGMAI SAN of the different formula selected is as follows:
Formula 1 Radix Ginseng: Radix Ophiopogonis: Fructus Schisandrae Chinensis=9:9:6 (3:3:2)
Formula 2 Radix Ginseng: Radix Ophiopogonis: Fructus Schisandrae Chinensis=9:18:9 (1:2:1)
Formula 3 Radix Ginseng: Radix Ophiopogonis: Fructus Schisandrae Chinensis=9:5.4:5.4 (5:3:3)
First use distilled water immersion 20 minutes, then slow fire boils, and decocts 30min, standardize solution, centrifugal, discards precipitation, by supernatant aseptic filtration, puts 4 DEG C of refrigerators stand-by, dilute different concentration during use.
embodiment two Chinese medicine compound SHENGMAI SAN to Caenorhabditis elegans MT2124(let-60 excessive activation sudden change) effect
Ras is a kind of small molecular protein, participates in a lot of important steps in regulation and control animal development process.Ras signal path is from nematicide to people's high conservative, and the Ras albumen that let-60 gene code is conservative in Caenorhabditis elegans, it has the homology of 83% compared with the Ras albumen of the mankind.If ras suddenlys change, nematicide can be caused in Caenorhabditis elegans to produce many vaginal orifices, be then the malignant proliferation causing cell in the mankind, lead oncogenic generation.Therefore, model organism Caenorhabditis elegans (
caenorhabditis elegans) Ras/MAPK signal path excessive activation type mutant is widely used in as test population screening the antitumor drug suppressing proto-oncogene ras path excessive activation.
MT2124 is the Caenorhabditis elegans of let-60/ras excessive activation, can produce the phenotype of many vaginal orifices.Chinese medicine compound is diluted certain concentration, act on Caenorhabditis elegans MT2124, if the many vaginal orifices of tested nematode population can be suppressed to produce individual ratio, and only have the individual ratio of the wild type nematicide of a Kidney-Yin door to rise, then illustrate that drug effect is in ras path, lower the excessive activation of this path, there is anti-tumor activity.In colony, the ratio of wild type nematicide is higher, then drug effectiveness is more remarkable.As represented then contrary with mutant ratio, wherein, WT ratios (%)=100%-mutant ratio (%)
Experiment material: Caenorhabditis elegans MT2124, genotype: let-60 (n1046) IV, purchased from CGC(Caenorhabditis Genetics Center); Escherichia coli OP50, uracil leaky mutant, as the food of Caenorhabditis elegans, purchased from CGC.
Reagent: solid NGM medium component and making (being upgraded to example with 1):
| Composition | Content |
| NaCl | 3.00g |
| K 2HPO 4 | 2.34g |
| KH 2PO 4 | 17.23g |
| Peptone | 2.50g |
| Agar | 17.00g |
| H 2O | 1000ml |
After solid NGM culture medium prepares, high-pressure constant temp sterilizing 20min at 121 DEG C, adds 5mg/ml cholesterol 1ml, 1M MgSO under aseptic operating platform
41ml, 1M CaCl
21ml, shakes up, and pours sterilized 9cm culture plate while hot into, about 20ml/ plate.Leave standstill and wait for culture medium solidifying, for subsequent use.
Specific experiment step:
The cultivation of nematicide: be connected on by nematicide and scribble on the solid NGM plate of escherichia coli OP50, then cultivates in the incubator being placed on 20 DEG C, carries out synchronization process when nematicide grows to adult.
Nematicide synchronization: with M9 buffer, the nematicide on flat board is swept away, flushing liquor is sucked centrifuge tube; The centrifugal 3min of 4000rpm/ min, removes supernatant; Add lysate (final concentration: 3.2% NaClO and 1M NaOH); Vortex instrument shakes 7min, the polypide of nematicide is broken release ovum; The centrifugal 3min of 4000rpm/ min, remove supernatant, collecting precipitation obtains a large amount of line eggs; M9 wash buffer twice, adds 1ml M9 buffer, is placed in 20 DEG C of incubator hatchings 48 hours.
Drug effect: Chinese medicine compound stock solution is diluted certain concentration, nematicide after synchronization is diluted to 80/20 μ about l, in 96 orifice plates, S liquid 120 μ l is added in each hole, the cholesterol 20 μ l of the escherichia coli OP50 20 μ l of worm liquid 20 μ l, 10mg/ml, 5mg/ml, the medicinal liquid 20 μ l of appropriate dilutions, each concentration arrange five parallel.Blank group does not add medicinal liquid, medicinal liquid is changed into S liquid.20 DEG C of constant temperature culture are ripe to nematicide, detect the WT ratios of nematicide under the microscope.Result is as shown in the table:
impact on Caenorhabditis elegans MT2124 WT ratios under table 1 SHENGMAI SAN formula 1 variable concentrations
impact on Caenorhabditis elegans MT2124 WT ratios under table 2 SHENGMAI SAN formula 2,3 variable concentrations
Note: * compares P < 0.05 with blank group
As can be seen from the above table, SHENGMAI SAN makes the WT ratios of Caenorhabditis elegans MT2124 significantly increase, and in concentration-dependent relation, illustrates to act on ras path in SHENGMAI SAN, there is obvious downward effect to ras proto-oncogene excessive activation, make mutant become wild type.Simultaneously in experimentation, the quantity of Caenorhabditis elegans does not reduce, and illustrates that the toxicity of SHENGMAI SAN is little, can't cause the death of Caenorhabditis elegans.
embodiment three Chinese medicine compound SHENGMAI SAN is to Caenorhabditis elegans MT8189(lin-15 mutant inactive) effect
Experiment material: Caenorhabditis elegans MT8189, many vaginal orifices phenotype, genotype: lin-15b (n765) X, purchased from CGC(Caenorhabditis Genetics Center); Escherichia coli OP50, uracil leaky mutant, as the food of Caenorhabditis elegans, purchased from CGC.
Specific experiment step: with embodiment two.
table 3 SHENGMAI SAN is to Caenorhabditis elegans MT8189(lin-15 mutant inactive) effect
Be positioned at the upstream of ras at lin-15 (n765) X, suppress the expression of ras gene under normal circumstances, in MT8189 nematicide strain, this gene inactivation causes nematicide to be voluminous egg apparatus phenotype.
It is as shown in the table, SHENGMAI SAN can not suppress many vaginal orifices phenotype of lin-15 (n765) X, the principle upper according to heredity, since many vaginal orifices phenotype that the let60/ras proto-oncogene excessive activation in its downstream of SHENGMAI SAN reversible causes, so also suppress many vaginal orifices phenotype of MT8189, but do not observe expected results in this experiment, namely SHENGMAI SAN can not suppress many vaginal orifices phenotype of let60/ras upstream gene lin-15 (n765) X.This may be that wild type let60/ras copies due to what comprise in lin-15 (n765) x-ray worm, SHENGMAI SAN only works to the let60/ras of process LAN, insensitive to wild type let60/ras, namely SHENGMAI SAN only acts on the Ras albumen of excessive activation, insensitive to wild-type Ras protein, what this illustrated that this Chinese medicine composition only acts on ras excessive activation causes tumor cell, and does not act on normal cell.
In addition, many vaginal orifices phenotype that arteries and veins formula 2 and 3 can not suppress let60/ras upstream gene lin-15 (n765) X is too given birth to.
embodiment four Chinese medicine compound SHENGMAI SAN is to Caenorhabditis elegans CB1275(lin-1 mutant inactive) effect
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, purchased from CGC.
Specific experiment step: with embodiment two.
table 4 SHENGMAI SAN is to Caenorhabditis elegans CB1275(lin-1 mutant inactive) effect
Lin-1 is a gene in ras downstream, is by the nuclear factor of MAPK phosphorylation, to be the negative growth factor that vaginal orifice is grown, regulates the signal path that the vaginal orifice in let-60 downstream is grown.Lin-1
mutant inactiveafter, nematicide is many vaginal orifices phenotype.Because lin-1 is nuclear factor, regulate the cytocerastic process of vaginal orifice.If the significant inhibitory action of many vaginal orifices phenotype tool that medicine suddenlys change to this gene inactivation, then illustrate that medicine directly can suppress the growth of vaginal orifice precursor, stop it to form many vaginal orifices and be non-specific cytotoxicity.
The result of the present embodiment is the impact that many vaginal orifices phenotype of CB1275 strain nematicide is not subject to SHENGMAI SAN, and this shows the false vaginal orifice that drug effect can suppress ras excessive activation to be formed in ras signal path, can not suppress lin-1
mutant inactivethe false vaginal orifice phenotype formed, illustrate that SHENGMAI SAN acts on ras and do not act on lin-1, prove that medicine has been lowered the excessive activation of ras instead of directly inhibit the propagation of cell to work, namely raw arteries and veins formula 1 works is that ras pathways relies on, but not cytotoxicity widely.
Raw arteries and veins formula 2 and 3 can not suppress many vaginal orifices phenotype of CB1275 strain nematicide too.
embodiment five Chinese medicine compound SHENGMAI SAN is on the sudden change of Caenorhabditis elegans MT2124(let-60 excessive activation) impact on wild type nematicide offspring after effect
Experiment material: with embodiment two
Experimental procedure: by the method for embodiment three, by drug effect in Caenorhabditis elegans MT2124, after nematicide maturation, the Caenorhabditis elegans of picking wild type is cultivated under not adding medical condition, from each drug treating concentration picking 5, arrange three parallel.20 DEG C, the offspring that 130rpm constant-temperature shaking culture produces to picking nematicide reaches maturity, and detects its offspring's nematode population WT ratios under the microscope.
Experimental result shows, wild type nematicide caused by drug treating transfers to the fresh CMC model not adding medicine, its offspring's WT ratios (17.5 ± 2.4) % drops to blank level (15.2 ± 1.5) %(P<0.05), illustrate that SHENGMAI SAN suppresses many vaginal orifices phenotype to be not because mutagenesis produces, show that SHENGMAI SAN is not work at gene level, not there is teratogenesis.
The zoopery of above embodiment shows, the Chinese medicine compound SHENGMAI SAN with anti-tumor activity involved in the present invention has obvious downward effect to the excessive activation caused by ras Oncogene Mutation, and do not act on wild type ras proto-oncogene, therefore toxicity is low, and without teratogenesis, illustrate that SHENGMAI SAN is suitable for applying preparing in decorrelation malignant tumor medicine, the pharmaceutical preparation of the associated malignancies that the treatment ras Oncogene Mutation being expected to be developed to a new generation causes.
Claims (5)
1. SHENGMAI SAN is suppressing the application in ras proto-oncogene process LAN.
2. SHENGMAI SAN is preparing the application in antitumor drug, and described tumor is caused by process LAN after ras Oncogene Mutation.
3. SHENGMAI SAN according to claim 2 is preparing the application in antitumor drug, it is characterized in that the Radix Ginseng contained by SHENGMAI SAN, Radix Ophiopogonis, Fructus Schisandrae Chinensis mass ratio be 4.5 ~ 9:4.5 ~ 9:3 ~ 6.
4. SHENGMAI SAN according to claim 3 is preparing the application in antitumor drug, and the mass ratio of the Radix Ginseng contained by it is characterized in that in SHENGMAI SAN, Radix Ophiopogonis, Fructus Schisandrae Chinensis is 9:9:6.
5. SHENGMAI SAN according to claim 3 is preparing the application in antitumor drug, it is characterized in that Radix Ginseng in SHENGMAI SAN, Radix Ophiopogonis, Fructus Schisandrae Chinensis mass ratio be 9:5.4:5.4.
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| WO2002007732A2 (en) * | 2000-07-26 | 2002-01-31 | Cambridge University Technical Services Limited | Use of panaxatriol for stimulation angiogenesis |
| CN102119958A (en) * | 2010-01-11 | 2011-07-13 | 李红玉 | Medicinal composition containing realgar |
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