CN102366416B - Pharmaceutical application of 12-dehydroxy-21-hydroxy protopanoxadiol - Google Patents
Pharmaceutical application of 12-dehydroxy-21-hydroxy protopanoxadiol Download PDFInfo
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- CN102366416B CN102366416B CN 201110362134 CN201110362134A CN102366416B CN 102366416 B CN102366416 B CN 102366416B CN 201110362134 CN201110362134 CN 201110362134 CN 201110362134 A CN201110362134 A CN 201110362134A CN 102366416 B CN102366416 B CN 102366416B
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Abstract
The invention belongs to the fields of chemical engineering and medicaments and relates to application of 12-dehydroxy-21-hydroxy protopanoxadiol in preparation of antitumor medicaments. The invention provides the application of the 12-dehydroxy-21-hydroxy protopanoxadiol in preparation of the antitumor medicaments. Tumor cells comprise liver cancer, leukaemia, adenocarcinorma of lung or pancreatic cancer. The 12-dehydroxy-21-hydroxy protopanoxadiol belongs to natural products and has a certain clinical utilization value. A micromolecular compound provided by the invention as a new antitumor medicament or an assistant component of the medicament is developed; the antitumor effect is obvious; the micromolecular compound is environmental friendly; and the invention provides a new way and means for treating and curing tumors.
Description
Technical field
The invention belongs to chemical field and field of medicaments, relate to 12-and go the application of hydroxyl-21-hydroxyl protopanoxadiol in the preparation antitumor drug.
Background technology
12-removes hydroxyl-21-hydroxyl protopanoxadiol, and structural formula is as follows.
1967, Chen etc. to its structure done parsing (Dryobalanone, a 21-hydroxydammarane triterpene.Cheung, Hee-Tai.Et al.Tetrahedron.Letters (1967), (29), 2807-9.).
Next year, there is again report to introduce the method for from borneo camphor tree, extracting this chemical compound.Get the vateria fat extract, through aluminium oxide (V.M.. naphtha solvent, benzene and chloroform) chromatography, obtain the Borneolum Syntheticum lactone, then obtain 12-with the methanol effect that contains sodium borohydride and go hydroxyl-21-hydroxyl protopanoxadiol (compound III, R=OH.Constituents of Dipterocarpaceae resins.II.Structure of dryobalanone from Dryobalanops aromatica.Cheung.H.T.Journal of the Chemical Society[Section] C:Organic (1968), (21), 2686-9.).
Borneo camphor tree belongs to Dipterocarpaceae.Eight or nine zhang of the height of trees greatly can six or seven be enclosed.Originate in the ground such as Sumatera, Persian, the ancient South Sea and China Fujian is wide.Resin claims Borneolum Syntheticum, is famous and precious spice.Borneolum Syntheticum: a kind of organic compound in the Dipterocarpaceae trunk.White crystal has the camphoraceous fragrance of class.Also available synthetic makes.Claim on the Chinese medicine " Borneolum Syntheticum ".Optical activity is arranged.Main hyoscine has heat clearing away, analgesic effect.But, go hydroxyl-21-hydroxyl protopanoxadiol report less about 12-of the present invention.
It is natural product that 12-removes hydroxyl-21-hydroxyl protopanoxadiol, has certain clinical use value.Along with people to the going deep into of the chemistry and biology of this compounds research, its molecular mechanism of action will progressively clear and definite, this will further promote chemical constitution modification and the structure activity study of this compounds, and help to improve the medical value of this compounds.
Summary of the invention
The purpose of this invention is to provide 12-and go the application of hydroxyl-21-hydroxyl protopanoxadiol in the preparation antitumor drug.
The invention provides 12-and go the application of hydroxyl-21-hydroxyl protopanoxadiol in the preparation antitumor drug.Described antitumor drug can be medicines resistant to liver cancer or anti-leukemia medicine.This antitumor drug can be injection or tablet.
The invention provides a kind of method that suppresses tumor cell in vitro propagation, go hydroxyl-21-hydroxyl protopanoxadiol to add in the culture fluid of tumor cell 12-.Described tumor cell can be hepatoma carcinoma cell, blood cell, lung adenocarcinoma cell or pancreatic cancer cell.The hepatoma carcinoma cell that adopts in one embodiment of the present of invention is QGY.Generally speaking, adding 12-, to remove the final concentration of hydroxyl-21-hydroxyl protopanoxadiol be 1-100 μ M.For example, 1-5 μ M, 1-10 μ M, 1-20 μ M, 1-50 μ M, 5-10 μ M, 5-20 μ M, 5-50 μ M, 5-100 μ M, etc.
The present invention also provides a kind of antitumor drug, and the active component of described antitumor drug is that 12-removes hydroxyl-21-hydroxyl protopanoxadiol.Described tumor can be hepatocarcinoma or leukemia.
Micromolecular compound 12-of the present invention goes hydroxyl-21-hydroxyl protopanoxadiol available from Chinese Academy of Sciences institute of materia medica, and high performance liquid chromatography is surveyed purity, HPLC 〉=98%.
Micromolecular compound of the present invention also can adopt the preparation method preparation of various routines.For example, adopt the synthetic method of artificial chemistry.
Utilize micromolecular compound of the present invention, by various conventional screening techniques, can filter out with 12-and go hydroxyl-21-hydroxyl protopanoxadiol that interactional material occurs, such as receptor, inhibitor or antagonist etc.
The present invention and inhibitor, antagonist etc. when when (administration) used in treatment, can provide different effects.Usually, but these materials are formulated in nontoxic, inertia with pharmaceutically acceptable aqueous carrier medium in, wherein pH is about 5-8 usually, preferably pH is about 6-8, although pH value can change to some extent with the character that is formulated material and disease to be treated.The pharmaceutical composition for preparing can carry out administration by conventional route, comprising (but being not limited to): intramuscular, intraperitoneal, subcutaneous, Intradermal or topical.
Go hydroxyl-21-hydroxyl protopanoxadiol as example take 12-of the present invention, can be with itself and suitable pharmaceutically acceptable carrier coupling.This class pharmaceutical composition contains chemical compound and pharmaceutically acceptable carrier or the excipient for the treatment of effective dose.This class carrier comprises (but being not limited to): saline, buffer, glucose, water, glycerol, ethanol and combination thereof.Pharmaceutical preparation should be complementary with administering mode.12-of the present invention goes hydroxyl-21-hydroxyl protopanoxadiol can be made into the injection form, for example is prepared by conventional method with normal saline or the aqueous solution that contains glucose and other adjuvant.Pharmaceutical composition such as Tablet and Capsula can be prepared by conventional method.Pharmaceutical composition such as injection, solution, Tablet and Capsula should be made under aseptic condition.The dosage of active component is the treatment effective dose, for example every day about 1 microgram/kg body weight-Yue 5 mg/kg body weight.In addition, 12-of the present invention goes hydroxyl-21-hydroxyl protopanoxadiol also can use with the other treatment agent.
When 12-of the present invention removes hydroxyl-when 21-hydroxyl protopanoxadiol is used as medicine, can go hydroxyl-21-hydroxyl protopanoxadiol to be applied to mammal the 12-for the treatment of effective dose, wherein should treat effective dose usually at least about 10 micrograms/kg body weight, and in most of the cases be no more than about 8 mg/kg body weight, preferably this dosage is about 10 micrograms/kg body weight-Yue 1 mg/kg body weight.Certainly, concrete dosage also should be considered the factors such as route of administration, patient health situation, and these all are within the skilled practitioners skill.
The invention provides 12-and go the application of hydroxyl-21-hydroxyl protopanoxadiol in the preparation antitumor drug.It is natural product that 12-removes hydroxyl-21-hydroxyl protopanoxadiol, obviously the propagation of inhibition tumor cell.Micromolecular compound of the present invention is developed as new antitumor drug or its auxiliary element, and tumor killing effect is obvious, environmental protection, and will and cure tumor for treatment provides a kind of new approach and means.
The specific embodiment
Experimental technique:
1. cell recovery
1) from liquid nitrogen container, takes out cryopreservation tube, directly drop in 37 ℃ of warm water, and frequently shake and make it melt as early as possible.
2) take out cryopreservation tube from 37 ℃ of water-baths, with suction pipe sucking-off cell suspension, inject centrifuge tube and add culture fluid more than 10 times, low-speed centrifugal after mixing is abandoned supernatant, repeats to wash once with culture fluid again.
3) with after the suitable dilution of culture fluid, the inoculated and cultured bottle is placed on 37 ℃ of incubators and leaves standstill cultivation, changes culture fluid next day, continues to cultivate.Go down to posterity when being cultured to finite concentration.The QGY cell culture is in containing the DMEM high glucose medium of 10% hyclone, and the k562 cell culture contains 100U/ml penicillin and 100 μ g/ml streptomycins in the culture medium in containing 1640 culture medium of 10% hyclone.
2. passage is cultivated
The situation of observation of cell growth every day goes down to posterity when converging when cell grows to about 90% in culture bottle, goes down to posterity once every 2-4 days approximately.One bottle goes down to posterity into three bottles, or a 25cm
2Go down to posterity in a 75cm
2Culture bottle in.Method:
1) with 1 * phosphate buffer washed cell once.
2) add the digestion of 2-3ml trypsinization liquid, place 37 ℃ of incubator numbers minute.Pat Tissue Culture Flask with hands, make cell separation.
3) suitable culture medium with the Gibico hyclone that contains 10-15% stops trypsinization.Cell is sub-packed in the new culture bottle, continues to cultivate.
3. cell cryopreservation
1) get the cell tryptase protease digestion that is cultured to exponential phase, be collected in the centrifuge tube and counting, centrifugal.
2) reject trypsin and old culture fluid add the frozen culture fluid (containing 10%DMSO, 40%DMEM and 50%Gibico hyclone) that configures, and the ultimate density of cell is 0.5-1 * 10 in the cryopreserving liquid
7/ ml.Blow and beat gently with suction pipe and to make cell even, then be distributed in the aseptic cryopreservation tube, every pipe adds 1-1.5ml.
3) cryopreservation tube is put into freezing storing box and put-80 ℃ of quick-freezings, move in the liquid nitrogen container after 5 hours and preserve.
4. medicine is prepared:
12-goes hydroxyl-21-hydroxyl protopanoxadiol to be dissolved in DMSO (dimethyl sulfoxide), and the mother solution that is mixed with 100mM or 50mM is for subsequent use.
Embodiment 1MTS method is measured 12-and is gone hydroxyl-21-hydroxyl protopanoxadiol to the growth inhibited effect of hepatoma carcinoma cell
3000/hole of QGY cell (available from Chinese Academy of Sciences's cell bank) is seeded to 96 orifice plates, cultivate and made it adherent rear adding 12-in 24 hours and remove hydroxyl-21-hydroxyl protopanoxadiol (available from Shanghai Pharmaceutical Inst., Chinese Academy of Sciences), if 6 Concentraton gradient, each concentration are established 3 multiple holes.Cell is at 37 ℃, 5%CO
2Cultivate after 72 hours under the condition, outwell culture fluid, measure cell survival rate with MTS test kit (Promega company).
Method of testing is: cell is washed one time with serum-free medium, added the MTS chromophoric solution (adding 2ml solution 1 and 100 μ l solution 2 in the 10ml serum-free medium, fully mixings) for preparing in advance according to the amount of 100 μ l/well.Do not have the hole of cell to be made as this bottom outlet with one, absorb in order to the bias light of calibration solution.Cell is put into cell culture incubator to be continued to cultivate 2～4 hours, then read absorbance value (reference wavelength 630-700nm with microplate reader, measure wavelength 490nm), calculate cell survival rate, to measure hole absorbance value/control wells absorbance value as the numerical value of cell survival rate.According to cell survival rate, calculate 12-and go hydroxyl-21-hydroxyl protopanoxadiol to the IC50 value of QGY cell.
IC50 refers to a suppressed half
InhibitorConcentration.Here be the concentration that the QGY cell quantity removes hydroxyl-21-hydroxyl protopanoxadiol for a half 12-who contrasts.The calculating of IC50 generally need to be measured the dosage effect more than 5, obtains function calculation by curve fitting again and gets.
It is 46.89 μ M to the IC50 value of QGY cell that result: 12-removes hydroxyl-21-hydroxyl protopanoxadiol.
Embodiment 212-goes hydroxyl-21-hydroxyl protopanoxadiol to human leukemia cell's growth inhibited effect
Utilize cck-8 test kit (Japanese colleague's chemistry institute) to detect.
Step:
1) k562 cell (available from ATCC) is planted in 96 orifice plates uniformly, every porocyte number is 10000.
2) treat adherent, the rear dosing of spending the night, (12-goes hydroxyl-21-hydroxyl protopanoxadiol concentration to be respectively 50,16.67,5.56,1.85,0.62 μ M, and each concentration has 3 multiple holes in dosing.
3) cultivate 48 hours, complete medium is replaced to the mixture (10: 1) of serum-free medium and CCK8, hatched 2 hours in 37 ℃ of incubators.
4) take 450nm as measuring wavelength, take 650nm as the contrast wavelength, on microplate reader, measure reading.
It is 5.22 μ M to the IC50 value of k562 cell that result: 12-removes hydroxyl-21-hydroxyl protopanoxadiol.
Claims (4)
1.12-go the application of hydroxyl-21-hydroxyl protopanoxadiol in the preparation antitumor drug, described tumor is hepatocarcinoma or leukemia.
2. application as claimed in claim 1 is characterized in that, this antitumor drug is injection.
3. a method that suppresses tumor cell in vitro propagation is characterized in that, goes hydroxyl-21-hydroxyl protopanoxadiol to add in the culture fluid of tumor cell 12-; Described tumor is hepatocarcinoma or leukemia.
4. method as claimed in claim 3 is characterized in that, it is 1-100 μ M that adding 12-removes the final concentration of hydroxyl-21-hydroxyl protopanoxadiol.
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Non-Patent Citations (6)
| Title |
|---|
| Constituents of Dipterocarpaceae Resins. Part II.l Structure of Dryobalanone from Dryobalanops aromatica;H. T. Cheung et al.;《J. Chem. SOC.》;19681231;第2686-2689页 * |
| DRYOBALANONE, A 21-HYDROXYDAMMARANE TRITERPENE;H. T. Cheung;《Tetrahedron Letters》;19671231(第29期);第2807-2809页 * |
| H. T. Cheung et al..Constituents of Dipterocarpaceae Resins. Part II.l Structure of Dryobalanone from Dryobalanops aromatica.《J. Chem. SOC.》.1968,第2686-2689页. |
| H.T.Cheung.DRYOBALANONE A 21-HYDROXYDAMMARANE TRITERPENE.《Tetrahedron Letters》.1967 |
| 刘娜等.原人参二醇及其衍生物的化学与抗癌活性研究进展.《中国药物化学杂志》.2008,第18卷(第5期),第384-388页. |
| 原人参二醇及其衍生物的化学与抗癌活性研究进展;刘娜等;《中国药物化学杂志》;20081031;第18卷(第5期);第384-389页 * |
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