CN103127057A

CN103127057A - Application of fraxinellone in preparing of antineoplastic medicines

Info

Publication number: CN103127057A
Application number: CN2011103832256A
Authority: CN
Inventors: 余龙; 唐丽莎; 刘祖龙; 胡立宏
Original assignee: Fudan University
Current assignee: Fudan University
Priority date: 2011-11-25
Filing date: 2011-11-25
Publication date: 2013-06-05

Abstract

The invention belongs to the field of chemical industry and medicine, and relates to application of fraxinellone in preparing of antineoplastic medicine. According to the application of the fraxinellone in the preparing of the antineoplastic medicine, tumor cells comprise leukemia cells and pancreatic cancer cells. Fraxinellone belongs to natural products, is high in biological utilization degree, stable in quality, and is of clinic using values. Small molecule compounds of the application of the fraxinellone in the preparing of the antineoplastic medicine are used as novel antineoplastic medicine or being used as an auxiliary component for developing, and are significant in tumor-restraining effect, environmental-protecting. The application of the fraxinellone in the preparing of the antineoplastic medicine provides a novel approach and means of treatment for tumors.

Description

The application of fraxinellone in the preparation antitumor drug

Technical field

The invention belongs to chemical field and field of medicaments, She is Ji the application of fraxinellone in the preparation antitumor drug.

Background technology

The Chinese medicine Cortex Dictamni is the dry root bark of rutaceae shaggy-fruited dittany (D ictamnusdasy carpus Turcz), has and dispels the wind, dampness, heat clearing away, the effects such as detoxifcation.Modern pharmacology studies show that Cortex Dictamni has the various active such as antioxidation, antiviral, antibacterium, antiinflammatory, antitumor.Studies show that in recent years, separating De from Cortex Dictamni is the main pharmacodynamics composition of Cortex Dictamni Dao De fraxinellone, dictamine, obacunone, has multiple stronger biological activity.Wherein, fraxinellone has good the liver protecting and ALT lowering and suppresses the hepatic fibrosis isoreactivity, and having antibacterial, parasite killing isoreactivity as a kind of novel hepatosis treating medicine.

Zhu Danni etc. have measured fraxinellone content in the high effective liquid chromatography for measuring Cortex Dictamni (fraxinellone content in the high effective liquid chromatography for measuring Cortex Dictamni, China Medicine University's journal, 1998,29 (4): 319～320) by HPLC.

The structural formula of fraxinellone is:

Studies show that both at home and abroad, dictaminine is with fraxinellone is active substance main in Cortex Dictamni, and it has the trapping effect to the demodicid mite class, to insecticide have the function that suppresses growth and to 3 age mythimna separata stronger cytotoxicity is arranged.

2005, former Herba Cymbidii Goeringii etc. were further reported the insecticidal activity (a kind of insecticidal activity of Novel botanical pesticide ketone, plant protection, the 31st the 5th phase of volume) of fraxinellone.2009, former Herba Cymbidii Goeringii etc. studied in great detail the extraction process of fraxinellone in Cortex Dictamni, had obtained a kind of simple and practical separating technology.Adopt this technique, the fraxinellone yield reaches 94.7%, for the industrial separation of fraxinellone provides a kind of effective method (the 26th the 7th phase of volume, in July, 2009, the Study on extraction of the property thing matter fraxinellone of living in Cortex Dictamni, fine chemistry industry).Li Xiang etc. have also reported and put forward the method (making simultaneously the method for standby fraxinellone, dictamine, obacunone monomer from Cortex Dictamni, Sichuan University's journal (engineering science version), V o.l 39 No.4) of getting fraxinellone from Cortex Dictamni.

Summary of the invention

The new medicinal usage of purpose Shi Ti Gong fraxinellone of the present invention.

The application of this invention Ti Gong fraxinellone in the preparation antitumor drug.Described antitumor drug is injection or tablet.Described antitumor drug can be anti-leukemia medicine or anti-cancer of pancreas medicine.This antitumor drug can be injection or tablet.

The invention provides a kind of method ， that suppresses tumor cell in vitro propagation adds fraxinellone in the culture fluid of tumor cell.Described tumor cell is leukaemia or pancreatic cancer cell.Generally speaking, the final concentration of Jia Ru fraxinellone is 0.1-50 μ M, 0.1-0.5 μ M for example, 0.1-5 μ M, 0.1-10 μ M, 0.1-20 μ M, 0.1-50 μ M, 0.3-5 μ M, 0.3-50 μ M, 0.3-20 μ M, 0.3-10 μ M, 5-10 μ M, 5-20 μ M, 5-50 μ M, etc.

The present invention also provides a kind of antitumor drug, the active component Shi fraxinellone of described antitumor drug.Described tumor can be leukemia or cancer of pancreas.

Micromolecular compound of the present invention 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 the Yu fraxinellone interactional material occurs, as receptor, inhibitor or antagonist etc.

The present invention and inhibitor, antagonist etc. when using (administration) in treatment, can provide different effects.Usually, these materials can be formulated in nontoxic, inertia with pharmaceutically acceptable aqueous carrier medium in, wherein pH is about 5-8 usually, preferably pH is about 6-8, 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.

Take the fraxinellone of Ben Faming as example, can be with itself and suitable pharmaceutically acceptable carrier coupling.This class pharmaceutical composition contains 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.The fraxinellone of Ben Faming 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 5 mg/kg body weight of 1 microgram/kg body weight-Yue.In addition, the fraxinellone of Ben Faming also can use together with the other treatment agent.

When the fraxinellone of Ben Faming is used as medicine, treatment effective dose De fraxinellone can be applied to mammal, wherein should treat effective dose usually at least about 10 micrograms/kg body weight, and in most of the cases be no more than approximately 8 mg/kg body weight, preferably this dosage is the about 1 mg/kg body weight of 10 micrograms/kg body weight-Yue.Certainly, concrete dosage also should be considered the factors such as route of administration, patient health situation, and these are all within the skilled practitioners skill.

The application of this invention Ti Gong fraxinellone in the preparation antitumor drug.Fraxinellone is natural product, 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) take out cryopreservation tube from liquid nitrogen container, 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, then is repeated to wash once with culture fluid.

3) with after the suitable dilution of culture fluid, the inoculated and cultured bottle is placed on 37 ℃ of standing cultivations of incubator, changes culture fluid next day, continues to cultivate.Go down to posterity when being cultured to finite concentration.Cell culture contains 100U/ml penicillin and 100 μ g/ml streptomycins in culture medium in containing the DMEM high glucose medium of 10% hyclone.

2. passage is cultivated

The situation of observation of cell growth every day is grown to approximately 90% and is gone down to posterity when converging in culture bottle when cell, approximately went down to posterity once every 2-4 days.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 2-3ml trypsinization liquid digestion, be placed in 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 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 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 in cryopreserving liquid, the ultimate density of cell is 0.5-1 * 10 ⁷/ ml.Blow and beat gently with suction pipe and make cell even, then be distributed in 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 liquid nitrogen container after 5 hours and preserve.

Embodiment 1MTS method Ce is Dinged the growth inhibited effect of fraxinellone to pancreatic cancer cell

PANC-1 cell (available from Chinese Academy of Sciences's cell bank) 1 * 10 ³/ hole is seeded to 96 orifice plates, cultivates to make it adherent Hou Jia Ru fraxinellone (available from Shanghai Pharmaceutical Inst., Chinese Academy of Sciences) in 24 hours, establishes 5 Concentraton gradient, and each concentration is established 3 multiple holes.Adding consistency is respectively 50,16.67,5.56,1.85,0.62 μ M.Cell is at 37 ℃, 5%CO ₂Cultivate after 72 hours under 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 mixing) 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, the IC50 value of Ji Suan fraxinellone to the PANC-1 cell.

IC50 refers to the concentration of a suppressed half inhibitor.Here be the PANC-1 cell quantity and be the concentration of a half fraxinellone of contrast.

The: fraxinellone is 0.32 μ M to the IC50 value of PANC-1 cell as a result.

The growth inhibited effect of embodiment 2 fraxinellone to the human leukemia cell

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, adding consistency is respectively 50,16.67,5.56,1.85,0.62 μ M, each concentration has 3 multiple holes.

3) cultivated 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, measure reading on microplate reader.

The: fraxinellone is about 5.66 μ M to the IC50 value of K562 cell as a result.

Claims

1. the application of fraxinellone in the preparation antitumor drug.

2. application as claimed in claim 1, is characterized in that, described tumor is leukemia or cancer of pancreas.

3. application as claimed in claim 1, is characterized in that, described antitumor drug is anti-leukemia medicine.

4. application as claimed in claim 1, is characterized in that, described antitumor drug is anti-cancer of pancreas medicine.

5. a method that suppresses tumor cell in vitro propagation, is characterized in that, fraxinellone added in the culture fluid of tumor cell.

6. method as claimed in claim 5, is characterized in that, described tumor cell is leukaemia or pancreatic cancer cell.

7. method as claimed in claim 5, is characterized in that, the final concentration of Jia Ru fraxinellone is 0.1-50 μ M.

8. an antitumor drug, is characterized in that, the active component of described antitumor drug is fraxinellone.

9. antitumor drug as claimed in claim 8, is characterized in that, described tumor is leukemia or cancer of pancreas.