CN1304007C - Application of acetyl boswellic acid in preparing antitumor agent - Google Patents

Abstract

The present invention relates to a new application of acetyl boswellic acid (BC-4) which is extracted from traditional Chinese medicine Boswellia carterii Birdw. and used as an antitumor drug, that is to say, the secondary use of the medicine. The present invention discovers that BC-4 has the new action of resisting the proliferation of tumor cells, inducing the apoptosis of malignant cells, suppressing the proliferation of human umbilical vein vascular endothelial cells, the growth of animal grafted tumors, and the metastasis of malignant tumors, etc. In vivo and in vitro research indicates that BC-4 has low toxicity, and is an excellent antitumor drug and tumor metastasis inhibitor.

Description

The application of acetyl boswellic acid in preparation neoplasm metastasis inhibitor

Technical field

The present invention relates to the pentacyclic triterpene constituents that extracts in the Chinese medicine Olibanum, the new purposes of acetyl boswellic acid (BC-4) in the preparation antitumor agent is used promptly used the second time of medicine.

Background technology

Cancer serious threat human life, the combination of operation, radiotherapy and chemotherapy has successfully improved the cure rate of multiple malignant tumor, has reduced the mortality rate of tumour patient.But recurrence after the Invasion and Metastasis of tumor and the treatment that causes thus, the toxicity that cancer therapy drug is stronger, the multiple factors such as drug resistance of cancerous cell still limit the therapeutic effect of malignant tumor.The Invasion and Metastasis behavior is the most essential characteristic of malignant tumor, also is the fatefulue the very crux of malignant tumor.Prevent that with chemicals the Invasion and Metastasis of cancer in situ from being to reduce most economical, the most effective means of tumor mortality rate.

Effects such as Chinese medicine Olibanum (Boswellia carterii Birdw.) tool promoting blood circulation and stopping pain, removing toxic substances and promoting subsidence of swelling.Acetyl boswellic acid (BC-4) is the pentacyclic triterpene constituents that extracts from the Chinese medicine Olibanum, and molecular weight is 498.3, and colourless acicular crystal is made up of by 1: 1 chemical compound α-BC-4 and β-BC-4, and its chemical constitution is as follows:

In research in the past, pay attention to the discussion of antiinflammatory action, and think its non-steroidal anti-inflammatory activity with its inhibition 5-lipoxygenase (5-LO) thus active synthetic relevant (the Safayhi H that suppresses leukotriene, Mack T, Sabieraj J, et al.J Pharmacol Exp Ther, 1992,261:1143-1146.).In to the BC-4 screening active ingredients, find that it has stronger differentiation-inducing action (Jing YK to HL-60 leukaemia, Xia LJ and Rui H.Chinese Medical Sciences J, 1992,7:12.), and when daidzein and BC-4 use in conjunction, the inhibitory action and the differentiation-inducing action of on cell proliferation obviously strengthen (Jing Yongkui, Han Rui. Acta Pharmaceutica Sinica, 1993,28:11.).Early stage discovers that it also is a topoisomerase enzyme inhibitor, and the effect of this respect has been disclosed in the United States Patent (USP) 5064823, and the open date is on November 12nd, 1991; And among the Chinese patent CN1041204C, date of declaration is December in 1998 16 days.In recent years, the boswellic acid anti-tumor activity has caused extensive attention.Result of study shows during the BC-4 low concentration multiple myelocytic leukemia cell to be had induction of differentiation widely, then can induced apoptosis in leukemia cell lines during high concentration (Jing Y, Nakajo S, Xia L, et al.Leuk Res, 1999,23:43-50.).

Summary of the invention

An object of the present invention is to provide the application of acetyl boswellic acid (hereinafter to be referred as BC-4) in the preparation antitumor drug.

Another object of the present invention provides the application of acetyl boswellic acid (hereinafter to be referred as BC-4) in the neoplasm metastasis inhibitor.

1. the structure of preparation method and chemical compound

Crude drug Olibanum pearl with 95% ethanol percolation, must be infiltrated and filters; Decompression and solvent recovery gets ethanol extract.The ethanol extract that obtains is dissolved in the petroleum ether-ethyl acetate mixed solvent, add sodium carbonate liquor and transfer to pH 10, petroleum ether-ethyl acetate mixed extractant solvent three times, water reuse 4N hydrochloric acid transfers to pH3, petroleum ether-ethyl acetate mixed extractant solvent three times, and organic facies washes with water twice, anhydrous sodium sulfate dehydration, filter, be concentrated into driedly, add methanol and concentrate foaming and obtain Olibanum total acid foaming thing.Then the Olibanum total acid is carried out silica gel column chromatography, the petroleum ether-ethyl acetate gradient elution obtains acetyl boswellic acid α, beta isomer crude mixture, carries out recrystallization again, obtains acetyl boswellic acid α, beta isomer mixture (1: 1) crystallization.Its molecular weight is 498.3, colourless fine needle crystal, and its chemical constitution is as follows:

HPLC detects the retention time that contains BC-4-α and BC-4-β and was respectively 11.70 minutes and 12.79 minutes (Fig. 1).

2. pharmacological action

1) effect of BC-4 in the prior art

In the prior art, BC-4 is a topoisomerase enzyme inhibitor and cell-differentiation inducers, can suppress topoisomerase I (Fig. 2) and topoisomerase II (Fig. 3) activity, human promyelocytic leukemia HL-60 cell is after BC-4 induces, to mature cell differentiation (Lu.Y andHan R.Acta Academia Medica Sinica, 1986,8 (37): 211-214).With BC-4 act on the HL-60 cell after 5 days the NBT reducing power of cell see Table 1.

Table 1:BC-4 is to HL-60 human leukemia cell's differentiation-inducing action

Concentration (μ g/ml)	NBT reducing power (%)
		1 8 10	2.5 27.0 51.0

2) new pharmacologic application of the present invention

The effect of a.BC-4 anti-tumour cell proliferative

The exponential phase cell is inoculated in 96 orifice plates by 1200/hole.Abandon culture medium next day, change the fresh culture that contains variable concentrations medicine and coordinative solvent contrast, every group of parallel 6 holes, continue cultivation after 4 days in 37 ℃, abandon supernatant, every hole adds the serum-free medium of the freshly prepared 0.4mg/mlMTT of containing of 100 μ l, continues to cultivate 4hrs, abandon culture supernatant, add 100 μ l DMSO dissolving MTT first hairpin precipitation,, on Bio-Rad 550 type microplate reader, measure 550nm place optical density value with the microoscillator mixing that vibrates, be calculated as follows the tumor cell survival rate, and calculate IC ₅₀

Studies show that, BC-4 can suppress the growth (comprising KB people's epithelial cancer cells, A2780 Proliferation of Human Ovarian Cell, MCF-7 human breast cancer cell, SHG-44 human glioma cell, HCT-8 human colon cancer cell and A549 people's non-small cell lung cancer cell) of the malignant cell of different tissue sources, the results are shown in Table 2, its IC ₅₀From 5.73 μ M to 16.44 μ M.

Table 2:BC-4 is to the growth inhibited effect of malignant cell

Cell line	IC 50μg/ml(μM)
		KB (HEP's cancer) A2780 (HOC) MCF-7 (human breast carcinoma) SHG-44 (human glioma) HCT-8 (human colon carcinoma) A549 (Non-small cell lung carcinoma) CHL (Chinese hamster pneumonocyte)	5.356(10.71) 6.159(12.32) 2.866(5.73) 8.219(16.44) 6.695(13.39) 6.159(12.32) ＞10(＞20)

B.BC-4 induces the malignant cell apoptosis

Detect the apoptosis-induced effect of BC-4 by following multiple technologies to malignant cell:

On coverslip, evenly coat poly-D-lysine, place six well culture plates, behind the dried 3-5min, add 70% ethanol sterilization 30min, inhale and remove ethanol, wash 2 times with PBS.With 5 * 10 ⁴Cell inoculation is in culture hole, after the overnight incubation, add the contrast of two fun gi polysaccharides and solvent, behind the effect certain hour, PBS washes once, takes out coverslip, be fixed in cell movement observation ward (preparing the same), fill apoptosis and detect liquid (containing 1 μ lAnnexin V-EGFP and 1 μ l iodate pyridine in the 1ml binding buffer liquid) in cell, behind the lucifuge effect 5min, observation of cell apoptosis under laser confocal microscope (Leica TCS 4D type) is inverted under the room temperature.

Cell is washed once with PBS after digesting, and gets the cell precipitation smear, and is air-dry rapidly, and with dye liquor Wright dyeing 1min, reuse dye liquor Giemsa dyes 10min, and distilled water flushing is air-dry, puts observation of cell apoptosis under the oily mirror.

Cell preparation becomes 1 * 10 ⁷/ ml cell suspension is got 95 μ l cell suspension and is added 5 μ l acridine orange solution (0.1mg/ml PBS, pH 6.8), and point sample covers observation of cell apoptosis under the rearmounted fluorescence microscope of coverslip behind the mixing on microscope slide.

Cell is with after the PBS washing, and with 2.5% glutaraldehyde fixedly more than the 1h, with 1% osmic acid (Osmic acid .) fixing 1h again, the centrifugal fixative of abandoning is washed 2 times with distilling after PBS washes 2 times.Dye 1h in advance with 2% acetic acid uranium, the centrifugal dye liquor of abandoning is with distillation washing 2 times.Use the 35%-50%-75%-90% dehydration of alcohol successively, each 5min soaks with epon 812, embedding, and after the polymerization, piece of tissue ultrathin section, lead citrate dyeing, observation of cell apoptosis under Hitachi800 type Electronic Speculum.

Scrape cell with the cell sleaker, PBS washes once, adds 50 μ l cell pyrolysis liquid (50mMTris-HCl, pH8.0,10mM EDTA, 0.5%SDS, 0.5mg/ml E.C. 3.4.21.64), put 40min in the ice, take out, add RNA enzyme 0.15mg/ml, 37 ℃ of incubations are 1h at least, adds E.C. 3.4.21.64 1mg/ml again, 37 ℃ of incubations are 1h at least, add sample-loading buffer after, with sample in 24V, 1.7% agarose gel electrophoresis, the DNA damage that the observation of cell apoptosis of taking pictures under the uviol lamp of EB dyeing back causes.

Collecting cell is washed once with fresh culture, cell precipitation is in the dark added binding buffer liquid 500 μ l (10mM HEPES, pH7.4,140mMNaCl, the 2.5mM CaCl that contains 0.5 μ lAnnexin V-EGFP ₂), behind the room temperature effect 10min, adding equal-volume 4% formalin in the sample cell, room temperature is fixedly behind the 15min, and 12.0 * 10 ³The centrifugal 1min of rpm, PBS washes once, adds 100 μ g/ml RNA enzyme A, 100 μ l in the cell precipitation, and 37 ℃ of effect 30min add the binding buffer liquid 400 μ l that contain 0.5 μ l iodate pyridine, and flow cytometer detects apoptosis behind the room temperature effect 10min.Set up blank simultaneously, only add 500 μ l binding buffer liquid in the sample; Annexin V-EGFP contrast only adds the binding buffer liquid 500 μ l that contain 0.5 μ l Annexin V-EGFP in the sample.Flow cytometer (Becton Dickinson FACSCalibur type) detects the apoptosis signal, and (Ex=488nm Em=530nm) uses FITC signal detector (FL1), and the FL2 signal detector is used in the dyeing of iodate pyridine in Annexin V-EGFP dyeing.

Above testing result is as follows:

I. human bladder cancer cell

Acted on the T24 human bladder cancer cell 8 hours with BC-475 μ M, promptly visible apoptotic generation shows as Phosphatidylserine and is turned to the skin (Fig. 4) of phospholipid bilayer film by the internal layer of phospholipid bilayer film, and the dna ladder band occurs.Flow cytometer detects and shows that act on the T24 cell after 24 hours with BC-475 μ M, percentage of cell apoptosis can reach 77.2% (Fig. 5).

Ii. human breast cancer cell

Result of study shows, the apoptosis that the MDA/HER-2 of MDA-MB-468, transfection pCMV-HER2 plasmid and the negative control cell MDA/NEO that imports blank plasmid thereof cause BC-4 is responsive (Fig. 6) all.

Iii.IFN _γApoptosis sensitization to the MV3 human melanoma cell

IFN _γ500U/ml acted on the MV3 cell after 72 hours, can reduce the expression of HMW-MAA, surface antigens such as α v, TAA, through IFN _γ72 hours MV3 cell of 500U/ml effect is but comparatively responsive to the apoptotic signal that BC-4 causes.Act on IFN with BC-4 _γThe MV3 cell of handling can cause 49.2% apoptosis (Fig. 7) after 8 hours.

Iv. human fibrosarcoma cell

BC-4 is under low concentration, but dose dependent ground suppresses human fibrosarcoma cell HT-1080 emiocytosis metalloproteases MMP-2 and MMP-9 (Fig. 8).Acted on the HT-1080 cell 24 hours with the above concentration BC-4 of 50 μ M, obvious cell death inducing, show as: cellular morphology is complete, but cyto-chromatin pyknosis, nucleus cracking are divided into fragment.Apoptotic body (Fig. 9) all appears in Wright-Giemsa dyeing, acridine orange fluorescence staining and electron microscopic observation in the HT-1080 nucleus, DNA analysis detects dna ladder band (Figure 10).The HT-1080 apoptosis that BC-4 causes has dosage and time dependence, and when with BC-450 μ M effect after 36 hours, the apoptotic cell percentage rate increases to 59.3% by 0.42% of matched group; When with BC-475 μ M effect after 24 hours, the apoptotic cell percentage rate then increases to 76.5% (Figure 11).

C.BC-4 is to the effect of human umbilical vein endothelial cell

Utilize [ ³H]-TdR mix DNA, [ ³H]-UdR mix RNA and [ ³H]-Leu mixes protein and observes the influence of BC-4 to human umbilical vein endothelial cell propagation.The trophophase cell of taking the logarithm, the digestion counting makes 1 * 10 ⁵/ ml cell suspension is inoculated in 96 porocyte culture plates, 37 ℃ of 5%CO by every hole 200 μ l ₂Cultivate 12hrs in the incubator, discard culture medium, change the culture medium that contains variable concentrations medicinal liquid and solvent control, every group of parallel 4 holes are in 37 ℃ of 5%CO ₂Continue to cultivate 24hrs in the incubator.4hrs before termination, every hole adds the precursor 1 μ Ci of radiosiotope labelling.Discard and contain isotopic culture medium, every hole adds 200 μ l pancreatin in 37 ℃ of digestion 20min, be collected in every porocyte on the glass fibre membrane respectively with the automated cell catcher, distilled water washes 20 times repeatedly, take out film and place 80 ℃ of infrared baking boxs to do roasting 15min, check the number and cut each diaphragm, place 4ml scintillator (0.4%PPO is housed, the xylene solution of 0.01%POPOP) liquid dodges in the cup, and the dpm value is measured with Beckman LS-9800 type liquid scintillation counter after placing 15min in the dark place.

Act on former being commissioned to train with BC-475 μ M and supported human umbilical vein endothelial cell 24 hours, do not cause apoptosis, show that BC-4 is to the somatic toxicity of normal person less (Figure 12).The EC-304 Human umbilical vein endothelial cells that acts on the BC-4 of variable concentrations can suppress to some extent [ ³H]-TdR, [ ³H]-UdR, [ ³H]-Leu mixes cell, shows that DNA, RNA and protein synthesis have inhibitory action (table 3) in the pair cell.

Table 3:BC-4 closes EC-304 human umbilical vein endothelial cell DNA, RNA and protein

The influence that becomes

Group	[ 3H]-TdR mixes DNA dpm	[ 3H]-UdR mixes RNA dpm	[ 3H]-Leu mixes protein d pm
				Contrast BC-4 5M 10M 20M 40M	19734.50±2431.74 15446.03±5290.44 9480.58±3220.91 ** 3513.33±887.52 ** 2466.33±1486.20 **	5743.67±782.79 4729.00±688.91 4845.00±517.95 2437.33±185.04 ** 553.67±150.60 **	3586.33±684.36 2949.67±360.15 2436.67±991.04 2340.67±497.46 1827.67±78.64 **

Compare with matched group ^*, p＜0.01

D.BC-4 is to the inhibitory action of animal transplanting tumor

The selection tumor growth is good, and general body state is lotus tumor BDF1 mice preferably, and the cervical vertebra dislocation is put to death.Fixing back iodine tincture alcohol disinfecting, aseptic condition takes out the tumor piece down, weighs, grinding, homogenate, filtration, with normal saline dilution in 1: 3, count the oncocyte number in every milliliter of serosity after the homogenate, inoculates 0.2ml tumor liquid (2 * 10 for every mice oxter ⁶Cell).Next day is with animal random packet and administration.In inoculation 24hrs pneumoretroperitoneum drug administration by injection, establish 25,50,100mg/kg three dosage groups, establish cyclophosphamide positive controls and blank group simultaneously, successive administration is after 10 days, and the cervical vertebra dislocation is put to death, and takes by weighing body weight respectively, tumor is heavy.Calculate inhibition rate of tumor growth (%), and the result is carried out statistical procedures.

The animal transplanting tumor scale-model investigation show BC-4 with 25, growth has certain inhibitory action to 100mg/kg dosage intraperitoneal injection to the S180 murine sarcoma, its suppression ratio is respectively 40.9% and 37.6%.Result of study shows that also BC-4 is lower to mouse toxicity, does not cause obviously the alleviating of mice body weight (table 4).

Table 4:BC-4 is to the influence of S180 murine sarcoma growth

Group	Number of animals		Body weight		Tumor is heavy	Suppression ratio (%)
							Beginning	Finish	Beginning	Finish
	Contrast endoxan 100mg/kg * 1 day BC-4 25mg/kg * 10 day 50mg/kg * 10 days 100mg/kg * 10 day	10 10 10 10 11	10 10 10 10 11	18.2±1.23 18.4±1.07 18.9±0.74 18.8±0.63 18.9±0.70							27.5±1.5 1 21.6±3.5 7 24.6±3.1 0 26.0±1.9 4 26.5±2.3 0	3.40±1.36 0.3±0.23 ** 2.00±0.70 ** 2.50±1.03 2.10±1.21 *	90.7 40.9 25.1 37.6

Compare with matched group ^*P＜0.05, ^*P＜0.01

The experimental treatment that e.BC-4 shifts B16BL6 murine melanoma spontaneous lung

Collect the B16BL6 mouse melanin tumor cell of exponential phase, be resuspended in the sterile saline, cell concentration is adjusted into 1 * 10 ⁷/ ml.In C57BL/6 mouse hind leg palmula subcutaneous injection 50 μ l oncocyte liquid, next day is with animal random packet and 3 weeks of successive administration.If BC-450,100mg/kg group, paclitaxel 2.5,5mg/kg group, BC-4 (50mg/kg) associating paclitaxel (2.5mg/kg) group are established negative control group, every group of 15 mices simultaneously.Inoculation tumor liquid was weighed after 3 weeks, used the etherization mice, and the ligation femoral artery is clipped lotus tumor lower limb, separates the tumor piece, weighs, and calculates inhibition rate of tumor growth (%).Drug withdrawal one day, in 3 weeks of successive administration, weighed in one week in the operation back, after 24 hours, the cervical vertebra dislocation is put to death, and weighs in the last administration, lung is got in dissection, uses that Bouin ' s is liquid-solid to be decided lung tissue and weigh, (10 times) counting lung surface tumours tuberosity number and measure diameter under the Olympus anatomic microscope, size fractionation by metastasis: I level, diameter＜0.5mm, II level, 0.5mm≤diameter＜1mm, the III level, 1mm≤diameter≤2mm, IV level diameter＞2mm.Total number=I level (tuberosity number) * 1+II level * 2+III level * 3+IV level * 4 of shifting.The result is carried out Mann-Whitney U check, and count utilization and simplify exact propability and carry out statistical test routine number and jump routine not shift to take place, adopt ChouShi merged index (CI) method to calculate the CI value to the curative effect after BC-4 and the paclitaxel use in conjunction, it when CI=1 summation action, when CI＜1 is synergism, is antagonism when CI＞1.

BC-4 and paclitaxel drug combination group reach 43.9% to the growth inhibition ratio of the former blackout melanoma of mice, and BC-4 50,100mg/kg group suppression ratio is respectively 26.3,33.3%, and paclitaxel does not have obvious inhibitory action (table 5) to former blackout melanoma.BC-4 100mg/kg group mouse melanin tumor metastasis lung weight significantly descends, and suppression ratio reaches 45.9% (table 6).Compare with model group, BC-4 administration group and paclitaxel administration group murine melanoma lung metastasis number all significantly reduce (table 7).

Therefore, according to one embodiment of the invention, the invention provides the application of acetyl boswellic acid in preparation neoplasm metastasis inhibitor, wherein said tumor does not comprise leukemia, and wherein said acetyl boswellic acid is made of α-acetyl boswellic acid and the β-acetyl boswellic acid proportion of composing according to 1: 1.

In another embodiment of the invention,, it is characterized in that described tumor comprises bladder cancer, breast carcinoma, melanoma and fibrosarcoma for aforesaid application.

Table 5:BC-4 is to the influence of B16BL6 murine melanoma growth in situ

Group	Number of animals		Body weight		Tumor heavy (g)	Suppression ratio (%)
							Beginning	Finish	Beginning	Finish
	Contrast BC-4 50mg/kg 100mg/kg Taxol 2.5mg/kg 5mg/kg BC-4 (50mg/kg)+Taxol (2.5 mg/kg)	15 15 15 15 15 15	15 15 15 15 15 15	19.5±0.67 19.7±0.82 20.1±0.85 19.2±1.01 19.3±0.88 19.5±1.12							19.6±1.05 19.8±1.05 19.8±1.13 19.0±1.05 19.3±0.86 19.6±1.05	0.57±0.21 0.42±0.10 * 0.38±0.14 * 0.51±0.13 0.46±0.11 0.32±0.18 **	26.3 33.3 10.5 19.3 43.9

Compare with matched group ^*P＜0.05, ^*P＜0.01

Table 6:BC-4 is to the influence of B16BL6 murine melanoma spontaneous lung transfer weight

Group	The animal number		Body weight		Lung weight (g)	Suppression ratio (%)
							Beginning	Finish	Beginning	Finish
	Contrast BC-4 50mg/kg 100mg/kg Taxol 2.5mg/kg 5mg/kg BC-4 (50mg/kg)+Taxol (2.5 mg/kg)	14 15 10 15 13 12	12 15 10 15 13 12	17.6±1.22 18.5±0.89 17.0±1.47 17.4±1.33 18.0±1.08 19.3±0.81							19.2±1.08 19.2±0.95 18.9±1.73 18.9±1.10 19.3±1.11 19.3±1.38	0.37±0.28 0.23±0.06 0.20±0.02 * 0.23±0.11 0.33±0.16 0.23±0.12	37.8 45.9 37.8 10.8 37.8

Compare with matched group ^*P＜0.05, ^*P＜0.01

Table 7:BC-4 is to the influence of B16BL6 murine melanoma spontaneous lung metastatic nodules

Group	Number of animals		Every Mus lung surface metastasis tuberosity number
				Shift	Do not shift
	Contrast BC-4 50mg/kg 100mg/kg Taxol 2.5mg/kg 5mg/kg BC-4 (50mg/kg)+Taxol (2.5 mg/kg)	12/12 14/15 8/10 14/15 9/13 7/12				0/12 1/15 2/10 1/15 4/13 5 */12	157，66，114，112，60，76，30，11，12，8，27，29 7，19，4，6，0，3，30，70，23，5，10，21，2，24，46 (**)0，8，3，5，34，0，44，3，29，3 (**) 31，1，6，26，0，1，9，69，16，17，4，13，15，4，34 (**)9，50，16，0，0，11，0，0，48，63，117，5，42 (*)0，0，2，0，0，0，5，1，8，36，35，1 (**)

Compare with matched group ^*P＜0.05, ^*P＜0.01

Advantage of the present invention and effect are anti-tumour cell proliferative, the propagation of inducing malignant cell apoptosis, inhibition human umbilical vein endothelial cell, the growth that suppresses animal transplanting tumor of having found BC-4, the new roles such as transfer that suppress malignant tumor.

According to the present invention, that the medication of described medicine has is for example oral, in Sublingual, intravenous, subcutaneous, transdermal, intramuscular, Intradermal, the sheath, epidural, ophthalmic, intracranial, suction, rectum, vagina administration etc.But can with cream, tablet, capsule, pill dispersion powder, granule, suppository, syrup, elixir, lozenge, injection solution, non-aqueous solution, suspension or emulsion, etc. form administration.Active component can be mixed with nontoxic pharmaceutically suitable carrier, such carrier comprises that glucose, lactose, arabic gum, gelatin, mannitol, starch are sticking, magnesium trisilicate, Pulvis Talci, corn starch, keratin, colloidal silica, potato starch, carbamide, glucosan etc.

For oral administration, contain for example calcium carbonate, lactose, calcium phosphate, sodium phosphate etc. and various granulation agent and disintegrating agent for example tablet, capsule, dragee, oil suspension dispersion powder or granule, emulsion, hard or soft capsule or syrup, elixir and the lozenge of tragcanth, corn starch, gelatin, arabic gum etc. of corn starch, potato starch, alginic acid etc. and binding agent for example of various excipient but can use.Can also add lubricant for example triethylamine magnesium stearate, triethylamine stearic acid, Talcum etc.Orally administered preparation can make according to any method of useful in preparing drug formulations known in the art, and such preparation can contain one or more and be selected from following adjuvant so that agreeable to the taste pharmaceutical preparation to be provided: sweeting agent is sucrose, lactose, glucide etc. for example, correctives is Herba Menthae, Ilicis Purpureae wet goods, coloring agent and antiseptic for example.Orally administered preparation can also contain suitable carriers, comprises emulsion, solution, suspension, syrup etc., and optional additive for example wetting agent, emulsifying agent and suspending agent, sweeting agent, correctives and the spice etc. of containing.Tablet can be a coating not, perhaps can be by known technology with its coating postponing its disintegrate and absorption in gastrointestinal tract, and provide continuous action in a long time thus.

For oral liquid, suitable carriers comprises solution, suspension, syrup etc., and optional additive for example wetting agent, emulsifying agent and suspending agent, sweeting agent, correctives and the spice etc. of containing.

For the parenteral administration liquid preparation, suitable carriers comprises non-aqueous solution, suspension or emulsion.For parenteral administration, the solution of implementing compound used therefor of the present invention also can comprise non-aqueous solution, suspension, emulsion etc.The example of nonaqueous solvent or carrier has for example olive oil and Semen Maydis oil, gelatin and injectable organic ester ethyl oleate etc. for example of propylene glycol, Polyethylene Glycol, vegetable oil.Such dosage form can also contain adjuvant for example antiseptic, wetting agent, emulsifying agent and dispersant.They can be sterilized, for example filter by keep filter via antibacterial, by in compositions, add biocide, by sterilizing with the compositions radiation treatment or by compositions is heated.Can also they be made the aseptic injection medium facing with preceding.

Can be suitable for intravenous, intramuscular, sheath is interior, subcutaneous and peritoneal injection.Used sterile media all is easy to make by the well-known standard technique of those skilled in the art.They can be sterilized, for example filter by keep filter via antibacterial, by in compositions, add biocide, by sterilizing with the compositions radiation treatment or by compositions is heated.Can also they be made sterile injectable medium facing with preceding.

Preferred dosage is different with the difference of clinical indication.According to treatment patient's disease, may must make some change, and under any circumstance, all determine the suitable dose of individual patient by the doctor to dosage.The effective dose of chemical compound depends on body weight, physiological situation and selected dosage regimen etc. in the per unit dosage.The chemical compound of unit dose is meant and does not comprise the weight of vehicle weight at the chemical compound of interior (when using carrier).

The route of administration that is used to implement chemical compound of the present invention depends on the position that disease and needs are treated.Because the pharmacokinetics of The compounds of this invention and pharmacodynamic profile have to a certain degree different, the most preferred method that therefore obtains treatment concentration in tissue is to increase dosage gradually and monitor clinical effectiveness.For such therapeutic dose of increase gradually, predose will depend on route of administration.

For any particular patient, concrete treatment effective dose level will depend on multiple factor, comprise activity, route of administration, this particular compound of the disease of being treated, disease severity, used particular compound removing speed, treatment persistent period, with this particular compound associating or the well-known factors in medical science field such as concrete medicine, patient's age, body weight, sex, diet and general health situation used simultaneously.Dosage level is generally about 10-200mg/kg/ days, is preferably about 50-200mg/kg/ days.

Description of drawings:

Fig. 1: the HPLC of BC-4 composition detects collection of illustrative plates,

Wherein: 1, BC-4-α; 2, BC-4-β

Fig. 2: BC-4 is to the active inhibitory action of topoisomerase I

Fig. 3: BC-4 is to the active inhibitory action of topoisomerase II

Fig. 4: BC-4 observes figure to the laser confocal microscope of the apoptosis-induced effect of T24 human bladder cancer cell,

Wherein A is BC-475 μ M effect 8 hours; B is BC-4 75 μ M effects 12 hours; C is BC-4 75 μ M effects 24 hours.

Fig. 5: BC-4 analyzes collection of illustrative plates to T24 human bladder cancer cell apoptosis-induced flow cytometer testing result and dna ladder band.

Wherein: ← be control cells; ↑ be the solvent control cells; → be 24 hours cells of BC-4 25 μ M effects; ↓ be 24 hours cells of BC-4 50 μ M effects; ° be 24 hours cell of BC-475 μ M effect; ± analyze collection of illustrative plates for the dna ladder band, wherein 1 is control cells, 2-4 is respectively BC-425,24 hours cell of 50,75 μ M effects.

The morphological observation that Fig. 6: BC-4 is apoptosis-induced to the MDA/HER-2 human breast cancer cell

Fig. 7: BC-4 is to IFN _γThe flow cytometer testing result (A) and the morphological observation (B) of the MV3 apoptosis-inducing effect of handling

Fig. 8: BC-4 is to the inhibitory action of HT-1080 human fibrosarcoma cell secrete metalloproteinases,

Wherein: 1 is control cells; 2 is the solvent control cells; 3-6 is BC-4 3.125,6.25,24 hours cell of 12.5,25 μ M effects.

Fig. 9: BC-4 is to the morphological observation of the apoptosis-induced effect of HT-1080 human fibrosarcoma cell,

Wherein: A is Wright-Giemsa colored light sem observation figure (left side is a control cells, and the right side is an apoptotic cell); B is acridine orange fluorescent staining microscopic examination figure (left side is a control cells, and the right side is an apoptotic cell); C is electron microscopic observation figure (left side is a control cells, and the right side is an apoptotic cell).

Figure 10: BC-4 analyzes collection of illustrative plates to the apoptosis-induced dna ladder band of HT-1080 human fibrosarcoma cell,

Wherein: 1 is control cells, and 2-4 is BC-425,24 hours cell of 50,75 μ M effects; 5 is 100bp dna molecular amount labelling.

The flow cytometer testing result that Figure 11: BC-4 is apoptosis-induced to the HT-1080 human fibrosarcoma cell,

Wherein: A is a control cells, and the apoptotic cell percentage rate is 0.42%; B is 24 hours a cell of BC-425 μ M effect, and the apoptotic cell percentage rate is 1.85%; C-D is 24,36 hours a cell of BC-450 μ M effect, and the apoptotic cell percentage rate is respectively 9.00%, 59.3%; E-G is 8,12,24 hours a cell of BC-475 μ M effect, and the apoptotic cell percentage rate is respectively 13.5%, 18.7%, 76.5%.

Figure 12: BC-4 supports the apoptosis-induced flow cytometer detection collection of illustrative plates of human umbilical vein endothelial cell to former being commissioned to train, and BC-4 does not cause apoptosis.

Wherein: ← be control cells; ↑ be the solvent control cells; → be 24 hours cells of BC-4 25 μ M effects; ↓ be 24 hours cells of BC-4 50 μ M effects; ° be 24 hours cell of BC-475 μ M effect.

Embodiment

Embodiment 1

With the ethanol percolation of 1 kilogram of usefulness 95% of crude drug Olibanum pearl, must infiltrate and filter; Decompression and solvent recovery gets ethanol ointment-like medicine for oral or plastering use 748.8 grams.The ethanol ointment-like medicine for oral or plastering use that obtains is dissolved in the petroleum ether-ethyl acetate mixed solvent, add sodium carbonate liquor (about 3000ml) and transfer to pH 10, petroleum ether-ethyl acetate mixed solvent 3000ml extraction three times, water reuse 4N hydrochloric acid transfers to pH 3, petroleum ether-ethyl acetate mixed solvent 3000ml extraction three times, organic facies water 1500ml washed twice, anhydrous sodium sulfate dehydration, filter, be concentrated into driedly, add methanol and concentrate foaming and obtain Olibanum total acid foaming thing 252.3.With Olibanum total acid 200-300 purpose silica gel column chromatography, the petroleum ether-ethyl acetate gradient elution, get acetyl boswellic acid α, beta isomer crude mixture 40.6g, carry out recrystallization again, obtain acetyl boswellic acid α, beta isomer mixture (1: 1) crystallization 23.8g.

Embodiment 2

Prepare the acetyl boswellic acid tablet by method well known in the art, make the acetyl boswellic acid that contains 6%-10% by actual needs in the wherein said tablet, also can strengthen or reduce the content of acetyl boswellic acid.

Claims (4)

1. the application of acetyl boswellic acid in preparation neoplasm metastasis inhibitor, wherein said tumor does not comprise leukemia, and wherein said acetyl boswellic acid is made of α-acetyl boswellic acid and the β-acetyl boswellic acid proportion of composing according to 1: 1.

2. application as claimed in claim 1 is characterized in that described tumor comprises bladder cancer, breast carcinoma, melanoma and fibrosarcoma.

3. application as claimed in claim 2 is characterized in that described tumor is a melanoma.

4. application as claimed in claim 1 is prepared into the form of cream, tablet, capsule, pill dispersion powder, granule, suppository, syrup, elixir, lozenge, injection solution, suspension or emulsion but its feature will be described neoplasm metastasis inhibitor.

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