CN105288121B - Pulse-activating powder alcohol extract with anticancer effect and preparation method thereof - Google Patents
Pulse-activating powder alcohol extract with anticancer effect and preparation method thereof Download PDFInfo
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Abstract
The invention belongs to the field of traditional Chinese medicines, and relates to a pulse-activating powder alcohol extract and application thereof in inhibition of ras proto-oncogene overexpression, in particular to application in preparation of an anti-tumor medicine for inhibiting ras proto-oncogene overexpression. Research has shown that about 90% of pancreas, 50% of colon cancer, 30% -40% of lung adenocarcinoma and 5% -40% of leukemia are caused by Ras proto-oncogene overexpression, and Ras protein has become a well-known target for screening drugs against relevant malignant tumors. Animal experiments show that the pulse-activating powder alcohol extract can specifically reduce the overexpression caused by ras proto-oncogene mutation, but does not act on the wild ras proto-oncogene. Meanwhile, compared with the traditional pulse-activating decoction, the pulse-activating decoction alcohol extract has low lethality rate and no teratogenic action, so that the pulse-activating decoction alcohol extract has lower toxicity and better effect and is expected to be developed into a new generation of antitumor drugs.
Description
Technical Field
The invention relates to a pulse-activating powder alcohol extract and application thereof in inhibition of ras proto-oncogene overexpression, in particular to application in preparation of an anti-tumor drug for inhibiting ras proto-oncogene overexpression, and belongs to the field of traditional Chinese medicines.
Background
Shengmai powder is a famous formula of Chinese medicine, and is first recorded in the section of Mai Dong of the enlightenment of medical enlightenment, Xiaguan of medical profession, written by jin Dynasty doctors as an element; the differentiation and treatment of internal and external injuries are also described in the patent literature. Is composed of ginseng, ophiopogon root and schisandra fruit, and aims to obtain the sources of ginseng for tonifying qi, ophiopogon root for bitter cold, purging heat and replenishing water, and schisandra fruit for clearing acid, purifying and drying gold.
The pulse-activating powder is firstly used for treating diseases of summer heat, hyperhidrosis and impairment of both qi and yin, which are manifested by symptoms of hyperhidrosis, body fatigue, tiredness, short breath, dry throat, pulse deficiency and the like. Ancient physicians also used for the symptoms of consumption of gold leaves, faint pulse, and the like, and as a tonic for patients with deficiency of both qi and yin. The formula is safe and effective after long-term use for hundreds of years and is durable. With the development of modern medicine, the clinical application of pulse-activating powder is continuously expanded, and the dosage forms of the pulse-activating powder, in addition to powder, also include pulse-activating decoction and pulse-activating injection; the prescription can be modified to develop SHENMAI decoction and SHENMAI powder radix Codonopsis. It is mainly used for treating cardiovascular diseases such as antishock, anti-heart failure, and anti-myocardial ischemia, diabetes, chronic bronchitis, various cardiogenic shock, coronary heart disease, and arrhythmia.
The shengmai powder is found to obviously inhibit the spontaneous metastasis of the lung cancer by Tangminxin and the like, but experiments prove that the anti-tumor effect is not achieved by directly acting the shengmai powder on cancer cells but indirectly by improving the immune function of an organism. Chinese patent 201010148044.0 discloses a composition with anti-tumor effect and application thereof in preparing drugs for treating tumor, the patent adds pulse-activating powder and other Chinese herbal compound into the anti-tumor drug composition containing main drug toad venom, the pulse-activating powder reduces the problem of cardiotoxicity caused by the toad venom; chinese patent 200510053562.9 discloses the use of SHENGMAI injection in preparing medicine for treating tumor with reduced toxicity, and the SHENGMAI powder can protect liver and kidney function during chemotherapy of tumor-bearing mice. Chinese patent 03142266.7 discloses the use of Shengmai injection in preparing synergistic medicine for treating tumor. The patent proves that the pulse activating powder injection has no effect on the anti-tumor effect at all times when being applied alone and only has stable synergistic effect when being used for treating colon cancer, breast cancer, liver cancer and pancreatic cancer cell strains. The anti-tumor application of the pulse-activating powder disclosed by the invention patent is limited to the application of the pulse-activating powder as an auxiliary medicine in the process of tumor treatment.
Approximately 30% of human tumors are due to increased Ras protein expression levels as a result of post-mutation activation of the Ras proto-oncogene. If the Ras protein is continuously in an activated state, it can bind with downstream effector proteins and transmit signals to downstream signaling elements, causing abnormal proliferation of cells, resulting in tumor development. Therefore, Ras has become a well-established target for screening drugs against relevant malignancies.
Researches show that the single medicines of ginseng, schisandra chinensis and radix ophiopogonis which form the pulse-activating powder have anti-tumor effects, but after the pulse-activating powder is formed by combining the medicines, people only find that the pulse-activating powder has stable synergistic effect on the anti-tumor application, so that the prescription can only be used as an auxiliary medicine for treating tumors. Experiments prove that the Shengmaisan has obvious inhibition effect on overexpression caused by ras protooncogene mutation, but does not act on wild ras protooncogenes.
However, the traditional pulse-activating water decoction has certain toxicity and poor safety, so that the pulse-activating water decoction is further separated and purified by macroporous resin on the basis of alcohol extraction of the pulse-activating water decoction, and the separated and purified components not only keep the function of the pulse-activating water decoction in inhibiting ras proto-oncogene overexpression, but also greatly reduce the toxicity of the pulse-activating water decoction, so that the effect is better, and the pulse-activating water decoction can be applied to preparation of drugs for resisting related malignant tumors and inhibiting ras proto-oncogene overexpression.
Disclosure of Invention
The invention aims to provide the pulse-activating powder alcohol extract which can effectively reduce the toxicity of the traditional pulse-activating powder water decoction and has better effect.
The invention also aims to provide a new application of the pulse-activating powder alcohol extract in pharmacy, relates to the application of the pulse-activating powder alcohol extract in inhibition of ras proto-oncogene overexpression, and particularly relates to the application in preparation of an anti-tumor medicament for inhibiting ras proto-oncogene overexpression, so as to provide an anti-tumor medicament with high anti-tumor activity, low toxicity and low price.
The preparation method of the pulse-activating powder alcohol extract comprises the following steps:
firstly, adding ginseng, dwarf lilyturf tuber and Chinese magnoliavine fruit into an ethanol solution for soaking, heating, refluxing, filtering, and concentrating under reduced pressure to obtain an extract, which is characterized in that: and then dissolving the extract completely by using distilled water, adsorbing by using a macroporous resin column, and then washing and collecting by using an ethanol solution.
Wherein the ratio of the ginseng to the ophiopogon root to the schisandra chinensis is 9:9:6, and the ethanol solution is absolute ethanol.
The model of the macroporous resin column is D101.
The pulse-activating powder alcohol extract can be applied to inhibition of ras proto-oncogene overexpression.
The pulse-activating powder alcohol extract can also be applied to the preparation of antitumor drugs, wherein tumors are caused by ras proto-oncogene overexpression.
The pulse-activating powder alcohol extract can be added with pharmaceutically acceptable carriers such as filler, lubricant, wetting agent, absorption promoting agent, adsorption carrier, diluent, excipient and the like, and can be added with sweetening agent, flavoring agent and the like as necessary to prepare various dosage forms of medicaments such as injection, powder, granules, powder, pills, oral liquid, tablets and the like, which can be understood by the technical personnel in the field.
The invention provides a pulse-activating powder alcohol extract and a new application thereof in preparing an anti-tumor medicament for inhibiting ras proto-oncogene overexpression. The pulse-activating powder alcohol extract and the application thereof disclosed by the invention have the following advantages:
(1) compared with the traditional pulse-activating powder water decoction, the pulse-activating powder alcohol extract has low fatality rate and no teratogenic action, thereby having lower toxicity and better effect and being expected to be developed into a new generation of antitumor drugs.
(2) The pulse-activating powder alcohol extract can obviously inhibit ras proto-oncogene overexpression and can be applied to the preparation of medicaments for treating tumors caused by ras proto-oncogene overexpression.
(3) The formula proportion among the ginseng, the ophiopogon root and the schisandra chinensis in the pulse-activating powder alcohol extract can be reasonably changed according to the traditional Chinese medicine formula principle, can still obviously reduce ras proto-oncogene over-activation, and has anti-tumor activity.
(4) The pulse-activating powder alcohol extract only acts on the overexpressed mutant Ras protein, and has no influence on the wild Ras protein. The pulse-activating powder alcohol extract is safe in application, and only acts on cancer cells, but not normal cells. In future, the pulse-activating powder alcohol extract is expected to be applied to preparation of related malignant tumor resisting medicines, wherein tumors are caused by overexpression of ras proto-oncogenes, so that better treatment is provided for patients with the tumors, and a new way is provided for treatment of some difficult cancers.
(5) The pulse-activating powder alcohol extract can obviously reduce ras proto-oncogene overexpression, is not caused by mutagenesis and cannot cause teratogenesis.
(6) Since the Ras signaling pathway is highly conserved from nematode to human, the let-60 gene in C.elegans encodes a conserved Ras protein with 83% homology to the human Ras protein. If the ras proto-oncogene is mutated, it causes multiple vulva production by caenorhabditis elegans and malignant proliferation of cells in humans, resulting in tumorigenesis. Thus, the model organism C.elegans (C.elegans) ((B))Caenorhabditiselegans) The Ras/MAPK signal channel overactivation type mutant is widely used as a test population to screen antitumor drugs for inhibiting Ras proto-oncogene channel overactivation. Therefore, the experiment adopts the caenorhabditis elegans mutant strain as a model for evaluating the drugs for inhibiting ras protooncogene pathway over-activation, and obtains the conclusion that the shengma powder alcohol extract has obvious down-regulation effect on ras protooncogene over-expression, has the function of inhibiting ras protooncogene pathway over-activation, has low lethality rate and no teratogenesis effect compared with the traditional shengma powder water decoction, and has smaller toxicity and better effect.
Specific examples are provided below to realize the pulse-activating alcoholic extract and its application in preparing antitumor drugs for inhibiting ras proto-oncogene overexpression, but are not limited to these examples.
Detailed Description
Example preparation method of a decoction for treating coronary artery disease
Materials: ginseng, Mai Dong and Wu Wei Zi are all purchased from Lanzhou Huanghe market
The preparation method comprises the following steps: soaking Ginseng radix, radix Ophiopogonis and fructus Schisandrae chinensis in distilled water, boiling, decocting, diluting to desired volume, centrifuging, removing precipitate, filtering supernatant under sterile condition, and placing in refrigerator at 4 deg.C. Wherein the ratio of the raw materials of ginseng, dwarf lilyturf tuber and Chinese magnoliavine fruit is 9:9: 6.
Example preparation method of Ershengmai alcoholic extract
Materials: ginseng, Mai Dong and Wu Wei Zi are all purchased from Lanzhou Huanghe market
(1) The preparation method of the pulse-activating powder extract comprises the following steps:
adding the ginseng, the radix ophiopogonis and the schisandra chinensis which are mixed according to the ratio of 9:9:6 into absolute ethyl alcohol for soaking, heating and refluxing, filtering, and concentrating under reduced pressure to obtain the pulse-activating powder extract.
(2) The preparation method of the pulse-activating powder alcohol extract comprises the following steps: dissolving the SHENGMAI powder extract with distilled water completely, adsorbing with macroporous resin column, washing with ethanol solutions of different concentrations of 30%, 50%, 70% and 100%, respectively, and collecting. The control was obtained by collecting the eluate after washing with water.
Wherein the model of the macroporous resin column is D101.
Example effects of Sanshengmai ethanol extract on improving toxicity of Shengmai powder decoction
Culture medium, drug:
NGM culture medium: NaCl 1.5g, K2HPO41.3g，KH2PO48.5g, peptone 1.4g, agar powder 8.5g, and distilled water was supplemented to 500 mL. After sterilization, 500 μ L of cholesterol (5 mg/mL, prepared in absolute ethanol) was added, 500 μ L of 1 mol/L MgSO sterilized by autoclaving4，500µL 1mol/L CaCl2。
LB liquid medium: NaCl 1g, peptone 1g, yeast extract 0.5g, make up distilled water to 100 mL. After dissolution, the pH value is adjusted to 7.0 by 1M NaOH, and high-temperature sterilization is carried out.
M9 buffer: na (Na)2HPO4·12H2O 15.12g，KH2PO43g，NaCl 5g，MgSO4·7H20.25g of O, and sterilizing at high temperature.
S base liquid (1L): NaCl 58.5g, K2HPO410g，KH2PO46g of a mixture; distilled water was added to 1000mL, and the mixture was sterilized at high temperature.
1M potassium citrate (1L): citric acid. H2O20 g, Potassium citrate H2O293.5 g, and distilled water was added to 1000mL, followed by high-temperature sterilization.
Trace(1L)：Sodium.EDTA 1.86g，FeSO4·7H2O 0.69g，MnCl2·4H2O 0.2g CuSO4·5H2O 0.29g，ZnSO4·7H2O0.025 g, adding distilled water to 1000mL, and sterilizing at high temperature.
S liquid (1L): 10mL of 1M potassium citrate, 10mL of Trace, 1M CaCl23mL，1M MgSO43mL, 5mg/mL cholesterol 1mL, supplement S base solution to 1L, just before the use preparation.
Instrument for measuring the position of a moving object
Experimental animals:
caenorhabditis elegans strain: caenorhabditis elegans mutant MT2124, genotype is: let-60(n1046sd, gf) IV, purchased from The Caenorhabditis Genetics Center (CGC).
Bacterial strains: uracil leak mutant E.coli OP50, purchased from The Caenorhabditis Genetics Center (CGC).
The specific experimental steps are as follows:
and (3) culturing nematodes: the nematodes were plated on solid NGM plates coated with E.coli OP50 and then incubated in an incubator at 20 ℃ to synchronize the nematodes as they grow to adults with eggs and nematodes on the plates.
Nematode synchronization: washing the nematodes on the flat plate down by using an M9 buffer solution, and sucking the washing solution into a centrifugal tube; centrifuging at 4000rpm/min for 3min, removing supernatant, adding M9 buffer solution, centrifuging, and repeating for several times to clean Escherichia coli; adding lysis solution (final concentration: 3.2% NaClO and 1M NaOH); vibrating on a vortex apparatus for 7min to break the body of the nematode to obtain eggs; centrifuging at 4000rpm/min for 3min, removing supernatant, and collecting precipitate to obtain a large amount of nematode eggs; the cells were washed twice with M9 buffer, added with 1ml of M9 buffer, and incubated in an incubator at 20 ℃ for 48 hours.
The medicine has the following functions: diluting the Shengmaisan alcohol extract to a certain concentration, synchronously diluting the nematodes to about 80 per 20ul, adding 120ul of S liquid, 20ul of worm liquid, 20ul of 10mg/mL escherichia coli OP50, 20ul of 5mg/mL cholesterol and appropriately diluted liquid medicine into each hole of a 96-well plate, and setting five parallels for each medicine concentration (the final medicine concentration is respectively 1.25mg/mL, 2.5mg/mL and 5.0 mg/mL). The blank control group was replaced with S solution without adding any solution. Culturing at 20 deg.C until the nematode is mature, and detecting the wild type ratio of nematode (namely the ratio of nematode with only one Eumycota) under microscope. The higher the proportion of wild type compared to the blank control group, the stronger the effect of the drug. The results are shown in the following table:
TABLE 1 measurement of ras channel overactivation inhibiting bioactivity of Shengmaisan alcohol extract and caenorhabditis elegans
Effect of growth of mutant MT2124
The experimental results in table 1 show that both the shengmai decoction and the shengmai ethanol extract can significantly inhibit ras channel over-activation and increase the wild type proportion of nematodes, but the shengmai decoction has high toxicity and can cause a great amount of nematode death compared with the shengmai ethanol extract obtained by eluting with absolute ethanol.
The pulse-activating powder alcohol extract greatly reduces the toxicity while keeping the effect of inhibiting ras pathway over-activation of a pulse-activating powder decoction, particularly, the alcohol extract obtained by eluting with absolute ethyl alcohol ensures higher drug effect and reduces the death rate of nematodes, and under the condition that the final concentration of the drug is 5.0mg/ml, the wild type proportion of the nematodes reaches 76%, but the death rate of the nematodes is only 2%. Therefore, compared with the former, the pulse-activating powder alcohol extract has lower toxicity and better effect.
Example Effect of Sishengmasan alcohol extract on caenorhabditis elegans MT8189 (lin-15 mutation inactivation)
Experimental materials: caenorhabditis elegans MT8189, multiple vulva phenotype, genotype lin-15(n765) X, purchased from CGC (caenorhabditis Genetics center); escherichia coli OP50 (uracil leaky mutant) was purchased from CGC (caenorhabditis Genetics center) as a feed for C.elegans.
The specific experimental steps are as follows: the same as the second embodiment.
In C.elegans, lin-15(n765) X, located upstream of ras, normally inhibits expression of the ras proto-oncogene, which inactivation in the MT8189 nematode strain results in the nematode being oviparous in a prolific ovulator phenotype.
The experimental results show that the mutation rate of the nematodes after the treatment of the pulverant extract is still 100%, therefore, the pulverant extract cannot inhibit the multiple vulvar phenotype of lin-15(n765) X, and according to the genetic principle, the pulverant extract can also inhibit the multiple vulvar phenotype of MT8189 since the pulverant extract can reverse the multiple vulvar phenotype caused by the excessive activation of the downstream let60/ras proto oncogene, but the expected result, namely the pulverant extract cannot inhibit the multiple vulvar phenotype of 60/ras upstream let gene lin-15(n765) X, is not observed in the experiment. This is probably because the lin-15(n765) X nematode contains wild type let60/Ras copy, and the Maillard alcohol extract only acts on over-expressed let60/Ras and is insensitive to wild type let60/Ras, i.e. the Maillard alcohol extract only acts on over-activated Ras protein and is insensitive to wild type Ras protein, which shows that the Maillard alcohol extract only acts on tumor cells caused by over-activation of Ras protooncogene and does not act on normal cells.
Example Effect of five-element Maisan alcohol extract on caenorhabditis elegans CB1275 (lin-1 mutation inactivation)
Experimental materials: caenorhabditis elegans CB1275 genotype: lin-1(e1275) IV was purchased from CGC (caenorhabditis Genetics center); coli OP50 (uracil leaky mutant) was purchased from CGC as a feed for C.elegans.
The specific experimental steps are as follows: the same as the second embodiment.
lin-1 is a gene downstream of ras, is a nuclear transcription factor that can be phosphorylated by MAPK, is a negative regulator of vulvar development, and regulates the signaling pathway of vulvar development downstream of let-60. Following inactivation of the lin-1 mutation, the nematode is of the polynegative phenotype. Since lin-1 is a nuclear transcription factor, it regulates the process of development of the vulvar cells. If the medicine has obvious inhibiting effect on the multiple vulva phenotype of the gene inactivation mutation, the medicine can directly inhibit the development of the vulva precursor cells and prevent the multiple vulva precursor cells from forming into non-specific cytotoxic effect.
The result of this example is that the polyvulval phenotype of the CB1275 strain nematode is not affected by the verapamil extract (the mutation rate is still 100%), which indicates that the drug acting on ras signaling pathway can inhibit the pseudovulval formed by ras overactivation and cannot inhibit the pseudovulval phenotype formed by lin-1 mutational inactivation, thus indicating that the verapamil extract acts on ras protooncogene and does not act on lin-1, demonstrating that the drug down-regulates the overactivation of ras protooncogene rather than directly inhibiting the proliferation of cells, i.e. the verapamil extract acts as ras pathway mechanism dependent rather than extensive cytotoxic action.
Animal experiments in the above examples show that the pulse-activating bulk alcohol extract of the invention can specifically reduce the overexpression of ras protooncogene after mutation, but does not act on wild-type ras protooncogenes. Meanwhile, compared with the traditional pulse-activating decoction, the pulse-activating decoction alcohol extract has low lethality rate and no teratogenic action, so that the toxicity is lower, the effect is better, and the pulse-activating decoction alcohol extract can be applied to the preparation of tumor drugs caused by ras protooncogene overexpression and is hopeful to be developed into a new generation of antitumor drugs.
Claims (1)
1. The application of an anhydrous ethanol extract of Shengmaisan in preparing a medicament for inhibiting ras proto-oncogene overexpression is characterized in that the extract is prepared from the following components in parts by weight: 9:6 soaking the ginseng, the dwarf lilyturf tuber and the Chinese magnoliavine fruit in an absolute ethanol solution, heating, refluxing, filtering, and concentrating under reduced pressure to obtain an extract; dissolving the above extract with distilled water completely, adsorbing with D101 type macroporous resin, washing with anhydrous ethanol solution, and collecting.
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