CN103127051A - Application of curcumenol in anti-tumor drug preparation - Google Patents

Abstract

The invention belongs to the field of chemical engineering and medicine, and relates to an application of curcumenol in anti-tumor drug preparation. The present invention provides an application of curcumenol in anti-tumor drug preparation, wherein tumor cells comprise liver cancer cells, leukemia cells and pancreatic cancer cells. The curcumenol belongs to natural products, and has characteristics of high bioavailability, stable property, and clinical use value. With application of the small molecule compound as a new anti-tumor drug or an auxiliary component thereof to be developed, advantages of significant tumor inhibition effect and green environmental protection are provided, and a new way is provided for treatment and cure of tumors.

Description

The application of curcumenol in the preparation antitumor drug

Technical field

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

Background technology

Curcumenol has another name called Curcumol, is the extract of zingiberaceous plant Rhizoma Curcumae, is colourless acicular crystal, is soluble in ether, chloroform, is dissolved in ethanol, is slightly soluble in petroleum ether, and is water-soluble hardly, and dissolubility is only 0.3% in water.

Relevant parameter is:

M.Wt：236.35

Formula：C ₁₅H ₂₄O ₂

Solubility：Unknown

Purity：＞99％

Storage：at-20℃?2years

CAS?No.：4871-97-0

traditional from Rhizoma Curcumae, extract the method for curcumenol in RADIX CURCUMAE, basically be with methanol or water extraction, then carry out steam distillation, obtain curcumenol [Hiroshi H through ethyl acetate or dehydrated alcohol recrystallization again, Kanji M, Yojiro S, et al.Structure of curcumol.[J] .Chem Pharm Bull, 1965, 13 (12): 1484, Xu Hongxia, Zheng Shuchen, Zuo Shixian, Deng. separating and evaluation [J] of the research-curcumenol of warm Rhizoma Curcumae antitumor effective ingredient and curdione. the Chinese herbal medicine communication, 1979, 10 (10): 11.], the productive rate and the purity that obtain are lower, need through subsequent purification such as silicagel column separation.

Recently, curcumenol crystalline technique improves to some extent.Meng Zhaoke etc. directly curcuma zedoary oil carry out vacuum topping, direct recrystallization prepares curcumenol to the fraction that obtains without silicagel column separates, and yield can reach 30%, purity 96%[Meng Zhao Mactra sulcatria Deshayes, Fu Meizhen. the extraction process of curcumenol [P]. Chinese patent: CN-1704417,2005-12-07.].The use supercritical extraction coupling swelling crystallization processs such as king's fine gold is auspicious extract content from the Rhizoma Curcumae decoction pieces [king's fine gold is auspicious higher than 90% fine powdered curcumenol crystal, Zhang Chaohui, the horse Chaoyang. the research of Crystallization of Curcmol With Scf [J]. He'nan University of Technology's journal (natural science edition), 2007,28 (3): 81.].Wang Ting etc. have set up take Oleum Curcumae as raw material, the new method of distilling under reduced pressure extraction curcumenol [Wang Ting, Li Tiefu, Lin Shaoqiang, etc. the isolation identification of curcumenol and assay [J]. Chinese Chinese medicine academic periodical, 2008,26 (5): 1 018.].

Biochip technology research is found, curcumenol can reduce the hepatic stellate cell transforminggrowthfactor-β1, Cytochrome P450 a expresses, potential [the Jiang Yuan that points out it to have anti-hepatic fibrosis, Li Zesong, Jiang Fusheng, Deng. the impact of curcumol on gene expression of cultured HSC-T 6 cells with DNA microarray [J]. Chinese combination of Chinese and Western medicine digestion magazine, 2005,13 (3): 144.].Add in the rat blood stasis models that epinephrine makes at hormone, curcumenol demonstrate certain anti thrombotic action [woods is former for Tang Zeyao, Zong Chengguo. the blood circulation promoting and blood stasis dispelling activity experiment research [J] of curcumenol. Pharmacology and Clinics of Chinese Materia Medica, 2003,19 (5): 15.].In addition, curcumenol also has obvious antifertility action, is used for doing bait formulation, reduce female Mus breeding potential and Mean litter size, with prevention and control the farmlands plague of rats [horse treasure, Zhang Danhua, Zhuan Wenjun, etc. curcumenol bait formulation control farmland mouse demonstration [J]. Chinese plant protection guide, 2007,27 (1): 34. imperial court woodss, Zhang Huidi, Zhao Riliang, etc. the control effect research [J] of antifertility agent curcumenol to regional bandicoot. the Shanxi Agricultural science, 2007,35 (9): 53.].In addition, curcumenol also is proved to be certain cytotoxicity.

Curcumenol is natural product, and bioavailability is higher, character is more stable, has clinical use value.To going deep into that this type of alkaloidal chemistry and biology 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 new medicinal usage that the purpose of this invention is to provide curcumenol.

The invention provides the application of curcumenol in the preparation antitumor drug.Described tumor can be hepatocarcinoma, leukemia, adenocarcinoma of lung or cancer of pancreas.Described antitumor drug is injection or tablet.Described antitumor drug can be medicines resistant to liver cancer or Antilung gland cancer medicine.

The invention provides a kind of method that suppresses tumor cell in vitro propagation, curcumenol is added in the culture fluid of tumor cell.Described tumor cell is hepatoma carcinoma cell, blood cell, lung adenocarcinoma cell, breast cancer cell or pancreatic cancer cell.The hepatoma carcinoma cell that adopts in one embodiment of the present of invention is QGY and HepG2, and the pancreatic cancer cell of employing is PANC-1, and the blood cell of employing is K562.Generally speaking, the final concentration that adds curcumenol is 0.5-100 μ M, preferably, can adopt 1-10 μ M, 1-100 μ M, even 0.5-1 μ M.For example, 0.1-5 μ M, 0.1-10 μ M, 0.1-20 μ M, 0.1-30 μ M, 1-5 μ M, 0.3-60 μ M, 0.3-50 μ M, 0.3-30 μ M, 0.3-20 μ M, 0.3-10 μ M, 0.3-5 μ M, 0.5-5 μ M, etc.

The present invention also provides a kind of antitumor drug, and the active component of described antitumor drug is curcumenol.Described tumor can be hepatocarcinoma, 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 and the interactional material of curcumenol generation, 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 curcumenol 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.Curcumenol 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, curcumenol of the present invention also can use together with the other treatment agent.

When curcumenol of the present invention is used as medicine, the curcumenol 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 curcumenol in the preparation antitumor drug, disclose especially first the concrete inhibition for hepatocarcinoma and leukaemia.Curcumenol belongs to 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.Simultaneously, also means are provided for further developing China's Chinese traditional herbs.

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.The PANC-1 cell culture is in containing the DMEM high glucose medium of 10%Gibico hyclone, and Hep G2 and QGY cell culture are in containing the DMEM high glucose medium of 10% hyclone, and the K562 cell culture is in containing 1640 culture medium of 10% hyclone.Contain 100U/ml penicillin and 100 μ g/ml streptomycins in culture medium.

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 (attached cell) 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 is measured curcumenol to the growth inhibited effect of hepatoma carcinoma cell

HepG2 cell (available from Chinese Academy of Sciences's cell bank) 1 * 10 ³/ hole is seeded to 96 orifice plates, cultivates to make it in 24 hours to add curcumenol (available from selleckchem company, S2407 after adherent, Curcumol, the network address http://www.selleckchem.com/products/Curcumol.html of company), establish 6 Concentraton gradient, 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 curcumenol to the IC50 value of HGC cell.

IC50 refers to the concentration of a suppressed half inhibitor.Here be the HepG2 cell quantity and be the concentration of a half curcumenol 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: curcumenol is 0.96 μ M to the IC50 value of Hep G2 cell.

Use same method, recording curcumenol is 0.60 μ M to the IC50 value of QGY cell (available from Chinese Academy of Sciences's cell bank).

The growth inhibited effect of embodiment 2 curcumenol to human pancreatic cancer cell

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

Step:

1) PANC-1 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, 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.

Result: curcumenol is 1429 μ M to the IC50 value of PANC-1 cell.

The growth inhibited effect of embodiment 3 curcumenol to people's blood cell

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

Step:

1) the K562 cell is planted in 96 orifice plates uniformly, every porocyte number is 10,000.

2) the rear dosing of spending the night, adding consistency is respectively 50,16.67,5.56,1.85,0.62 μ M, and each concentration has 3 multiple holes.

3) cultivated 48 hours, add the mixture (ratio of culture medium and cck8 is 10: 1) of 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: recording curcumenol is 1.70 μ M to the IC50 value of K562 cell (available from Chinese Academy of Sciences's cell bank).

Claims (9)

1. the application of curcumenol in the preparation antitumor drug is characterized in that described tumor is hepatocarcinoma or leukemia.

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 anti-leukemia medicine.

4. application as claimed in claim 1, is characterized in that, described antitumor drug is tablet or injection.

5. a method that suppresses tumor cell in vitro propagation, is characterized in that, curcumenol 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 leukaemia.

7. method as claimed in claim 5, is characterized in that, the final concentration that adds curcumenol is 0.5-100 μ M.

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

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