CN1692901A - Application of magnolol for preparing medicine compositions to treat and prevent tumor - Google Patents

Abstract

An application of honokiol in preparing the medicine for preventing and treating tumor with broad spectrum and high effect is disclosed.

Description

The application of honokiol in the pharmaceutical composition of preparation antitumor or prophylaxis of tumours

Technical field

The present invention relates to the application of a kind of Lignanoids compounds honokiol in the pharmaceutical composition of preparation antitumor or prophylaxis of tumours.

Technical background

The whole world has 1,000 ten thousand New Development cancer patients every year approximately, and because aged tendency of population, smoking, dietary habit and the change of diet structure and the raising of industrialization degree, the morbidity number of whole world cancer also will be with the speed increase of average annual 3%-5%, and mortality rate also is ascendant trend year by year.What the treatment of cancer was taked at present is a kind of Comprehensive Treatment that comprises operation, radiotherapy, chemotherapy and biology, immunization therapy etc., but no matter which kind of means of employing, the overall cure rate of cancer is still paced up and down about 30%, and long-term survival rate and quality of life are still undesirable.

Tumor is a kind of disease of cell proliferation and apoptosis dysequilibrium, is again the unusual disease of cell cycle simultaneously, and it comprises long-term aggregated and the biochemistry of cell and the change of gene that damages on several different biological levels.Being one and lasting the several years of tumor (average 15-20), complicated, multistage progressive process, its process can be divided into startups (initiation), progress (promotion) and urge cancer (progression) three phases.Precancerous cell further will be converted into cancer through the carcinogenic promoting agent effect, is autonomous growth, infiltration and the transfer of cancerous cell then, and preceding half stage of differentiation also is the link that might block.

Therefore, the method for ideal control tumor development is to take two hands policies, the one, and effective kill tumor cell, another is to take effectively to prevent intervening measure, suppresses the factor of short tumor development.

, most of known chemotherapeutics all have stronger toxic and side effects, as suppressing hemopoietic system, show as leukocyte, erythrocyte and thrombocytopenia, and immunity of organisms is low inferior.The cancer patient of quite a few finally may not be to die from cancer, but dies from the body function depletion of caused by chemotherapeutic medicines.Therefore, the development of the clear and definite new anti-cancer drug thing of high-efficiency low-toxicity mechanism of action is badly in need of by treatment of cancer.On the other hand, the chemopreventive agent that highly effective and safe is nontoxic is competitively carried out development in countries in the world.And screening and separating broad spectrum chemopreventive agent come into one's own day by day from natural product or traditional Chinese herbal medicine, and be main because its mechanism of action is extensive, and aboundresources has more safety and practicality and caused the extensive concern of researcher.Existing so far tens of kinds of natural products enter clinical preceding and clinical research, as the Polyphenols in the natural product, flavone and flavonoid, fragrant isosulfocyanate, terpenoid, carotene, organosulfur compound etc.; Microcomponent in the food is as vitamin Class A, vitamin C, vitamin E, calcium, selenium etc.; Other natural products such as curcumin, polyamines, cellulose etc. are also under study for action.Therefore, the exploitation of the intervention blocking-up reagent of the chemotherapeutics of high-efficiency low-toxicity and cancer generation development seems quite important.Certainly, even more ideal is to have two kinds of effects concurrently with a kind of medicine.

The molecular formula of honokiol (honokiol) is C ₁₈H ₁₈O ₂White crystal, 84.8～85.0 ℃ of fusing points, molecular weight are 266, its structural formula is:

Formula I

This chemical compound and its isomers all are to extract from the bark of Magnoliacea plant Cortex Magnoliae Officinalis or magnolia officinalis rehd.et wils.var.biloba rehd.et wils. or root bark, are used for the medicine of warming middle-JIAO, the therapeutic method to keep the adverse QI flowing downwards, dampness, expectorant on the traditional Chinese medical science.

Summary of the invention

The object of the present invention is to provide honokiol as the application of effective ingredient in the medicine of preparation treatment or preclude blood system tumor.

Another object of the present invention is to provide honokiol as the application of effective ingredient in the medicine of preparation treatment or prevention digestive tract tumor.

The The compounds of this invention honokiol is suc as formula shown in the I,

Formula I

This formula I chemical compound has antioxidation, removing free radical, reduces the effect of some carcinogen toxicity, prophylaxis of tumours generation development or recurrence, can be used for preparing the pharmaceutical composition of antitumor or prophylaxis of tumours.

The antitumor of formula I compound of the present invention or the pharmaceutical composition of prophylaxis of tumours are preferably the pharmaceutical composition of a kind of treatment or preclude blood system tumor.

The antitumor of formula I compound of the present invention or the pharmaceutical composition of prophylaxis of tumours are preferably the pharmaceutical composition of a kind of treatment or prevention digestive tract tumor.

The pharmaceutical composition of antitumor of the present invention or prophylaxis of tumours comprises formula I chemical compound and at least a pharmaceutically acceptable carrier or the excipient of effective dose.

The medicine that the present invention makes both can use as antineoplastic agent separately, also can unite use with other antitumor drug.

The medicine that the present invention makes both can also can use with other known chemical preventive 5 sides separately as the prophylaxis of tumours drug use.

Formula I chemical compound of the present invention can be made into the preparation of intestinal or non-intestinal combination medicine by this area inhibition method, as tablet, capsule, granule, injection etc.

According to the principle of the invention, formula I chemical compound honokiol can also be prepared into and be used for chemotherapy of tumors or chemoprophylactic auxiliary health product.

Chemical compound of the present invention is the phenyl ring class natural activity molecule that extracts among a kind of therefrom web Piao, various blood tumor cells and various solid tumor cell all had cytotoxicity,, be the natural broad-spectrum anti-cancer drug of a class mainly by causing that apoptosis of tumor cells reaches anticancer purpose.The pharmacokinetic data explanation, honokiol can be absorbed rapidly by gastrointestinal tract and abdominal cavity mucosa, and the time of Zhi Liuing is longer relatively in vivo.Animal vivo test explanation, honokiol have the activity of ideal inhibition ascites tumor and solid tumor growth, the life span of significant prolongation tumor animal.It is suitable with classical anticancer drugs, doxorubicin to press down tumor efficient in the honokiol body.When giving effective dose, no matter be former generation normal cell of In vitro culture, or feed mice in the body and all do not have obvious toxic and side effects.Honokiol is the chemotherapy of tumors drug candidate of a kind of safe, efficient, wide spectrum, convenient drug administration.

Chemical compound of the present invention has significant antioxidant role simultaneously, strengthens enzymatic activitys such as LDH, GST, GSH-px; Can directly combine and form complex, influence carcinogenic absorption, distribution and metabolism activation with carcinogen; Influence the dna adduct formation that carcinogen causes; Influence the activity of metabolic enzyme, thereby influence the degraded and the drainage of carcinogen metabolism and metabolite thereof; Significantly suppress telomerase activation; Can induce the leukaemia's who does not have differentiation and maturation further differentiation; Selectivity suppresses the expression of one of Cycloxygenase isomerase COX-2; Suppress tumor cell proliferation and can cause early stage cancerous tumor cell apoptosis.Animal experiment confirms that multiple mutagenic agent and carcinogen are had stronger antimutagenesis in the body, so honokiol also can be made high-efficiency low-toxicity intervention blocking-up combination of agents thing.

Description of drawings

Fig. 1 is that honokiol is to human fibroblasts, lymphocyte and huve cell toxicity inspection; Wherein For separating lymphocyte from people's fresh blood; is the former human fibroblasts of foster source from children's's foreskin of being commissioned to train;

Be the former foster Human umbilical vein endothelial cells of being commissioned to train.

Fig. 2 A detects through the RKO cell DNA ladder band that honokiol is handled; Wherein, contrast to not adding the honokiol group; H5 is a 5ug/ml honokiol processed group; H10 is a 10ug/ml honokiol processed group; H15 is a 15ug/ml honokiol processed group;

Fig. 2 B detects through the K562 cell DNA ladder band that honokiol is handled; Wherein, contrast to not adding the honokiol group; H5 is a 5ug/ml honokiol processed group; H10 is a 10ug/ml honokiol processed group; H15 is a 15ug/ml honokiol processed group.

Fig. 3 A is that the hypodiploid peak of the RKO cell of honokiol processing detects and cell cycle analysis; Wherein, A figure is not for adding the honokiol group; B figure is a 5ug/ml honokiol processed group; C figure is a 10ug/ml honokiol processed group; D figure is a 15ug/ml honokiol processed group. The expression apoptosis; The expression G1 phase; The expression S phase; represents the G2 phase;

Fig. 3 B is that the hypodiploid peak of the K562 cell of honokiol processing detects and cell cycle analysis; Wherein, A figure is not for adding the honokiol group; B figure is a 5ug/ml honokiol processed group; C figure is a 10ug/ml honokiol processed group; D figure is a 15ug/ml honokiol processed group.

The expression apoptosis; The expression G1 phase; The expression S phase; represents the G2 phase.

Fig. 4 A is Caspase-3 and-9 protein expressions in the RKO cell that the variable concentrations honokiol is handled; Wherein, contrast to not adding the honokiol group; H5 is a 5ug/ml honokiol processed group; H10 is a 10ug/ml honokiol processed group;

Fig. 4 B is Caspase-3 and-9 protein expressions in the K562 cell that the variable concentrations honokiol is handled; Wherein, contrast to not adding the honokiol group; H5 is a 5ug/ml honokiol processed group; H10 is a 10ug/ml honokiol processed group.

Fig. 5 is a honokiol lumbar injection pharmacokinetic curve.

Fig. 6 is the inhibition effect of honokiol to the lotus RKO of Balb/c nude mice institute solid tumor; Wherein,

Be matched group, only injecting normal saline; ■ is a group of solvents, only injects the solvent of equivalent and honokiol group corresponding dosage;

Be the amycin group, injection is dissolved in the 50ug/20g mice of normal saline; is the honokiol group, presses 100mg/kg dosage injection injection mice.

Fig. 7 detects the iNOS protein expression for Western blot; Wherein, matched group is not for adding honokiol; H5 handles for adding the 5ug/ml honokiol; H10 handles for adding the 10ug/ml honokiol.

Fig. 8 detects the proteic expression of Cox-2 for Western blot; Wherein, matched group is not for adding honokiol; H5 is for adding 5ug/ml honokiol processed group; H10 is for adding 10ug/ml honokiol processed group.

Fig. 9 is intestinal neoplasms and cancer effect due to the honokiol prevention DMH.Wherein is tumor incidence rate (%);

Expression carcinogenesis rate;

The percentage rate of expression lotus tumor number and lotus cancer number.

The specific embodiment

Embodiment 1-2 is to the evaluation of honokiol cytology effectiveness

The experiment of this part is mainly carried out effect assessment by honokiol for the lethal effect of multiple hematopathy cell and solid tumor cell, and utilizes primary cultured cell to carry out toxicity inspection.

Wherein, used cell is k562 (people's chronic myeloid leukemia cells system), HL-60 (people is children's grain acute leukemia cells system early), Aspc-1 (human pancreatic cancer cell), MG-63 (human osteosarcoma cell line), SW480 (former colon adenocarcinoma cell system of people), SW620 (people shifts colon adenocarcinoma cell system), RKO (human large intestine cancer cell line), ECV304 (immortal human huve cell), 293 (human renal carcinoma cell lines), LS174T (human colon adenocarcinoma cell line), NCI-H460 (human lung cancer cell line), RD (human rhabdomyosarcoma's cell line), Hela (human cervical carcinoma cell system), Bcap37 (MCF-7), HepG2 (people HBV hepatoma cell line), NB4 (children's grain leukaemia system in before the people is acute), BxPC-3 (human pancreas's oncocyte system), HCT-116 (people's colorectal cancer cell line), HT29 (people COX-2 positive colon-cancer cell system), U937 (people huge have a liking for cell lymphoma cell system), more than these human cell lines provide by the Zhejiang University institute of oncology.

Used reagent and instrument are as follows:

Honokiol solution: monocrystalline dissolves with DMSO, is made into the working solution of 5mg/ml, is made into working solution with RPMI 1640.RPMI 1640, MTT, calf serum, culture plate, CO2 gas incubator, microplate reader, flow cytometer (FACS).Cell culture is with RPMI-1640 (Gibco BRL) culture medium, wherein contains calf serum (Chinese Hangzhou Ilex purpurea Hassk.[I.chinensis Sims biotech company), 100u/ml penicillin and the 100ug/ml streptomycin of 10% deactivation, in 37 ℃, 5%CO ₂Incubator is cultivated.Caspase-3 ,-7 ,-9 and Pan-actin available from NeoMarkers, Fremont, CA, USA.Honokiol is taken at Chinese biological goods medicine and identifies institute, and nominal purity is greater than 98%.This medicinal DMSO is made into the storing solution of 10mg/ml, makes the DMSO final concentration less than 1% with the culture medium dilution during use.MTT is available from U.S. Sigma chemical company.The Balb/c Mus of experiment usefulness and Balb/c nude mice are all available from Zhejiang University's Experimental Animal Center.

Embodiment 1, mtt assay detect honokiol to be suppressed and IC different cell line cell propagation ₅₀Value

Method: cell suspension with three parallel multiple hole kinds in 96 orifice plates, every hole 100ul.Cultivate for 37 ℃ and made cell attachment in 24 hours, be made into final volume 200ul with the fresh culture that contains the variable concentrations honokiol, 37 ℃ hatch 68 hours after, every hole adds the MTT of 50ul 2mg/ml, continue to cultivate dull and stereotyped centrifugal 1000rpm * 10min, supernatant discarded 4 hours.Add 120ul DMSO dissolving MTT crystallization, dull and stereotyped vibration 10min, Elisa value of reading under the 570nm.Every kind of cell all carries out independent trials three times.

Computational methods: IC ₅₀=suppression ratio is 50% o'clock a drug level

Experimental result: list in table 1, by table 1 data as can be seen, honokiol suppresses all kinds of tumor cell proliferations external, and its cytotoxicity and drug level and action time are proportional, cause the drug level (IC of half death of neoplastic cells ₅₀) approximately be 5-15ug/ml, honokiol is a kind of phenolic compound medicine of natural broad spectrum anticancer.

Various cell line IC under table 1, the honokiol effect ₅₀

Cell line	??IC 50	Initial inoculating cell number	Cell line	??IC 50	Initial inoculating cell number
						????MG-63 ????293 ????NCI-H460 ????HL-60 ????MG-63 ????Bcap-37 ????HCT-116 ????NB4 ????Sw620	??10.31±1.02 ??7.75±1.01 ??9.42±0.53 ??0.51±0.12 ??12.19±1.36 ??14.51±1.25 ??11.99±0.77 ??2.97±0.62 ??13.17±1.43	????5×10 3????7×10 3????4×10 3????1×10 4????5×10 3????5×10 3????2.5×10 3????1×10 4????1×10 4	?ECV304 ?RD ?LS?174T ?Aspc-1 ?SW480 ?HT29 ?Bxpc-3 ?HepG2 ?RKO	??5.38±0.35 ??13.5±1.37 ??12.7±0.98 ??15.4±1.72 ??10.5±1.19 ??10.7±0.81 ??5.23±1.77 ??14.2±0.76 ??11.62±1.29	????2×10 3????5×10 3????1.5×10 4????1×10 4????1×10 4????2.5×10 3????2.5×10 3????2×10 4????1×10 4

Embodiment 2, carry out toxicity inspection with primary cultured cell

Method: former generation human fibroblasts (fibroblast) is taken from fresh children's's foreskin, is incubated among the DMEM; Human lymphocyte (lymphocyte) separates from Cord blood, is incubated among the IMDM that adds the import hyclone, and adds 5ug/ml phytohaemagglutinin (PHA); Human umblilical vein endothelial (HUVEC) is taken from new childbirth umbilical cord, and used culture medium is M199.The equal 5000/hole of all cells kind is in 96 orifice plates, and is adherent back with variable concentrations (0-80ug/ml) honokiol effect 24h, three parallel multiple holes of every concentration.Cytoactive is calculated with trypan blue dyeing.The equal triplicate of all toxicity tests.

Experimental result: as shown in Figure 1, honokiol is less to former normal person fibroblast of being commissioned to train foster and the lymphocytic toxicity of human blood, and safe dose can reach 40ug/ml.But Human umbilical vein endothelial cells shows supernormal low tolerance to honokiol.

Embodiment 3-6 is the Its Mechanisms to the chemical compound honokiol

Preferred RKO and K562 cell are as subjects, and the formation, the hypodiploid peak that detect the dna ladder band respectively exist and caspase family protein expression variation, confirm that its mechanism of action mainly is to pass through inducing apoptosis of tumour cell.

Embodiment 3, dna ladder band detect

Method: RKO or K562 cell are through 5,10, the honokiol effect 48h of 15ug/m, with containing 50mMTris-HCL (PH7.5), 20mM EDTA and the cracking of 1%NP-40 lysis buffer, add 1% SDS and Rnase (5ug/ml) then in supernatant, hatch 2h for 56 ℃, add E.C. 3.4.21.64 (2.5ug/ml) again and hatch 2h for 37 ℃.With ammonium acetate (3.3M) and ethanol (99.5%) dissolution precipitation, add sample-loading buffer.The dna ladder band detects through 1.5% agarose gel electrophoresis, the EB observation of dyeing.

Experimental result: according to honokiol to the Cytotoxic IC of RKO ₅₀, we with 5,10,15ug/ml is as detectable concentration.After above concentration acted on 48 hours respectively, the apoptosis form of RKO cell expression characteristics: cell space became circle, granule and cavity appear in endochylema, the after birth shrinkage, and final whole cell suspension is in culture fluid.Shown in Fig. 2 A, drug treating group cell genomic dna ruptures, and the ladder band of 180bp-200bp multiple occurs through agarose gel electrophoresis.These phenomenon promptings, honokiol may be induced the RKO cell generation apoptosis of In vitro culture.The result of K562 cell shows that equally apoptosis has taken place the K562 cell and ratio is bigger shown in Fig. 2 B.

Embodiment 4, dna content and cell cycle analysis

Method: variable concentrations (5,10 and 15ug/ml) honokiol is handled RKO cell 48h respectively, with not adding honokiol but add equivalent in the RKO cell of the DMSO of 15ug/ml honokiol group volume in contrast, also cultivates 48h, the grouping harvesting.4 ℃ with 70% alcohol fixation cell 15min, PI (1.0ug/ml) dyeing, flow cytometer 488nm wavelength detects cell cycle down to be changed, the result is with ModFIt 3.0 software analysis.Every duplicate samples detects 10,000 cells.The same method of K562 cell is handled.

Experimental result: the results are shown in table 2, the G relevant with dosage appears in the drug treating group ₀/ G ₁The z phase blocks.As shown in Figure 3A, along with honokiol concentration increases, the hypodiploid cell quantity increases; When concentration reached 10ug/ml and 15ug/ml, the DNA fragment obviously increased, and reached 14.10% and 20.31% respectively.These results further specify honokiol and cause phenomena of apoptosis.K562 increases with the honokiol concentration of treatment shown in Fig. 3 B, and the hypodiploid peak increases gradually, and apoptosis cell obviously rises.

Table 2, honokiol are to the period profile of RKO cell and the influence of apoptosis

The % cell cycle
					Handle (ug/ml)	????G0/G1	??S	????G2/M	The % apoptosis
Contrast 5 10 15	????44.25 ????48.03 ????54.04 ????58.94	??45.10 ??42.37 ??40.30 ??39.36	????10.14 ????9.60 ????5.66 ????1.70	????0.51 ????4.51 ????14.10 #????20.31 *

^#P＜0.05; ^*P＜0.01st compared with matched group.

Embodiment 5, Western blot analyze apoptosis molecule caspase family and express

Method: 5 * 10 ⁶The RKO cell is collected the back and is washed twice with ice PBS (PH7.4) with 0.4% trypsinization that contains 0.02%EDTA.Total protein of cell cell pyrolysis liquid { 150mM NaCL, 0.5M Tris-HCL (PH 7.2), 0.25M EDTA (PH 8.0), 1% Triton X-100,5% glycerol, 1.25% SDS} extracting.Get 30ug albumen at 12% SDS-PAGE electrophoresis, 250mA changes film 2h (transfering buffering liquid contains 25mM Tris-HCL (PH8.3), 192mM glycine and 20% methanol) to pvdf membrane.5% defatted milk room temperature sealing 2h adds caspase-3, and-9 one anti-(TBST was by dilutions in 1: 400) are spent the night for 4 ℃.Two anti-(dilutions in 1: 5000) that add the horseradish peroxidase labelling, room temperature effect 1h; Add colour developing liquid after the TBST washing for several times, exposure is developed a film.The K562 cell is a suspension cell, does not need trypsinization, directly collects and handles as stated above.

Experimental result: Caspase family is the important regulating and controlling factor of apoptosis path.Be the apoptosis that causes of explanation honokiol and the dependency of Caspase family, we have detected Caspase-3 and-9 expression variation with Western blot method.Shown in Fig. 4 A, after 48 hours, two kinds of proteic expressions of Caspase obviously increase the RKO cell in the cell, and are proportionate with drug dose at drug effect.Shown in Fig. 4 B, corresponding two kinds of albumen all have remarkable rise.

Effect assessment in the embodiment 6-9 animal body

This part embodiment then mainly analyzes honokiol in the intravital drug distribution parameter of mice, estimates the anti-tumor effect to relevant ascites tumor model and transplanted tumor model mouse.

Embodiment 6, pharmacokinetic

Method: press 7: 3 miscible honokiols of volume ratio with PEG400 (PEG400) and glucose, make into 20mg/ml stock solution.30 the every Mus lumbar injection of Balb/c Mus 250mg/kg honokiols, different intervals is collected blood sample, and every time point is got three Mus as sample, sample treatment reference literature [Proc.Natl.Acad.Sci.1997; 94:2620-2625 and Nucleic Acid Drug Dev.1998; 8:135-139].Blood plasma Chinese medicine concentration is analyzed with high performance liquid chromatography (HPLC), and the detection wavelength is 290nm, the anti-phase C18 post of chromatographic column adopting 5um * 100mm * 2.1mm (i.d), and mobile phase adopts methanol: 0.1% trifluoroacetic acid (volume ratio), flow velocity 0.2ml/min.Pharmacokinetic parameter Modkine software analysis (Biosoft, UK).

Experimental result: as shown in Figure 5, Babl/c mouse peritoneal injection honokiol 250mg/kg, maximum plasma concentration appears at injection back 27.179 ± 6.252 minutes.Blood plasma is eliminated curve and absorbed the one-compartment model description with one-level: half soak time 12.1 ± 2.761 minutes, eliminating the half-life is 5.218 ± 0.461 hours.

The life span of embodiment 7, prolongation lotus RKO ascites tumor nude mice

Method: Balb/c nude mice abdominal cavity inoculation 1 * 10 ⁷The RKO cell is divided into drug treating group and matched group at random.Honokiol is dissolved in PEG400/Tween20 (volume ratio 9: 1).The drug treating group respectively after inoculation 0,2,4,6,8,10 day every Mus irritate stomach 2mg honokiol every day, matched group is irritated stomach equal-volume solvent.Expand and body weight change in the periodic observation abdominal cavity.

Experimental result: the results are shown in table 3, honokiol 200mg/kg irritates stomach and obviously suppresses the formation of RKO ascites tumor, the life span of significant prolongation tumor animal.Abdominal tympanites appears successively behind all control animals (n=5) abdominal cavity inoculation RKO cell, and dead in 22 days.Have only 1 example to die from ascites and take honokiol group (n=5), other 4 routine survival rates were above 40 days.Therefore, honokiol is 80% to RKO one-tenth ascites tumor inhibition total effective rate.

Table 3, honokiol influence the nude mice existence of lotus RKO ascites tumor

Group	Mice with tumor (only)	Mean survival time (my god)	Dead Mus	Mus lives	Survival rate (%)
						Control treatment	????5 ????5	????20.7 ????44.3	????5 ????1	???0 ???4	????0％ ????80％

Embodiment 8, inhibition RKO cause the growth of solid tumor

Method: 5 * 10 ⁶The RKO cell inoculation is subcutaneous in Balb/c nude mice armpit.An about week, animal evenly was divided into four groups: amycin processed group, honokiol processed group, vehicle group and blank group when the tumor body is visible.The each lumbar injection 80mg/kg of honokiol group, vehicle group injection equivalent honokiol solvent (PEG400:dextrose, volume ratio 7: 3).Amycin group injection 5mg/kg amycin (being dissolved in normal saline), matched group injection equal-volume normal saline.All animals every two days injectable drugs or placebo once, and with kind of calliper tumor body size.Tumor body volume calculation adopts formula: volume=∏/6 * (length * wide ²), the minor axis of wide representative.

Experimental result: as shown in Figure 6, blank group and group of solvents mice are in 4 weeks behind inoculation RKO cell, and tumor body volume presents gradual growth, and rate of increase was respectively 1627.6% and 1408.2% in 28 days.The tumor body of drug treating group mice increases slower than corresponding control animals.28 days tumor bodies of honokiol processed group rate of increase is 968.9%, compares difference with group of solvents and has significance (P＜0.05).28 days rate of increase 709.9% of amycin group are compared significant difference with its matched group, but do not have significant difference (P＞0.05) with the honokiol group.The result shows that honokiol can effectively suppress the growth that RKO causes solid tumor in vivo, its anticancer function is suitable with classical anticarcinogen amycin.

The prolongation of embodiment 9, lotus RKO solid tumor nude mice life

Chemotherapeutics removes the growth that suppresses gross tumor volume, and another crucial index is an increase in life span.The compare average life of matched group, group of solvents and amycin group mice of the mice that honokiol is handled prolongs all detected.The result is that the honokiol group is close with amycin group effect, compares with matched group to have significant life prolongation, and is shown in Figure 8 as table 4, do not have significant difference between matched group and the group of solvents.

Table 4, honokiol are to the effect of lotus entity RKO tumor nude mice

Group	The Mus number	Mean survival time (my god)	Processed group/matched group (%)
				Control solvent amycin honokiol	????7 ????6 ????7 ????7	????28.8 ????29.7 ????46.2 ????50.9	100 104.5 160.4 *176.7 *

^*P＜0.05.

Embodiment 10-14 is the chemoprophylaxis Mechanism Study of external honokiol to tumor cell

Embodiment 10, oxidation and removing free radicals

Method:

1 〉. hydroxy radical response system: 2-deoxy-D-ribose (30mmol/L) 170ul is arranged, EDTA (10mmol/L) 40ul, FeSO in the 2ml reactant liquor ₄(2mmol/L) 40ul; Hypoxanthine (2mmol/L) 200ul; Phosphate buffer (0.1mol5/L, PH7.4) 1.5ml; H ₂O ₂(17.6mmol/L) 10ul, reaction begins to add xanthine oxidase (0.4u/ml) 40ul.The mixed liquid of reaction is cultivated 15min at 35 ℃.

2 〉. oxygen free radical reaction system: replace FeSO in the above-mentioned hydroxy radical response system with the phosphate buffer of 0.15mol/L, PH7.4 ₄And H ₂O ₂

3 〉. thiobarbituricacid (TBA) chromogenic reaction: get above-mentioned culture fluid 1ml and add TBA (1%W/V is in 0.05mol/L NaOH) 1ml and glacial acetic acid 1ml, in boiling water bath, heat 30min behind the mixing, A532 is measured in the cooling back on spectrophotometer, with the 1.1-3.3.-tetramethoxy propane as titer, 6 parts of every kind of sample replications.

Experimental result: list in table 5, honokiol can be removed hydroxy radical and oxygen-derived free radicals simultaneously, and general effect is seen than SOD and is eager to excel.The oxidation resistance that the bibliographical information honokiol is arranged is 500～700 times of VE, is used to 9.6 times of (Life Sciences including Phamacology Letters, 1997 that clinical tea polyphenols class has only VE; 61:1961-1971).This conforms to our result of the test, and it is very capable that it removes free radical.

Table 5, honokiol oxidation and removing free radicals effect

	The hydroxy radical response system		The oxygen free radical reaction system
					Scavenger contrast SOD (15u/ml) honokiol (10um)	A532nm 1.017±0.032 0.631±0.027 0.514±0.102	Clearance rate (%)-38% 49.5%	??A532nm ??0.482±0.016 ??0.349±0.009 ??0.311±0.011	Clearance rate (%)-27.6% 35.5%

Annotate: matched group: do not add scavenger (being SOD and honokiol) group; SOD group: superoxide dismutase processed group; Honokiol group: act on above-mentioned two reactions with the 10um honokiol.

Embodiment 11, inhibition telomerase activation

Method: handle HT29 cell 48h with the variable concentrations honokiol, collecting cell, counting get 50 * 10 respectively ², 25 * 10 ², 12.5 * 10 ², 6.25 * 10 ²Individual cell is with normal saline washing 3 times; 4 ℃, the centrifugal 10min of 12krpm abandon supernatant; After precipitation broken up, add pre-cooling lysate 50 μ l, mixing, 4 ℃ of 2h; 4 ℃, the centrifugal 10min of 12krpm; Get cell extract 3 μ l, add 25 μ l TRAP-PCR amplification liquid; Aseptic DEPC water is supplied volume to 50 μ l, and paraffin oil covers, the performing PCR amplification; Amplification condition: 25 ℃ of 30min, 94 ℃ of 5min:94 ℃ of 30sec, 50 ℃ of 30sec, 72 ℃ of 90sec; 30 circulations; 72 ℃ of 10min.Get PCR product 5 μ l, add degeneration liquid 20 μ l mixings, room temperature 10min: add hybridization solution 225 μ l, mixing; Take out 100 μ l bag by in the ELISA Plate of anti-digoxin, room temperature, lucifuge effect 2h; Cleaning mixture is washed 3～5 times, pats dry: add peroxidase working solution 100 μ l/ holes, room temperature, lucifuge 30min; Cleaning mixture is washed 3～5 times, pats dry; Add tmb substrate 100 μ l/ holes, room temperature, lucifuge 30min; Add stop buffer 100 μ l/ holes with cessation reaction; With its absorbance of analysis-e/or determining (A value), calculate with A=A460-A690 at 450nm, 690nm wavelength.Positive sample A value=A460-A690＞0.2.

Experimental result: list in table 6, the HT29 cell is the strain of telomerase high activity cell.When handling with the variable concentrations honokiol, telomerase activation is reduced thereupon, and when reaching 10 μ g/ml, its A value shows as negative sample less than 0.2.

The telomerase activation that table 6, honokiol are handled back HT29 cell changes

	Contrast	????H1	????H5	????H10
					The A value	????3.143	????2.985	????1.113	????0.127

Annotate: contrast to not adding honokiol but add the processed group of equivalent in the H10 DMSO of honokiol volume that group adds; H1 is a 1ug/ml honokiol processed group; H5 is a 5ug/ml honokiol processed group; H10 is a 10ug/ml honokiol processed group; The A value is the relative absorbance of expression telomerase activation.

Embodiment 12, induce the HL60 cell differentiation

Method: adopt the NBT reduction test to detect.Collect 1 * 106 in HL60 cell, add the normal saline that 0.5ml contains 1.34mmol/L NBT (nitro blue tetrazolium) and 200ug/L TPA (myristoyl Buddhist ripple acetate), 37 insulations 1 hour, the centrifugal supernatant of abandoning, the system cell smear, Wright-Giemsa dyeing, oily mirror is observed 200 cells down, calculates the NBT positive percentage.

Experimental result: matched group has 3.9% to be positive, after handling, the 3ug/ml honokiol have 27.3% cell to be positive, and the HL-60 cell that the 6ug/ml honokiol is handled has a large amount of cell deaths, do not have in the dead cell 56.1% cell positive (p＜0.01) is arranged, have utmost point significance.Experimental results show that honokiol has certain ability of inducing the HL-60 cell differentiation, but find that according to our further analysis honokiol can not carry out end Mo differentiation by cell guiding.

Embodiment 13, anti-nitric oxide (NO) effect

Method: the RKO cell is gathered in the crops after the variable concentrations honokiol is handled 24h, extracts total protein with 2 * SDS cell protein extracting solution.Western blot checks (detailed process is referring to embodiment 5).Matched group: do not add honokiol; H5: add the 5ug/ml honokiol and handle; H10: add the 10ug/ml honokiol and handle.

Experimental result: deriving from the arginic NO of L-has detrimental effect to some important biomolecule, and its overexpression takes place with cancer and develops relevant.Rao CV etc. discovers that inducibility NO synzyme (iNOS) may play a major role in the generation of colon cancer, azoxymethane (AOM) can produce to form colon distortion crypts (carcinogenesis, 1999,20 (1)) by inducing iNOS.Shown in Figure 7, the honokiol of 10ug/ml can obviously suppress the expression of iNOS, forms thereby suppressed inductive colon cancer, makes its incidence rate significantly reduce (P＜0.05).

Embodiment 14, selectivity downward modulation Cox-2 express

Method: HT29 (human large intestine cancer cell line) cell is handled 24h results, cell protein extracting solution extracting total protein, row western blot check (specifically referring to embodiment 5) through the variable concentrations honokiol.Matched group: do not add honokiol; H5: add 5ug/ml honokiol processed group; H10: add 10ug/ml honokiol processed group.

Experimental result: Cox-2 is one of Cycloxygenase isomerase, in normal structure, express hardly, and in the sporadic colorectal cancer more than 85%, there is overexpression, with the generation development of colorectal carcinoma substantial connection is arranged, its high expressed can promote cell proliferation, suppress apoptosis, increasing the aggressivity of malignant cell, is the catalyst that intestinal neoplasms takes place.HT29 is the strain of Cox-2 high expressing cell.As shown in Figure 8, after honokiol is handled the HT29 cell, can observe the remarkable downward modulation of the Cox-2 protein expression of HT29 cell.

Animal experiment in the body of embodiment 15, honokiol chemoprophylaxis effect

Material: Kunming kind white mice, male, body weight 18 ± 2.5g, Zhejiang University experimental animal center provides.DMH is available from Sigma company.DMH dissolves with sterile saline, faces the time spent preparation, and the sodium bicarbonate with 5% is transferred PH to 6.5-7.0.

Method: 100 mices are divided into 4 groups at random, 10 of negative control group (being called for short the NS group), weekly subcutaneous injection normal saline, continuous 20 weeks; 30 of positive controls (DMH group) are pressed 20mg/kg dosage subcutaneous injection DMH solution weekly, continuous 20 weeks; 60 of experimental grouies, the administration startup stage of luring cancer is divided into 2 groups by honokiol administration concentration 50mg/kg, 150mg/kg.DMH lures cancer once in a week, and honokiol is once irritated stomach every other day, continuous 20 weeks.Claim the mice body weight weekly once, to adjust dosage.

Put to death for the 20th weekend and respectively organize mice, dissect the mice large intestine, cut off large intestine to ileocecus from the anus stringer that makes progress, observe the form and the distribution position of large bowel neoplasm, counting is also measured the tumor maximum gauge.Clean whole section large intestine sweeps across method with holostrome whole large intestines is fixed in 10% formalin, ethanol dehydration, and dimethylbenzene is transparent, and the routine paraffin wax embedding is standby.Section: conventional section, roasting sheet is preserved standby.Haematoxylin-Yihong dyeing and microscopy: calculate mouse tumor incidence rate, average tumor number, tumour inhibiting rate, carcinogenesis rate, cancer percentage rate.Tumour inhibiting rate=(the average tumor number of the matched group-average tumor number of medication the group)/average tumor number of matched group * 100%.

Experimental result: as shown in Figure 9,

1〉.20 when week honokiol is to the influence of mouse tumor incidence rate: large bowel neoplasm (adenoma and cancer) all takes place in 30 mices of DMH group, and the tumor incidence rate is 100%, and processed group H1, H2 group is respectively 86% and 72%, and certain difference is arranged.The normal saline group does not have tumor.

Honokiol was organized under 30 mice mirrors the influence of mice intestinal cancer incidence rate: DMH and is all found cancer when 2〉.20 was all, and the intestinal cancer incidence rate is 100%, and the H1 group has only 59%, and the H2 group has 43%, and DMH group and processed group have notable difference (P＜0.01); Also variant between H1 and the H2.

3 〉. 358 of intestinal neoplasms take place to 30 mice meters of the influence of the average lotus tumor of mice number: DMH group in honokiol, and 8.95 in tumor takes place average every mice, and H1 and H2 group have only 3.67,3.01 respectively, and the DMH group is significantly higher than all the other each groups (P＜0.01).In addition, cancer is 324 in the DMH group, accounts for 90.5%, and H1 and H2 only 47% and 31%.

Do not find in the whole experiment that honokiol has tangible lethal toxic and side effects to mice, show that honokiol is safely and effectively, see that from result of the test the antimutagenesis of honokiol is sure, a kind of potential chemopreventive agent of can yet be regarded as.

Claims (7)

1, the application of the chemical compound honokiol shown in a kind of formula I in the pharmaceutical composition of preparation antitumor or prophylaxis of tumours.

Formula I

2, application as claimed in claim 1 is characterized in that, the pharmaceutical composition of described antitumor or prophylaxis of tumours is the pharmaceutical composition of treatment or preclude blood system tumor.

3, application as claimed in claim 1 is characterized in that, the pharmaceutical composition of described antitumor or prophylaxis of tumours is the pharmaceutical composition of treatment or prevention digestive tract tumor.

4, application as claimed in claim 1 is characterized in that, the pharmaceutical composition of described antitumor or prophylaxis of tumours comprises formula I chemical compound and at least a pharmaceutically acceptable carrier or the excipient of effective dose.

5, application as claimed in claim 1 is characterized in that, the pharmaceutical composition of described antitumor or prophylaxis of tumours is to use separately.

6, application as claimed in claim 1 is characterized in that, the pharmaceutical composition of described antitumor or prophylaxis of tumours is for uniting use with other antitumor drug.

7, application as claimed in claim 1 is characterized in that, the pharmaceutical composition of described antitumor or prophylaxis of tumours is the preparation of intestinal or non-intestinal combination medicine.

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