CN101103973A - Anti-tumor medicine - Google Patents

Abstract

Disclosed is an anti-tumor drug with nor-icariin as the effective ingredient. Pharmacological test effect proves that the nor-icariin has the activity to inhibit the production of cellular lipid, can reduce the production of cellular fatty acid by inhibiting the activity of FAS, has a function of inhibiting the production and causing withering of microvascular endothelial cells, shows a broad-spectrum anti-tumor function in a cytotoxicity test of thirty-three tumor cell strains of a human body, and shows a function in strongly inhibiting the production of tumor, such as prostate cancer cells LnCAP, lung cancer cells NCI-H23 and colon cancer cells HCT-116 of a human body in a tumor transplantation nude mice model test. A pharmacokinetics test of the nor-icariin in the body of a little mouse proves that the nor-icariin can be kept with a certain concentration, good for better anti-humor effect. The acute toxicity test of the nor-icariin in the body of the mouse shows nontoxicity; therefore the nor-icariin is proved to be a newly-developed broad spectrum anti-humor drug with broad development prospect.

Description

A kind of antitumor drug

Technical field

The present invention relates to antitumor drug, specifically is with the antitumor drug of desmethylicaritin as effective ingredient about a kind of.

Background technology

Herba Epimedii is common in Asia and Mediterranean Region, is that a kind of acidity is viewed and admired herbaceous plant.Herba Epimedii English is called as epimedium, is because it is similar to a kind of plant of Asia Media kingdoms in ancient times (an existing part for Iran).Herba Epimedii is a genus, comprises multiple corresponding plants, and some of them comprise arrow leaf Herba Epimedii, Berberidaceae Herba Epimedii, Flos Caryophylli Herba Epimedii and Herba Epimedii as medicinal.Arrow leaf Herba Epimedii is a kind of important traditional Chinese herbal medicine, is widely used as tonic and antirheumatic in China, Japan and Korea S, and is proved to be and can effectively treats osteoporosis, cardiovascular disease and tumor.

Icariin (ICA) (molecular weight is 676), a kind of flavonol glucosides is the main component of arrow leaf Herba Epimedii.Icariin is a kind of new biological response modifier and short differentiation agent.For further illustrating its reversing tumor malignant phenotype's mechanism, the someone has been ICA and has acted on the highly in vitro tests of transitivity lung carcinoma cell (PG).The result shows that ICA not only can influence the PG cell cycle and shorten the S phase, and can make cAMP level rising in the PG cell, and the cGMP level reduces, and cAMP/cGMP ratio improves.On the other hand, ICA can reduce the adhesion rate of PG cell to laminin substrate, suppresses the invasion and attack and the transfer ability of PG cell.Find that in addition ICA can improve the membrane fluidity of PG cell, increase the antigenic expression of film HLA-ABC.These Notes of Key Datas, ICA may be a kind of cancer therapy drug (Mao Haiting etc., Chinese crude drug, 1999,22 (1): 35-36; Mao Haiting etc., Chinese crude drug, 2000,23 (9): 554-556).Yet in another research, ICA does not show ability (Lin CC et al., Clin Exp Pharmacol Physiol, 2004,31 (1-2): 65-69) that suppress hepatoma cell line and leukaemia system.Therefore, whether ICA has antitumor action and unclear.

Bibliographical information is arranged, ICA is by biotransformation, at least be converted to 3 kinds of metabolites: icarisid II (molecular weight is 514), 3,5,7-trihydroxy-2-(4-methoxy-phenyl)-8-(3-methyl-but-2-enyl)-chromen-4-one (molecular weight is 368) and desmethylicaritin (molecular weight is 354) (Liu J et al., J Pharm Biomed Anal, 2004,36 (2): 365-370; Liu J et al., Pharmazie, 2005,60 (2): 120-125).The research report, desmethylicaritin has faint phytoestrogen sample effect, and this estrogen-like effects is mediated by estrogen receptor.But the desmethylicaritin Antitumor Effects is not appeared in the newspapers.

(Fatty Acid Synthase is the synthetic key enzyme of long-chain fatty acid FAS) to fatty acid synthetase, expresses low in most normal tissue cells.Because normal tissue cell utilizes the fatty acid growth of food intake usually, so FAS does not have critical function.Yet the quick growth of tumor cell mainly utilizes the synthetic fatty acid of cell endogenous, and therefore the FAS gene expression dose increases in the tumor cell of many types, and enzymatic activity strengthens.FAS further improve along with tumor development, and the prognosis of the tumor patient of high expressed FAS is relatively poor at the tumorigenic up-regulated that just begins in early days.And FAS micromolecule chemical inhibitor caerulomycin and C75 etc. can suppress growth of tumour cell and cell death inducing (Pizer ES et al., Cancer Res, 1996,56 (6): 1189-1193; Pizer ESet al., Cancer Res, 1998,58 (20): 4611-4615).Therefore, FAS has been a new oncotherapy molecular target by generally acknowledging now.Identify that new FAS inhibitor causes the interest that people are very big always.The research report is arranged recently, and epigallocatechin gallate (EGCG) in the Folium Camelliae sinensis (EGCG) is by suppressing tumor cell FAS activity, and it is synthetic to suppress cell endogenous fatty acid, causes growth of tumour cell to suppress and apoptosis.Document is reported again, comprises that the various plants chromocor compound of digicitrine, oak Beijing opera ketone, kaempferol all has the effect that suppresses tumor cell FAS.

In addition, document reports clearly that also tumor-blood-vessel growth has crucial effect in tumor growth, invasion and attack and transfer process.Therefore, the blocking-up neonate tumour blood vessel, the approach that cuts off tumor tissues acquisition nutrition has become new antineoplaston strategy, is the research contents of antineoplaston field hot topic always.

Desmethylicaritin also is a kind of flavone compound, and it is suppressed the FAS activity, suppresses the activity that cell fatty acid generates, and suppressing the research of human vascular endothelial growth effect and antitumor action also is a new research trend.

Summary of the invention

The invention provides a kind of with the antitumor drug of desmethylicaritin as effective ingredient.The desmethylicaritin and pharmaceutically suitable carrier and/or the excipient that contain the antitumor effective dose in the antitumor drug.

The inventor is a very effective FAS antagonist by the evidence desmethylicaritin, has the effect that suppresses the human vascular endothelial growth, also can extensively suppress multiple human tumour cell line's growth, the one-step inducing apoptosis of tumor cells of going forward side by side.In 3 kinds of human tumor xenograft models' test, desmethylicaritin is proved to be a kind of effective antitumour agent, and energy strong inhibition people pulmonary carcinoma, colon cancer and prostate gland cancer cell are grown in vivo.Desmethylicaritin has the great potential that is developed to the broad-spectrum anti-tumor new drug.

The preparation of desmethylicaritin, the list of references report (leaf swell etc. journal of Zhejiang university (medicine), 2005,34 (2): 131-136), be summarized as follows: arrow leaf Herba Epimedii is clayed into power, use solvent extraction, extract passes through silica gel column chromatography, use solvent elution, can make the precursor icariin of 3,5,7-trihydroxy-2-(4-methoxy-phenyl)-8-(3-methyl-but-2-enyl)-chromen-4-one and desmethylicaritin.According to the reaction scheme of following signal, icariin is made 3,5,7-trihydroxy-2-(4-methoxy-phenyl)-8-(3-methyl-but-2-enyl)-chromen-4-one by hydrolysis again, further make desmethylicaritin again by demethylation reaction.

The pharmacological testing of desmethylicaritin

1. in vitro tests

A) desmethylicaritin has the activity that suppresses the cytolipin generation

In order to confirm that desmethylicaritin suppresses the activity that cytolipin generates, handled prostate gland cancer cell LnCAP 5 hours, quantitative assay 2-with the desmethylicaritin of variable concentrations ¹⁴The incorporation of the acetate of C labelling in cytolipin.As shown in Figure 1,0.5 μ M desmethylicaritin suppresses the lipid generation greater than 50%.The higher concentration desmethylicaritin reduces cytolipin in the dose dependent mode and generates.Handle after 24 hours and then further reduce.

B) fatty acid generates desmethylicaritin in the cell to reduce by suppressing the FAS activity

In order to confirm that desmethylicaritin is a FAS inhibitor, with the desmethylicaritin pretreatment LnCAP cell protein extract of variable concentrations, quantitative assay 2-then ¹⁴The incorporation of the malonyl CoA of C labelling in fatty acid.As shown in Figure 2,0.5 μ M desmethylicaritin is in external reduction FAS enzymatic activity, and enzymatic activity is less than 50%.Western blot the analysis showed that desmethylicaritin does not influence the FAS protein level.Therefore confirm to suppress that fatty acid generates is the direct result that suppresses the FAS enzymatic activity, rather than express and realize by reducing FAS.

C) desmethylicaritin has the human microvascular endothelial cell (mvec) of inhibition HMVEC growth and causes effect of apoptosis

In order to confirm that desmethylicaritin suppresses the human vascular endothelial growth and causes endothelial cell apoptosis, has carried out the test of growth of vitro inhibition human microvascular endothelial cell (mvec) and tubular structure formation to desmethylicaritin.Result of the test shows that 10 μ M desmethylicaritins can suppress the human microvascular endothelial cell (mvec) growth and cause endothelial cell apoptosis, 8 μ M desmethylicaritins can suppress HMVEC cells in vitro blood vessel tubular structure to form.About its mechanism of action, the inventor thinks, because FAS is the molecular target of desmethylicaritin, desmethylicaritin can make vascular endothelial cell FAS activity be subjected to press down as the FAS inhibitor, fatty acid biosynthesis block, vascular endothelial growth factor receptor II (VEGF2/KDR/FLK1) can not normal expression on cell membrane, so vascular endothelial cell growth factor can not combine with it, cause relevant signal transmission blocking-up, thus inducing endothelial cell growth inhibited and apoptosis.

Desmethylicaritin suppresses the test of human microvascular endothelial cell (mvec) propagation and carries out (Koch AE, et al., Nature, 1995 Aug 10 according to the method for document description; 376 (6540): 517-519).The desmethylicaritin handler capillary endothelium HMVEC of application variable concentrations 72 hours measures cell growth survival condition, is contrast with the cell of handling without desmethylicaritin, calculates the HMVEC inhibitory rate of cell growth.The result as shown in Figure 3,10 μ M desmethylicaritins can suppress HMVEC cell growth fully, cause apoptosis.Because the cell number that this test is handled preceding plantation with desmethylicaritin is the benchmark contrast, so the data of curve below zero base line represent that then the survivaling cell number after desmethylicaritin is handled is lower than the cell number of handling preceding plantation among Fig. 3.

Desmethylicaritin inhibition human microvascular endothelial cell (mvec) forms the test of tubular structure and carries out (Fajardo LF, et al., Lab Invest., 1988 Jun according to the method for document description in external matrigel; 58 (6): 718-724).Use the desmethylicaritin of variable concentrations and handle the HMVEC cell, and, detect the situation that in matrigel, forms tubular structure with the positive contrast of Amebacilin (fumagilin, a kind of angiogenesis inhibitor of affirmation).The microscopically observed result is compared with the negative control excipient as shown in Figure 4, and the tubular structure number that desmethylicaritin is handled back formation significantly reduces.Through counting, statistics, the result as shown in Figure 5, desmethylicaritin can suppress the HMVEC cell and form tubular structure, and has concentration dependent.8 μ M desmethylicaritins can obviously suppress the HMVEC cell and form tubular structure, and its effect is equivalent to 218 μ M Amebacilins, and the EC50 that suppresses tubular structure formation is 3.2 μ M.

D) end user's tumor cell line carries out cell toxicity test

In containing the normal culture medium of variable concentrations desmethylicaritin, cultivate 33 kinds of tumor cell lines, use mtt assay after 3 days and measure the cell survival situation.Result's (table 1) shows that most of human body tumour cells are to the desmethylicaritin sensitivity.Some tumor cell is lower than 10 μ m to the IC50 (half-inhibition concentration) of this chemical compound.And showing that the human breast cancer cell compares with estrogen-dependent, estrogen dependent/non-dependent human breast cancer cell is more responsive to desmethylicaritin.But (MCF10a) is insensitive to desmethylicaritin for the normal human mammary epithelial cell, even if this compound concentrations up to 50 μ m, these cells are well-grown still.In vitro tests proof desmethylicaritin can extensively suppress the human body tumour cell strain, but to the not effect of normal cell strain.

2. the tumour transplatation nude mice model of should choosing carries out the antitumor efficiency assay of desmethylicaritin

Effectively suppress at desmethylicaritin on the basis of kinds of tumor cells in-vitro multiplication, the tumour transplatation nude mice (subcutaneous vaccination) of should choosing is estimated the antitumor action of desmethylicaritin, and the tumor cell that is tried comprises Human Prostate Cancer Cells LnCAP, human lung carcinoma cell NCI-H23 and human colon cancer cell HCT-116.Result's (seeing Fig. 6-8) shows, these 3 kinds of tumor growths of desmethylicaritin strong inhibition, and its TGI value is respectively 12%, 44.7% and 20%.Simultaneously do not find that desmethylicaritin has significant side effects, comprise the variation of body weight.These data show that desmethylicaritin can obviously suppress tumor growth in vivo, are the good candidate compounds of the new antitumor drug of exploitation.

Carried out following test in addition:

Desmethylicaritin is in the intravital pharmacokinetics test of mice

The desmethylicaritin that mouth is raised administration sees Table 2 at 4 intravital pharmacokinetic parameters of mice, and these experimental data explanation desmethylicaritins can keep certain drug level in vivo, help the antineoplastic effect.

Desmethylicaritin is to the acute toxicity test of mice after the administration

Tried to carry out pathological analysis after mice is put to death, the result shows, gives desmethylicaritin with single dose 1000mg/kg and multidose 200mg/kg (Q2D * 15), does not all observe toxicity, and all are tried the mice well-grown, none death.

Effect by above pharmacological testing proves that the desmethylicaritin that derives from natural materials medium-height grass poisoned arrow leaf Herba Epimedii has the activity that suppresses the cytolipin generation; Can be by suppressing the FAS activity to reduce fatty acid generation in the cell; Also have the growth of the human microvascular endothelial cell (mvec) of inhibition and cause effect of apoptosis; To the cell toxicity test of 33 kinds of tumor cell lines, the result shows the broad-spectrum antitumor action; Should choose tumour transplatation nude mice model test, desmethylicaritin demonstrates the effect that Human Prostate Cancer Cells LnCAP, human lung carcinoma cell NCI-H23 or human colon cancer cell HCT-116 is had intensive inhibition tumor growth, these 3 kinds of tumors all belong to malignant solid tumor, do not show significant side effects simultaneously.In addition, the pharmacokinetics test shows that also desmethylicaritin can keep certain drug level in vivo, helps antitumous effect, and desmethylicaritin is not observed toxicity to the acute toxicity test of mice yet, and all are tried the mice well-grown, none death.Therefore desmethylicaritin is a kind of broad-spectrum anti-tumor new drug with very great development prospect, desmethylicaritin can be used as the crude drug of antitumor drug, preferentially as the crude drug of anti-malignant entity tumor medicine, desmethylicaritin also can be made antitumor drug with pharmaceutically suitable carrier and/or excipient.Described pharmaceutically suitable carrier and/or excipient be Captisul for example, corn oil, sodium carboxymethyl cellulose etc.

General oral recommended dose is 750mg/ body surface area m every day ², in continuous three weeks, the week of having a rest is a course of treatment.Every day, accumulated dose divided sooner or later twice, and oral half an hour after the meal, concrete case can be by the doctor according to state of an illness adjustment.

Table 1 desmethylicaritin vitro inhibition growth of human tumor cells

Human tumor cell line	Cell type	Desmethylicaritin IC50 (μ M)
			?LnCAP	Prostate	6.1
?D145	Prostate	9.8
			?PC3	Prostate	10.6
?HCT-116	Colon	6.9
			?Widr	Colon	11.3
?HT29	Colon	16.8
			?LoVo	Colon	10.5
?CCL-225	Colon	8.9
			?CCL-247	Colon	17.6
?NCI-H23	Lung	6.5
			?A549	Lung	10.7
?MDA-MB-231	Mammary gland	6.5
			?MDA-MB-435	Mammary gland	12.7

?AU-565	Mammary gland	8.9
			?BT-549	Mammary gland	9.6
?MCF-7	Mammary gland	24.8
			?Caki-1	Kidney	7.5
?ACHN	Kidney	9.7
			?786-0	Kidney	10.9
?SN12C	Kidney	15.9
			?SKOV3	Ovary	10.4
?IGROV1	Ovary	11.2
			?Mid?PaCa-2	Pancreas	8.6
?U-251	Glioblastoma	7.3
			?SK-MEL-5	Skin	10.8
?G-361	Skin	7.4
			?MCF-10a	The normal breast epithelial cell	＞50
?HepG2	Liver	9.9
			?SGC-7901	Stomach	9.1
?EC109	Esophagus	8.1
			?CNE-2Z	Nasopharynx	7.2
?Raji	Lymphoma	5.3
			?K562	The leukaemia	6.7

Table 2 desmethylicaritin is at the intravital pharmacokinetic parameter of mice

Pharmacokinetic parameter	Mean+/-standard deviation
		?T1/2(min)	355+/-43
?Tmax(min)	62+/-9
		?Cmax(μM)	56+/-5

Description of drawings

Fig. 1 is the lipid generation-desmethylicaritin concentration curve of LnCAP cell after desmethylicaritin is handled.

Fig. 2 is the fatty acid synthetase-desmethylicaritin concentration curve of LnCAP cell after desmethylicaritin is handled.

Fig. 3 is the inhibitory rate of cell growth-desmethylicaritin concentration curve of HMVEC cell after desmethylicaritin is handled.

Fig. 4 is that desmethylicaritin, excipient or Amebacilin suppress the microscopic examination result that the HMVEC cells in vitro forms tubular structure.

Among the figure: 4a negative control excipient

The bent toxin (218 μ M) of 4b cigarette

4c desmethylicaritin (1 μ M)

4d desmethylicaritin (8 μ M)

Fig. 5 handles the back with desmethylicaritin, Amebacilin or negative control excipient to suppress the tubular structure number that the HMVEC cells in vitro forms.

Handled thing numbering among the figure:

1, Amebacilin (218 μ M); 2, Amebacilin (108 μ M);

3, Amebacilin (21.8 μ M); 4, the negative control excipient;

5, desmethylicaritin (0.25 μ M); 6, desmethylicaritin (0.5 μ M);

7, desmethylicaritin (1 μ M); 8, desmethylicaritin (2 μ M);

9, desmethylicaritin (4 μ M); 10, desmethylicaritin (8 μ M).

Fig. 6 is a desmethylicaritin, the gross tumor volume of the human prostata cancer of treatment of control group subcutaneous vaccination (LnCAP) tumor-transplanting number curve day after tomorrow figure.

Fig. 7 is a desmethylicaritin, the gross tumor volume of people's pulmonary carcinoma (NCI-H23) tumor of treatment of control group subcutaneous vaccination-transplanting number curve day after tomorrow figure.

Fig. 8 is a desmethylicaritin, the gross tumor volume of the human colon carcinoma of treatment of control group subcutaneous vaccination (HCT-116) tumor-transplanting number curve day after tomorrow figure.

The specific embodiment

Embodiment 1

The preparation of desmethylicaritin is (referring to leaf swell etc., journal of Zhejiang university (medicine), 2005,34 (2): 131-136).

The arrow leaf Herba Epimedii 2kg that is taken at the collection acquisition of SOUTHERN CHINA mountain area clays into power, and uses 95% ethanol (10L) extraction then.Obtain 250g and concentrate raw material, further extract with 2.5L chloroform, ethyl acetate and n-butanols then.With 8g acetic acid ethyl ester extract and the 10g n-butanols extract silica gel column chromatography of packing into, use CHCl ₃-MeOH-HCOOH (150: 1: 0.5) and CHCl ₃-MeOH (8: 2) is eluting respectively.By this process, can make 300mg 3,5,7-trihydroxy-2-(4-methoxy-phenyl)-8-(3-methyl-but-2-enyl)-chromen-4-one and desmethylicaritin precursor icariin.Then according to the method for following concise and to the point description with the precursor hydrolysis, make 3,5,7-trihydroxy-2-(4-methoxy-phenyl)-8-(3-methyl-but-2-enyl)-chromen-4-one earlier.Solution A: with 80mg precursor ultrasonic dissolution in the 18ml methanol solution; Solution B: 500U (0.433g) cellulase is dissolved in 180ml (0.1mol/L), in the pH5.0 acetate buffer.While stirring solution A is slowly added in the solution B,, be connected glucose and rhamnose on the precursor with abundant hydrolysis then with gained mixture overnight incubation in 37 ℃ shaking bath.Second day ethyl acetate extraction with three parts of equal-volumes (about 200ml), and organic matter layer vacuum drying on Rotary Evaporators got 3,5,7-trihydroxy-2-(4-methoxy-phenyl)-8-(3-methyl-but-2-enyl)-chromen-4-one (150mg).Reuse method as described below makes the 3,5,7-trihydroxy-2-(4-methoxy-phenyl)-8-(3-methyl-but-2-enyl)-chromen-4-one demethylation promptly get desmethylicaritin.Under drying condition, the solution with 20mg 3,5,7-trihydroxy-2-(4-methoxy-phenyl)-8-(3-methyl-but-2-enyl)-chromen-4-one and the fresh distillatory dichloromethane preparation of 10ml joins in the solution of 1ml Boron tribromide and 5ml dichloromethane preparation, 80 ℃ of reactions 1 hour, is transferred to room temperature continuation reaction then and spends the night.Add water (10ml) cessation reaction, to remove the boride that excessive Boron tribromide and hydrolysis generate.With the ethyl acetate extraction of three parts of equal-volumes (about 25ml), and organic matter layer vacuum drying on Rotary Evaporators got desmethylicaritin (130mg).

Embodiment 2

With (2- ¹⁴C) the acetate method of mixing is measured the inhibition activity that desmethylicaritin pair cell lipid generates

Handle the LnCAP cell after 5 hours or 24 hours with the desmethylicaritin of variable concentrations, 2- ¹⁴Acetate (the 57mCi/mmol of C labelling; 2 μ Ci/dish; Amersham Biosciences) adds in the LnCAP cell culture medium.After hatching 4 hours, collecting cell and culture medium, the cell of resuspended centrifugal collection is in 0.8ml PBS.Use Bligh Dyer method (Swinnen J.V.et al., Endocrinology, 1996; 137:4468-4474) extracting lipid is by (the 2-of scintillation counting technique quantitative assay cell lipid ¹⁴C) acetate incorporation.The result who obtains is standardized as sample protein matter content.Lipid generation-desmethylicaritin concentration curve was seen Fig. 1 after desmethylicaritin was handled the LnCAP cell.

Embodiment 3

Desmethylicaritin is to the active test that suppresses of LnCAP cell extract fatty acid synthetase

Utilize LnCAP cell protein extract to measure FAS enzymatic activity (Brusselmans K.et al., JBiol Chem, 2005 Feb 18; 280 (7): 5636-5645).By centrifugal collection LnCAP cell, be resuspended in that (1mM EDTA, 20mM Tris-HCl pH7.5), use BCA method (Pierce) working sample protein content then in the hypotonic buffer liquid.The desmethylicaritin (0.03-30 μ M) of the albumen of equivalent (50 μ g) and variable concentrations place the 2.5ml phosphate buffer (100mM, pH7.0), 37 ℃ of preincubates 30 minutes.Add 20 μ l reactant liquor [2.5mM NADPH, 1.25mM acetyl-coenzyme A, 1.25mM malonyl-coenzyme A, 0.02mM[2-afterwards ¹⁴C] malonyl-coenzyme A (60mCi/mmol; PerkinElmer Life Sciences)], hatched again 15 minutes for 37 ℃, and 1M hydrochloric acid/methyl alcohol mixed liquor that adding 3ml is ice-cold (6: 4, V/V) cessation reaction, and, mix 2-by the scinticounting analysis with petroleum ether extracting fatty acid ¹⁴The malonyl coenzyme A of C.The result as shown in Figure 2.

Embodiment 4

The test of desmethylicaritin inhibition human microvascular endothelial cell (mvec) propagation (referring to Koch AE, etal., Nature, 1995 Aug 10; 376 (6540): 517-519)

HMVEC is available from ATCC (U.S.'s cell, strain library).EBM culture medium with containing 2%FBS (hyclone) is containing 5%CO ₂37 ℃ of incubators in cultivate.The cell that trypsinization converges, counting after the culture fluid washing.In every hole of 96 well culture plates, add 2.5 * 10 ³Individual cell, adhere-wall culture 4 hours.The desmethylicaritin that adds variable concentrations then in the hole, matched group add desmethylicaritin solvent 0.5%DMSO.Continue to cultivate after 72 hours, drug treating group cell and cellular control unit are carried out MTT (tetrazole indigo plant) test, measure cell growth survival condition, calculate the HMVEC inhibitory rate of cell growth.The results are shown in Figure 3.

Embodiment 5

The test of the external formation tubular structure of desmethylicaritin inhibition human microvascular endothelial cell (mvec) (referring to Fajardo LF, et al., Lab Invest, 1988 Jun; 58 (6): 718-724).

Endotheliocyte can form the blood capillary tube chamber spline structure that is surrounded by endotheliocyte in matrigel, carry out quantitative counting then, and this is to measure a kind of method that extracorporeal blood vessel generates.In brief, the matrigel of 90 μ l (10mg/ml) is coated with is layered on 96 well culture plates, in 37 ℃ of incubations 30 minutes, to promote gelling.HMVECs is resuspended in growth medium (1% serum of simplification, 5 units heparin/ml), every hole adds 10,000 cell, use the desmethylicaritin of variable concentrations and handle the HMVEC cell, and with the positive contrast of Amebacilin (fumagilin, a kind of angiogenesis inhibitor of affirmation), making whole volume is 100 μ l.After 18 hours, culture plate is placed the situation of test under microscope at matrigel formation tubular structure, the results are shown in Figure 4.

Embodiment 6

Cell toxicity test (referring to Carmichael J et al., Cancer Research, 1987,47:943-946)

End user's tumor cell line carries out cell toxicity test.Human tumor cell line is available from ATCC (U.S.'s cell, strain library) and NCI (national cancer institute), and the DMEM (the Eagle culture medium of improvement) with containing 10%FBS (hyclone) is containing 5%CO ₂37 ℃ of incubators in cultivate.With the cell that trypsinization converges, counting after the culture fluid washing.In every hole of 96 well culture plates, add 3000～6000 cells, hatched 16 hours or 24 hours.The desmethylicaritin that in the hole, adds variable concentrations then.Continue to cultivate after 72 hours, drug treating group cell and cellular control unit are carried out MTT (tetrazole indigo plant) test.The results are shown in Table 1.

Embodiment 7

The tumor of should choosing xenotransplantation nude mice model carries out the antitumor efficiency assay of desmethylicaritin

(1) set up people's tumor xenotransplantation nude mice model (referring to Harrison SD Jr., et al., J Natl Cancer Inst, 1991,83 (20): 1509)

(T lymphocyte function defective, nu/nu) male, 6 ages in week are available from Chinese Academy of Sciences's Shanghai Experimental Animal Center, in the feeding and management of SPF level Animal Lab. for the BALB/c nude mouse.With 5 * 10 ⁶Individual LnCaP, NCI-H23, HCT-116 cell be suspended in respectively 0.2ml HBSS (Hanks ' balanced salt solution) or matrigel (50: 50, V/V) in, the flank portion zone of subcutaneous vaccination to 36 mice.When tumor average diameter reached 7～8mm, therefrom selecting 16 tumor sizes was 100～200mm ³Mice, be divided into desmethylicaritin processed group (8) that gives 15%Captisol (U.S. CyDex company exploitation a kind of carrier) preparation and the matched group that only gives carrier (15%Captisol).For guaranteeing that desmethylicaritin processed group and matched group gross tumor volume when handling beginning distributes about equally, is divided into three classes with mice: gross tumor volume little (length＜4mm), gross tumor volume medium (length 4～8mm), the big (length＞8mm) of gross tumor volume.

(2) administration of nude mouse tumour transplatation model

In the oral administration test, desmethylicaritin is dissolved among the 15%Captisol.Filling every day is raised and is given single dose 200 μ l medicinal liquids (with the 15%Captisol preparation, containing the 5mg desmethylicaritin), and continuous 5 days, had a rest 2 days, so continued for 3 weeks; Matched group is by same method afford carrier (Captisol).Mice is raised separately, allows free choice feeding.

(3) Graft Versus Tumor evaluation

Along two vertical direction tumor was measured in per 3～4 days.Gross tumor volume calculates according to following formula:

V＝(a ²×b)/2

Wherein a is tumor width (than a minor diameter), and b is length (than a major diameter).The ratio of gross tumor volume when the relative tumour volume of each tumor (RTV) is defined as the gross tumor volume of some preset time and handles beginning.Each processed group is all calculated average RTV and SE.Calculate tumor growth inhibiting value (TGI) according to following formula, judge anti-tumor activity:

TGI(％)＝T/C×100％

Wherein T is the meansigma methods of processed group test endpoint (4 week) RTV, and C is the meansigma methods of matched group test endpoint RTV.Adopt the minimum anti-tumor activity standard (TGI≤42%) of national cancer institute.During off-test,, carry out histological observation with tumor resection and fixing in formaldehyde.Result of the test is seen Fig. 6-Fig. 8.

Embodiment 8 3,5,7-trihydroxy-2-(4-methoxy-phenyl)-8-(3-methyl-but-2-enyl)-chromen-4-ones are in the intravital pharmacokinetics test of mice

Get 48 week BALB/c mouse in age (2 male and 2 female), single dose 100mg/kg give 3,5,7-trihydroxy-2-(4-methoxy-phenyl)-8-(3-methyl-but-2-enyl)-chromen-4-one.Respectively at after the administration 0.5,1,3,6,12,24,48,72 hour, get blood and make blood plasma through vena ophthalmica, to measure the plasma concentration of 3,5,7-trihydroxy-2-(4-methoxy-phenyl)-8-(3-methyl-but-2-enyl)-chromen-4-one.The 3,5,7-trihydroxy-2-(4-methoxy-phenyl)-8-(3-methyl-but-2-enyl)-chromen-4-one that mouth is raised administration sees Table 2 at 4 intravital pharmacokinetic parameters of mice, and these experimental data explanation 3,5,7-trihydroxy-2-(4-methoxy-phenyl)-8-(3-methyl-but-2-enyl)-chromen-4-ones can keep certain drug level in vivo.

Desmethylicaritin is to the acute toxicity test of mice after embodiment 9 administrations

Get the BALB/c mouse in two group of 8 age in week, every group 10 (5 male and 5 female), give desmethylicaritin (with corn oil or carrier preparation) with single dose 1000mg/kg and multidose 200mg/kg (Q2D * 15) respectively, observed for 1 week and 4 weeks then respectively.Weighed in per two days.After the off-test, will be tried mice and be put to death, be carried out pathological analysis.The result shows, gives 3,5,7-trihydroxy-2-(4-methoxy-phenyl)-8-(3-methyl-but-2-enyl)-chromen-4-one with single dose 1000mg/kg and multidose 200mg/kg (Q2D * 15), does not all observe toxicity, and all are tried the mice well-grown, none death.

Claims (2)

1. an antitumor drug is characterized in that this medicine is as effective ingredient with desmethylicaritin.

2. antitumor drug as claimed in claim 1 is characterized in that containing in this medicine desmethylicaritin and the pharmaceutically suitable carrier and/or the excipient of antitumor effective dose.

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