CN103127039A - Application of magnolol in preparation of antitumor drug - Google Patents

Abstract

The invention belongs to the chemical industry field and the medicine field, and relates to an application of magnolol in preparation of antitumor drugs. The invention provides the application of magnolol in preparation of antitumor drugs, and the tumor cells comprise liver cancer cells, and cervical cancer cells. The magnolol belongs to a natural product, has little toxic and side effect, high bioavailability, and stable properties, and has clinical application value. The micromolecular compound of the invention can be developed as a new antitumor drug or its auxiliary component, has obvious tumor suppression effect, is green and environment-friendly, and provides a new approach and means for treating and curing tumors.

Description

The application of magnolol in the preparation antitumor drug

Technical field

The invention belongs to chemical field and field of medicaments, relate to the application of magnolol in the preparation antitumor drug.

Background technology

Magnolol be sepia to the Cortex Magnoliae Officinalis extract of white fine powder, the effective ingredient of antibacterial action in the Cortex Magnoliae Officinalis skin has special, lasting myorelaxant effects and stronger antibacterial action, but anticoagulant.Clinically mainly as antibacterium bacterium, antifungal agent.

The magnolol basic parameter is as follows:

[English name] Magnolol

[another name] 5 ', 5-diallyl-2,2 '-'-biphenyl diphenol

[chemical name], 6 ', 7,12-Tetramethoxy-2,2 '-dimethyl-1-beta-berbaman

[molecular formula and molecular weight] C18H1802,266.32

[No. CAS] 528-43-8

[structural formula]

Li Jiaxiao etc. use supercritical CO ₂Fluid technique carries out extract and separate to Cortex Magnoliae Officinalis and obtains magnolol.With OSI method and oven method measuring the antioxidation of magnolol to Adeps Sus domestica, soybean oil and 10% emulsion.Result shows: magnolol in soybean oil and Adeps Sus domestica anti-autoxidation ability a little less than; In 10% emulsion system, magnolol almost do not show antioxidant activity (Chinese grain and oil journal, in March, 2007, the 22nd the 2nd phase of volume, 71-74).

Magnolol is natural product, and bioavailability is high, stable in properties, has clinical use value.To going deep into that the chemistry and biology of this compounds is studied, its molecular mechanism of action will be progressively clear and definite along with people, and this chemical constitution that will further promote this compounds is modified and structure activity study, and helps to improve the medical value of this compounds.

Summary of the invention

The purpose of this invention is to provide the application of magnolol in the preparation antitumor drug.

The invention provides the application of magnolol in the preparation antitumor drug.Described antitumor drug can be medicines resistant to liver cancer or medicament for resisting cervical cancer.This antitumor drug can be injection or tablet.

The invention provides a kind of method that suppresses tumor cell in vitro propagation, magnolol is added in the culture fluid of tumor cell.Described tumor cell can be hepatoma carcinoma cell, blood cell, cervical cancer cell or pancreatic cancer cell.The hepatoma carcinoma cell that adopts in one embodiment of the present of invention is Hep-G2.Generally speaking, adding the final concentration of magnolol is 1-100 μ M.For example, 1-5 μ M, 1-15 μ M, 1-50 μ M, 1-70 μ M, 1-80 μ M, 10-80 μ M, 15-80 μ M, 15-50 μ M, 20-70 μ M, 20-50 μ M, etc.

The present invention also provides a kind of antitumor drug, and the active component of described antitumor drug is magnolol.Described tumor can be hepatocarcinoma or cervical cancer.

Micromolecular compound magnolol of the present invention is 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 and interactional material occurs magnolol, as receptor, inhibitor or pick up anti-dose etc.

The present invention and inhibitor thereof, pick up anti-dose etc., when using (administration) 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.

Take magnolol of the present invention 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.Magnolol of the present invention 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, magnolol of the present invention also can use together with the other treatment agent.

When magnolol of the present invention is used as medicine, the magnolol for the treatment of effective dose 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 invention provides the application of magnolol in the preparation antitumor drug.Magnolol 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.HepG2 and HELA cell culture contain 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.

4. medicine is prepared:

Magnolol is dissolved in DMSO (dimethyl sulfoxide), and the mother solution that is mixed with 100mM or 50mM is standby.

Embodiment 1MTS method is measured magnolol to the growth inhibited effect of hepatoma carcinoma cell

3500/hole of Hep-G2 cell (available from Chinese Academy of Sciences's cell bank) is seeded to 96 orifice plates, cultivates to make it in 24 hours to add magnolol (available from Shanghai Pharmaceutical Inst., Chinese Academy of Sciences) after adherent, establishes 6 Concentraton gradient, and each concentration is established 3 multiple holes.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, calculate magnolol to the IC50 value of HGC cell.

IC50 refers to the concentration of a suppressed half inhibitor.Here be the Hep-G2 cell quantity and be the concentration of a half magnolol of contrast.The calculating of IC50 generally need to be measured the dosage effect more than 5, then obtains function calculation by curve fitting and get.

Result: according to the method described above, detect magnolol to the effect of HepG2 cell (available from ATCC), magnolol is 76.51 μ M to the IC50 value of Hep-G2 cell.

The growth inhibited effect of embodiment 2 magnolol to human cervical carcinoma cell

Utilize cck-8 test kit (Japanese colleague's chemistry institute) to detect.

Step:

1) HELA cell (available from ATCC) is planted in 96 orifice plates uniformly, every porocyte number is 3000.

2) treat adherent, the rear dosing of spending the night, dosing (magnolol concentration 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.

Result: magnolol is 15.89 μ M to the IC50 value of HELA cell.

Claims (10)

1. the application of magnolol in the preparation antitumor drug is characterized in that described tumor is hepatocarcinoma or cervical cancer.

2. application as claimed in claim 1, is characterized in that, described antitumor drug is medicines resistant to liver cancer.

3. application as claimed in claim 1, is characterized in that, described antitumor drug is medicament for resisting cervical cancer.

4. application as claimed in claim 1, is characterized in that, the dosage form of described antitumor drug is suppository, lotion, tablet or capsule.

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

6. method as claimed in claim 5, is characterized in that, described tumor cell is hepatoma carcinoma cell or cervical cancer cell.

7. method as claimed in claim 6, is characterized in that, the final concentration that adds magnolol is 1-100 μ M.

8. method as claimed in claim 6, is characterized in that, the final concentration that adds magnolol is 15-80 μ M.

9. an antitumor drug, is characterized in that, the active component of described antitumor drug is magnolol.

10. antitumor drug as claimed in claim 9, is characterized in that, described tumor is hepatocarcinoma or cervical cancer.