CN109394739A - Schizandrin A is used to prepare the purposes of anti-tumor drug - Google Patents
Schizandrin A is used to prepare the purposes of anti-tumor drug Download PDFInfo
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- CN109394739A CN109394739A CN201810749179.9A CN201810749179A CN109394739A CN 109394739 A CN109394739 A CN 109394739A CN 201810749179 A CN201810749179 A CN 201810749179A CN 109394739 A CN109394739 A CN 109394739A
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- schizandrin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/075—Ethers or acetals
- A61K31/085—Ethers or acetals having an ether linkage to aromatic ring nuclear carbon
- A61K31/09—Ethers or acetals having an ether linkage to aromatic ring nuclear carbon having two or more such linkages
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
Abstract
Can targeting in the traditional Chinese medicine monomer schizandrin A of apj receptor.It is able to suppress four kinds of human hepatocarcinoma BEL-7402, HeLa Cells, human breast carcinoma MDA-MB-231 cell and human pulmonary epithelial cells tumor cell proliferations, and most strong to the inhibiting effect of human pulmonary epithelial cells.The discovery of schizandrin A novel targets and the purposes in terms for the treatment of tumour.
Description
Technical field
The present invention relates to the discovery of traditional Chinese medicine monomer schizandrin A novel targets and antitumor functions and purposes.
Background technique
O ' Dowd in 1993 etc. has found that a kind of 1 receptor of Angiotensin II is related using the method that homology is cloned
Albumen is named as APJ (AGTRL-1, APLNR), be g protein coupled receptor (G protein coupled receptor,
GPCR) family member.People's APJ Gene A PLNR is located in the region q12 of No. 11 chromosomes, encodes 380 amino acid residue compositions
Receptor.APJ is distributed mainly on the tissue such as lung, heart, kidney, central nervous system and fat, participates in a variety of pathology of body
Physiology course, including inflammatory reaction, angiogenesis, maintenance myocardial contractive power, adjusting isohydria etc. and the cardiovascular disease of participation
The occurrence and development of disease, tumour and metabolic disease, play an important role in a variety of diseases, it is verified that becoming a kind of new
The potential target spot of more disease treatments.Have now been found that 2 kinds of endogenic ligands (Apelin, ELABELA) can activate apj receptor.
Up to the present, it although there is more and more APJ lead compounds to report, but still does not develop for clinical apj receptor
Drug.This laboratory is gone out using membrane flexibility technology screening can be with the traditional Chinese medicine monomer in conjunction with APJ --- schizandrin A.And benefit
With Discovery Studio2.5 software, quasi-medicated property analysis is carried out to schizandrin A, the results show that schizandrin A has
ADME property belongs to quasi-medicated property molecule.Schisandra chinensis is the dry of perennial fallen leaves liana magnoliaceae schisandra section Schizandra
Dry ripening fruits, it is sweet, sour, pungent, bitter, salty bittersweet, therefore claim Schisandra chinensis.Lignanoids is as activity most important in Schisandra chinensis
Ingredient, including schizandrin A, deoxyschizandrin etc..It is very extensive to the research of schizandrin A pharmacological action, it is related to digesting
The various aspects such as system, cardiovascular system, central nervous system, reproductive system and urinary system.And for its effect target
It puts and to human hepatocarcinoma BEL-7402, HeLa Cells, human breast carcinoma MDA-MB-231 cell and human lung adenocarcinoma
The inhibiting effect of these four tumor cell proliferations of A549 cell, is not found.
Summary of the invention
Present inventor passes through years of researches, it has unexpectedly been found that traditional Chinese medicine monomer schizandrin A has antitumor
New function, and specifically find that it can combine apj receptor.Schizandrin A can inhibit human hepatocarcinoma BEL-7402, people
Four kinds of Cervical Cancer HeLa Cells, human breast carcinoma MDA-MB-231 cell and human pulmonary epithelial cells tumor cell proliferations, and it is right
The inhibiting effect of human pulmonary epithelial cells is most strong.
According to an aspect of the present invention, the purposes that schizandrin A is used to prepare drug is provided, wherein the drug is used for
Treat human cervical carcinoma, human liver cancer, human breast carcinoma and human lung adenocarcinoma.
The drug is for inhibiting and treating HeLa Cells proliferation, human hepatocarcinoma BEL-7402 proliferation, human milk
The caused tumour of gland cancer MDA-MB-231 cell Proliferation, human pulmonary epithelial cells proliferation.
According to another aspect of the present invention, a kind of antineoplastic pharmaceutical compositions are provided, it includes schizandrin A.The medicine
Compositions are for treating human cervical carcinoma, human liver cancer, human breast carcinoma and human lung adenocarcinoma.
Traditional Chinese medicine monomer schizandrin A can targeting in apj receptor.Disclosed herein as well is the schizandrin As to exist
Treat the application in the basic research and drug research of tumour.
The present invention relates to the discovery of schizandrin A antineoplastic new function and its discoveries of related mechanism.By the present invention in that
Being gone out with membrane flexibility technology screening and can find that it can inhibit with the traditional Chinese medicine monomer in conjunction with apj receptor -- schizandrin A
Human hepatocarcinoma BEL-7402, HeLa Cells, human breast carcinoma MDA-MB-231 cell and human pulmonary epithelial cells
Four kinds of tumor cell proliferations, and it is most strong to the inhibiting effect of human pulmonary epithelial cells.The invention discloses the Schisandra chinensis first
Application of the element in the basic research and drug research for the treatment of tumour.
The experiment in vitro of drug of the invention:
Preferred concentration are as follows:
Advantageous effects of the invention
1, schizandrin A of the invention is able to suppress human hepatocarcinoma BEL-7402, HeLa Cells, human milk
Four kinds of tumor cell proliferations of gland cancer MDA-MB-231 cell and human pulmonary epithelial cells, and the suppression to human pulmonary epithelial cells
It is most strong to make use.
2, schizandrin A of the invention can be used in preparing antineoplastic pharmaceutical compositions.
3, the novel targets (apj receptor) of schizandrin A of the invention can be used in preparing bonding agent or the targeting of the target spot
Drug.
Detailed description of the invention:
Fig. 1-5 is the testing result of construction recombination plasmid pEGFP-N1-APJ.
Fig. 1 is PCR amplification APJ genetic fragment electrophoretogram.
M is Marker DL2000, and swimming lane 1 is the APJ gene of PCR amplification.
Fig. 2 is APJ and pEGFP-N1 double digestion electrophoretogram.
M is Marker DL2000, and swimming lane 1 is the APJ after double digestion, and swimming lane 2 is the carrier pEGFP-N1 after double digestion.
Fig. 3 is recombinant plasmid pEGFP-N1-APJ electrophoretogram.
M1 is Marker DL5000, and swimming lane 1-12 is recombinant plasmid, and M2 is Marker DL2000.
Fig. 4 is recombinant plasmid pEGFP-N1-APJ base sequence.
Fig. 5 is recombinant plasmid pEGFP-N1-APJ sequence alignment.
Fig. 6-10 is the search result of stable transfection recombinant plasmid.
Fig. 6 is that transfection cell screening GFP fluorescence is taken pictures figure.
A is the cell for transfecting empty carrier pEGFP-N1, and B is the cell for transfecting recombinant plasmid pEGFP-N1-APJ.
Fig. 7 is transfection cell total rna electrophoretogram.
M is Marker DL2000, and swimming lane 1-5 is cell APJ-HEK 293, and swimming lane 6 is the unloaded pEGFP-N1- of transfection
293 cell of HEK.
Fig. 8 is RT-PCR solubility curve.
APJ Tm=84 DEG C, GAPDH Tm=90 DEG C.
Fig. 9 is APJ mRNA expression in 293 cell of APJ-HEK.
N=6, * * p < 0.01APJ-HEK 293vs pEGFP-N1-HEK 293, control group are 293 groups of cells of HEK.
Figure 10 is APJ protein expression level in APJ-HEK293 cell.
A:Western Blot histogram, B: gray scale scanning statistical chart；N=3, * * p < 0.01APJ-HEK 293vs
pEGFP-N1-HEK 293。
Figure 11-12 is membrane flexibility technology screening apj receptor drug
Retention time of the 16 kinds of traditional Chinese medicine monomers of Figure 11 in APJ-HEK-293/CMC and HEK-293/CMC.
Figure 12 schizandrin A PJ-HEK-293/CMC and HEK-293/CMC chromatogram (RT schizandrin A=
27.35min)
Figure 13-16 is the inhibition tumor cell proliferation of schizandrin A
Figure 13 is that (Mean ± SEM, n=5, X-axis are drug concentration to schizandrin A inhibition human pulmonary epithelial cells proliferation
(μM), p < 0.01 * p < 0.05, * *)；
Figure 14 is that schizandrin A inhibits HeLa Cells to be proliferated (Mean ± SEM, n=5, X-axis
For drug concentration (μM), p < 0.01 * p < 0.05, * *)；
Figure 15 is that (Mean ± SEM, n=5, X-axis are drug to schizandrin A MDA-MB-231 cell Proliferation
Concentration (μM), p < 0.01 * p < 0.05, * *)；
Figure 16 be schizandrin A inhibit human hepatocarcinoma BEL-7402 proliferation (Mean ± SEM, n=5,
X-axis is drug concentration (μM), p < 0.01 * p < 0.05, * *).
Specific embodiment
Screening can with the traditional Chinese medicine monomer schizandrin A in conjunction with apj receptor and detect its function, shown in following examples.With
It is lower to implement for illustrating the present invention, but should not be used to limit the scope of the invention.
The screening of embodiment 1 can be with the traditional Chinese medicine monomer in conjunction with APJ
Membrane flexibility technology using drug in conjunction with cell-membrane receptor when specific affinity, by high-efficient liquid phase color
Spectrum, cell biology, molecular biology, biochemistry and receptor pharmacology combine, by drug in vivo act on chromatographic column
Enterprising Mobile state simulation.By the way that competent cell film is fixed on immobilization carrier surface, it is prepared into cell membrane stationary phase (CMSP), with
Buffer solution is mobile phase, cell membrane or cell-membrane receptor on the technique study drug or compound and stationary phase of chromatograph
Between interaction.
1. construction recombination plasmid pEGFP-N1-APJ
Source of people APJ genetic fragment is 1143bp.PCR specific amplification is carried out by template of target gene fragment, passes through 1%
Agarose gel electrophoresis is detected, and the purpose condition (referring to Fig. 1) that molecular weight is 1100bp or so is obtained, big with APJ cDNA
It is small to meet.Carrier pEGFP-N1 is a kind of carrier for expression of eukaryon that molecular weight is 4733bp, has multiple cloning sites, facilitates purpose
The insertion of gene.The APJ target gene point that pEGFP-N1 and PCR amplification are obtained with restriction enzyme Hind III and XhoI
Not carry out double digestion (37 DEG C, 4h), respectively obtain molecular size range be 4700bp and 1100bp purpose band (referring to fig. 2).
After carrier pEGFP-N1 and APJ to be attached to (overnight, 4 DEG C), with E. coli competent DH5 α to connection
Plasmid converted.By conversion, monoclonal choose bacterium, amplification, plasmid extract and etc. after, obtained plasmid is tested
Card obtains the double bands identical respectively at carrier and target gene containing molecular size range (referring to Fig. 3).To the bacterium for extracting plasmid
Liquid is sequenced (referring to fig. 4), obtained sequence is compared with Blast gene pool (referring to Fig. 5), the fund of recombinant plasmid
Sequence and APJ cDNA sequence are completely the same, and no base mutation shows that target gene fragment has been successively inserted into carrier, recombinate matter
Grain pEGFP-N1-APJ is constructed successfully.
2. stable transfection recombinant plasmid
Recombinant plasmid pEGFP-N1-APJ is transfected into 293 cell of HEK with liposome lipofectamine 2000, it is real
It tests and sets two groups: 1. pEGFP-N1,2. pEGFP-N1-APJ.After transfecting 6h, two groups of cell fluorescence expressions are observed, are detected glimmering
Light shows plasmid Successful transfection into cell (referring to Fig. 6).After screening two weeks by G418, luciferase expression is observed, finds fluorescence
It is located in cell membrane surface, is that cell membrane surface receptors feature is consistent with APJ, prompts successfully to obtain APJ-HEK293 cell.Through
It crosses G418 drug screening, after the processing such as monoclonal is selected, finally obtains the transfection cell for stablizing high expression APJ.
When cell it is long to 90% when, extract cell total rna (referring to Fig. 7).By total serum IgE reverse transcription, using cDNA as template into
There is unimodal (referring to Fig. 8) in the expression of APJ mRNA in row Real_time quantitative detection cell, solubility curve.Compared with the control group,
Higher levels of APJ mRNA is expressed in APJ-HEK293 cell (referring to Fig. 9).To the APJ albumen table in APJ-HEK293 cell
It is detected up to level, it is found that APJ protein expression level is apparently higher than control group in APJ-HEK293 cell (referring to Figure 10).
3. membrane flexibility technology screening apj receptor drug
The APJ high expressing cell obtained using screening, takes cell film preparation stationary phase, according to the phase between receptor-drug
Interaction carries out drug screening to 100 kinds of Chinese medicine standard items with membrane flexibility technology.Using water as mobile phase, with 0.2mL/min
Flow velocity carry out high performance liquid chromatography sample introduction is analyzed, obtain 16 kinds of active constituents with a grain of salt.And among these, have including asarum
Rouge element, waste Hu Jiasu, saikoside A, CAULIS MARSDENIAE TENACISSIMAE glycosides H, 6 kinds of retention times of schizandrin A and piperine 10min with
On active constituent (referring to Figure 11).With the 293 cell film preparation stationary phase of HKE of blank, 16 kinds of activity that screening is obtained at
Divide and carry out false positive exclusion experiment, experimental result is compared with APJ-HEK 293/CMC.By screening, sky is repeated several times carefully
Born of the same parents' negative control experiment finally obtains and the active constituent schizandrin A retained occurs (referring to figure in APJ-HEK 293/CMC
12)。
The inhibition tumor cell proliferation of embodiment 2APJ acceptor compound schizandrin A is studied
Effect of the schizandrin A to tumour cell is inquired into, handles human liver cancer cell SMMC- with schizandrin A respectively
7721 cells, HeLa Cells, human breast carcinoma MDA-MB-231 cell and four kinds of tumours of human pulmonary epithelial cells are thin
Born of the same parents.Drug treatment for 24 hours after, with mtt assay detect cell absorbance value, calculate drug cell proliferation inhibiting rate.
Schizandrin A plays the role of inhibiting tetra- kinds of tumor cell proliferations of SMMC-7721, HeLa, A549, MDA-MB-231,
But action intensity and specificity have differences.In these four tumour cells, schizandrin A is most strong to A549 cytosis,
IC50 is 60.27 μM (referring to Figure 13).In remaining three kinds of cells, HeLa cytosis is slightly stronger, IC50 value is 123.31 μ
M (referring to Figure 14)；Followed by MDA-MB-231 cell, IC50 are 215.63 μM (referring to Figure 15)；To SMMC-7721 cytosis
Most weak, IC50 is 370.68 μM (referring to Figure 16).
In conclusion schizandrin A of the invention has tetra- kinds of SMMC-7721, HeLa, A549, MDA-MB-231 of inhibition to swell
The effect of tumor cell proliferation can be used in preparing relevant drug.
Claims (5)
1. schizandrin A is used to prepare the purposes of drug, wherein the drug is for treating human cervical carcinoma, human liver cancer, human breast carcinoma
And human lung adenocarcinoma.
2. purposes according to claim 1, wherein the drug is for treating HeLa Cells proliferation, human liver cancer
SMMC-7721 cell Proliferation, human breast carcinoma MDA-MB-231 cell Proliferation, human pulmonary epithelial cells proliferation are caused to swell
Tumor.
3. a kind of antineoplastic pharmaceutical compositions, it includes schizandrin A.
4. pharmaceutical composition according to claim 3, the pharmaceutical composition is for treating human cervical carcinoma, human liver cancer, human milk
Gland cancer and human lung adenocarcinoma.
5. purposes of the schizandrin A as the target agent medicine for acting on apj receptor.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710563204X | 2017-07-11 | ||
| CN201710563204 | 2017-07-11 |
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| CN109394739A true CN109394739A (en) | 2019-03-01 |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102048827A (en) * | 2010-01-25 | 2011-05-11 | 浙江大学 | Application of shiandra for preparing medicaments for resisting tumor invasion and metastasis |
| CN104825442A (en) * | 2015-04-30 | 2015-08-12 | 浙江鼎辉医药科技有限公司 | Application of schisandrin B in preparation of drugs used for preventing colitis or colorectal carcinomas |
-
2018
- 2018-07-10 CN CN201810749179.9A patent/CN109394739A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102048827A (en) * | 2010-01-25 | 2011-05-11 | 浙江大学 | Application of shiandra for preparing medicaments for resisting tumor invasion and metastasis |
| CN104825442A (en) * | 2015-04-30 | 2015-08-12 | 浙江鼎辉医药科技有限公司 | Application of schisandrin B in preparation of drugs used for preventing colitis or colorectal carcinomas |
Non-Patent Citations (3)
| Title |
|---|
| HYE-YOUNG MIN 等: "Antiproliferative effects of dibenzocyclooctadiene lignans isolated from Schisandra chinensis in human cancer cells" * |
| XUEQIN MA 等: "Phenylpropanoids fromPodocarpium podocarpum" * |
| 于洋 等: "五味子提取物对HepG2细胞凋亡及Bcl-2、Bax基因转录的影响" * |
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