CN1283254C - Use of 23-hydroxy betulic acid in inhibiting blood vessel formation - Google Patents
Use of 23-hydroxy betulic acid in inhibiting blood vessel formation Download PDFInfo
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- CN1283254C CN1283254C CN 200510038469 CN200510038469A CN1283254C CN 1283254 C CN1283254 C CN 1283254C CN 200510038469 CN200510038469 CN 200510038469 CN 200510038469 A CN200510038469 A CN 200510038469A CN 1283254 C CN1283254 C CN 1283254C
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Abstract
The present invention discloses the application of 23-hydroxy betulinic acid to angiogenesis inhibition, which relates to the technical field of traditional Chinese medicine application. The present invention discloses a new application of the 23-hydroxy betulinic acid the angiogenesis inhibition, and researches indicate that the 23-hydroxy betulinic acid has the action of inhibiting the angiogenesis. The effective dose of the 23-hydroxy betulinic acid applied to the preparation of curing and/or preventing medicine for inhibiting the angiogenesis is 3 to 50 mg per kg of body weight per day. The present invention slickly takes the angiogenesis as a target for curing diseases, and the present invention has the advantages of specificity for the cure is carried out by aiming at already started new vessels, low dose, high curative effect and small side effect because vascular endothelial cells are exposed in blood flow and the medicine directly takes effects, and no generation of drug resistance because gene expression of endothelial cells is relatively stable.
Description
Technical field
The 23-hydroxyl radical white birck acid suppresses the application in the medicine for the treatment of and/or preventing of angiogenesis in preparation, relates to the new purposes of 23-hydroxyl radical white birck acid.
Background technology
The growth of primary tumo(u)r and transfer depend on the generation that is derived from the new vessels of both depositing blood vessel, and this process is called angiogenesis (angiogenesis).Tumor both can be obtained nutrition and oxygen from the host by tumor vessel, can carry transitional cell to the host continuously by tumor vessel again, and formed at other position continued growths and the induction of vascular of body, caused neoplasm metastasis.If there is not angiogenesis, the growth of primary tumo(u)r can not surpass 1～2mm, can not occur soaking into and shifting yet.It is closely related with generation, development, infiltration and the transfer of blocking-up tumor to suppress tumor-blood-vessel growth, and can stop the pernicious transformation of precancerous lesion to cancer.The not only growth of entity tumor, infiltration and transfer rely on new vessels and generate, and the growth of blood system malignant tumor (as malignant lymphoma, Lymphocytic leukemia etc.) and transfer are also closely related with angiogenesis.Growth of tumor, transfer, recurrence, prognosis and angiogenesis are closely related, angiogenesis with tumor is a target spot, the exploitation angiogenesis inhibitor, not only can be used for the treatment of most of entity tumors, also can be used for the prevention of cancer and the treatment of blood system malignant tumor, simultaneously to the disease of other and associated angiogenesis as: the prevention and the treatment of diabetic renal papillary necrosis, rheumatic arthritis, psoriasis, hemangioma, atherosclerosis etc. all have important theory and realistic meaning.Therefore, the vascular system of tumor has become brand-new, an antineoplaston target spot likely.
R and D can destroy or suppress angiogenesis, and (angiogenesis inhibitor, tumor angiogenesis inhibitor TAI) are present research focus to stop the medicine of tumor growth and transfer effectively.TAI has many advantages by cutting off source of nutrition and the migrating channels that tumor is rely growth and shifted: when 1. treatment took place, angiogenesis was activated, so the TAI treatment has good specificity.2. vascular endothelial cell is exposed in the blood flow, and medicine can directly play a role, so dosage is little, curative effect is high.3. the endothelial cell gene expression is relatively stable, is difficult for producing drug resistance.4. the growth rate of tumor vascular endothelial cell is than the normal fast manyfold of vascular endothelial cell, so TAI is minimum to normal tissue toxicity.In recent years, the research of this class inhibitor makes progress, is expected to become at the beginning of 21 century that a class is brand-new, antineoplaston medicine likely, and wherein the exploitation of natural drug receives much concern.
The Radix Pulsatillae is a Chinese medicine, tool heat-clearing and toxic substances removing, blood circulation invigorating efficacies, and tcm clinical practice is mainly used in the treatment of intestinal canal tumours such as colon cancer, rectal cancer and cervical cancer, hypophysis cerebri tumor, thyroid tumor, pulmonary carcinoma.The 23-hydroxyl radical white birck acid is from the isolated a kind of pentacyclic triterpene saponins of the Chinese medicine Radix Pulsatillae (Pulsatilla Chinensis) root, is the characteristic chemical constituent of the Chinese medicine Radix Pulsatillae, and its structural formula is:
Molecular formula is C
30H
48O
4, the off-white color powder, molecular weight is 472, is soluble in organic solvents such as dimethyl sulfoxine, chloroform, ethyl acetate, ethanol, and is water insoluble.It can obtain by taking " application of 23-hydroxyl radical white birck acid in preparation treatment or prophylaxis of tumours and AIDS-treating medicine " (application number is to have done detailed narration in 03152904.6 the Chinese invention patent application documents), but this is not unique method and the unique source that obtains the 23-hydroxyl radical white birck acid.
The 23-hydroxyl radical white birck acid has anti-tumor activity widely.The inside and outside presses down the tumor experiment and shows, it is stronger than other derivant melanoma effect of extracting in the Radix Pulsatillae, (10-20ug/mL) has tangible differentiation-inducing action to the melanin tumour b16 cell during low dosage, (mouse stomach 300-600mg/kg) inducing apoptosis of tumour cell during high dose has the obvious suppression effect to inside and outside melanoma propagation.In addition, it is different from the composition Betula platyphylla Suk. acid of extracting of reports such as Pisha in Cortex Betulae Luminiferae, and the latter only has inhibitory action to melanoma and other tumor cell is not acted on.It all has inhibitory action to kinds of tumor cells system as people's gastric cancer SGC-7901 cell line, human ovarian cancer A0 cell line, human leukemia HL-60 cell system, human leukemia K562 cell line, human cervical carcinoma Hela cell system and mouse ascites tumor S180 cell line etc.Chinese patent (application number is 03152904.6) reports that also it all has inhibitory action to pulmonary carcinoma, hepatocarcinoma, glioma brain tumour, intestinal canal tumour, HIV (human immunodeficiency virus) etc.But, limited the performance of its antitumaous effect because its antitumaous effect mechanism is also indeterminate.
Summary of the invention
A kind of new purposes that the purpose of this invention is to provide the 23-hydroxyl radical white birck acid, the 23-hydroxyl radical white birck acid suppresses the application in the medicine for the treatment of and/or preventing of angiogenesis in preparation.
The inventor studies show that, the 23-hydroxyl radical white birck acid has the effect that suppresses angiogenesis.Therefore technical scheme of the present invention is: the 23-hydroxyl radical white birck acid preparation suppress angiogenesis treat and/or prevent application in the medicine time, its effective dose is 3～50mg/kg body weight/day.
When needing, suppress treating and/or preventing of angiogenesis in the preparation of 23-hydroxyl radical white birck acid and can also add one or more pharmaceutically acceptable carriers in the medicine.Described carrier comprises diluent, excipient, filler, binding agent, wetting agent, disintegrating agent, absorption enhancer, surfactant, absorption carrier, lubricant, emulsifying agent, osmotic pressure regulator of pharmaceutical field routine etc., can also add flavouring agent, sweeting agent etc. in case of necessity.
Medicine of the present invention can be made various ways such as injection, tablet, powder, granule, capsule, oral liquid, unguentum, cream, Emulsion.The medicine of above-mentioned various dosage forms all can be according to the conventional method preparation of pharmaceutical field.
Of the present invention studies show that, except treating and/or preventing cancer, with the 23-hydroxyl radical white birck acid be active component the inhibition angiogenesis treat and/or prevent medicine, for belonging to diseases such as disease, hemangioma, atherosclerosis and have definite treatment and preventive effect with the disease of associated angiogenesis such as diabetes, retinopathy, retinopathy of prematurity, retinal vein occlusion, the degeneration of old plate-like speckle, rheumatic arthritis, silver, formation also has significant inhibitory effect to the spot in the wound healing simultaneously.
Beneficial effect of the present invention: the present invention with the target spot of angiogenesis as the treatment disease, has the following advantages dexterously: 1, treatment is carried out at the new vessels that has started, and has specificity; 2, because vascular endothelial cell is exposed in the blood flow, and medicine directly plays a role, so dosage is little, the curative effect height, side effect is little; 3, because the endothelial cell gene expression is relatively stable, so be difficult for producing drug resistance.
Description of drawings
Fig. 1 23-HBA is to the effect of HMEC cells in vitro inhibition of proliferation.
Fig. 2 23-HBA is to the morphology influence of HMEC cell.
The influence that Fig. 3 23-HBA forms the external tubule of HMEC cell Matrigel.
The specific embodiment
Embodiment 1:23-hydroxyl radical white birck acid is to the effect of human vascular endothelial inhibition of proliferation.
1.1 23-hydroxyl radical white birck acid (23-HBA) is to the in-vitro multiplication test of human vascular endothelial (HMEC)
With human vascular endothelial (HMEC, France national health Medicine Research Inst. is so kind as to give) place and contain the 1mmol-glutamine, 1 μ g/mL hydrocortisone is cultivated (37 ℃, 5%CO in the MCDB-131 culture fluid of 10ng/mL epidermal growth factor (EGF) and 15% calf serum
2, 95% humidity).The trophophase cell of taking the logarithm is with 10
5The cell/mL cell inoculation is in 96 orifice plates, and 4 parallel holes are established for every group in 100 μ L/ holes, are positioned over 37 ℃, 5% humidifying CO
2Incubator spend the night.The 23-HBA that adds variable concentrations (10～100 μ g/mL), with the negative contrast of solvent dimethyl sulfoxine (DMSO), cultivate after 48 hours, remove supernatant, every hole adds 10% trichloroacetic acid, 100 μ L lightly and fixes, leave standstill and move on to 4 ℃ behind the 5min and outwell fixative after placing 1h, wash 5 times air drying with deionized water.Every hole adds 0.4% sulphonyl rhodamine B (SRB), 100 μ L room temperatures and places 30min, washes 5 times with 1% acetate solution, adds the dissolving of 150 μ L 10mM Tris alkali liquor (pH10.5), and spectrophotometer is measured the absorbance (A) of each aperture at 531nm wavelength place automatically.Above-mentioned experiment repeats 3 times.Suppression ratio (%) computing formula is as follows:
The result as shown in Figure 1, the 23-hydroxyl radical white birck acid is the dose-dependent inhibition effect to the propagation of human vascular endothelial, its half amount of suppression is 40.44ug/mL.
1.2 23-HBA is to the morphology influence of HMEC cell
Add sheet glass in 24 orifice plates earlier, add exponential phase HMEC cell again, every hole 2 * 10
5Individual cell, 37 ℃, 5% humidifying CO
2Cultivate 24h.The 23-HBA that adds variable concentrations (1～60 μ g/mL) handles 72h, takes out sheet glass, 0.001M phosphate buffer (PBS) (pH 7.4) washing, and cold acetone is fixed, HE dyeing, the morphological change of observation of cell is also taken pictures.The result is as shown in Figure 2: normal cell is dyed still to keep original growth form, and nucleus is complete, dyes the blue or redness more slightly of homogeneous.After adding 1 μ g/mL 23-HBA, the part cell begins apoptosis, attached cell shrinkage occurs, become circle, comes off, the smaller volume of cell, distortion, cell membrane is complete but foamed phenomenon occurs, and it is black-and-blue that nucleus is, and endochylema is pale red or peony, chromatin concentrates, marginalisation, and nuclear membrane cracking, chromatin are divided into bulk.Bring up to more than the 10 μ g/mL when 23-HBA concentration, the apoptosis phenomenon of HMEC cell is more obvious.
Embodiment 2:23-hydroxyl radical white birck acid forms the inhibitory action of ability to the human vascular endothelial pipe.
2.1 external tubule forms test
Add 50 μ L growth minimizing type Matrigel in 96 well culture plates bottom under 4 ℃, pave the back incubator and hatch 4h for interior 37 ℃, 23-HBA and 15% calf serum MCDB-131 culture fluid and HMEC cell (4 * 10
4Cells/well, every pore volume are 0.25ml) add culture plate behind the mixing, the 23-HBA final concentration is 1～60 μ g/mL, with the negative contrast of solvent.37 ℃, 5% humidifying CO
2After cultivating 24h, use inverted microscope, observe the influence of 23-HBA endotheliocyte external structure blood vessel.The visible 23-HBA of result is formed with significant inhibitory effect to HMEC cells in vitro Matrigel tubule.When 23-HBA concentration is 1 μ g/mL, HMEC cell tubule formed promptly has a significant effect, tubule decreased number not only, and also tube chamber is imperfect; When concentration is 10 μ g/ml, does not almost have behind the HMEC cell attachment and move, do not have tubule at all and form; Under 60 μ g/ml concentration, the cell attachment difficulty.23-HBA concentration is that 10 μ g/ml, 20 μ g/ml, the 40 μ g/ml area that 4 experimental grouies form tube chambers during with 60 μ g/ml has been compared notable difference (Fig. 3) with matched group.
2.2 Matrigel plug method blood vessel inhibition test in the body
Get liquid Matrigel under 4 ℃ and mix respectively with the 23-HBA of variable concentrations (1～200 μ g/mL), getting 0.4mL, to be injected into the armpit of BALB/c mouse subcutaneous, and 4 mices are one group.Put to death mice behind the 7d, take out the Matrigel parcel, 10% neutral formalin is fixed, embedding, and section, HE dyeing, microscopically is taken the photograph sheet, amplifies 200 times, migrates into the quantity of Matrigel parcel endotheliocyte in 5 visuals field of picked at random.The results are shown in Table 1.Mirror is observed down 23-HBA concentration when being higher than 1 μ g/mL, can obviously reduce the endotheliocyte quantity of invading the Matrigel parcel, and with the rising of concentration, the number that cell is invaded descends, and compares with matched group, and difference has significance (P＜0.001).
Table 1 23-HBA is to the influence of Matrigel parcel endotheliocyte quantity
| Drug level (μ g/mL) | n | The migrating cell number |
| The normal control group | 5 | 502.98±31.93 |
| 1 | 5 | 350.00±32.00 |
| 5 | 5 | 207.50±22.73 |
| 10 | 5 | 134.46±15.97 |
| 50 | 5 | 80.02±14.72 |
| 100 | 5 | 18.20±4.71 |
| 200 | 5 | 9.4±1.95 |
Compare with matched group: P＜0.001
Embodiment 3:23-hydroxyl radical white birck acid is to the inhibitory action of human vascular endothelial transfer ability.
3.1 suppress the migration of vascular endothelial cells test
24 orifice plates are 60 ° of inclinations, add 1% agarose, wait to solidify the back, do not have a side of agarose to add the HMEC cell, every hole 10 with the culture medium washing at 1/2 place of bottom, every hole
5Individual cell.The 23-HBA that adds negative control and variable concentrations (1～60 μ g/ml) behind the cell attachment respectively writes down the quantity of each group endotheliocyte to second half migration with inverted microscope behind the 72h.The result shows that do not have the migration quantity of 23-HBA intervention group cell higher, the cell migration suppression ratio is concentration dependent, and 23-HBA may suppress the generation (table 2) of new vessels by the migration that suppresses vascular endothelial cell.
Table 2 23-hydroxyl radical white birck acid is to the influence of migration of vascular endothelial cells number
| Drug level (μ g/mL) | n | HMEC cell migration number |
| The normal control group | 4 | 289.8±25.4 |
| 1 | 4 | 61.5±12.9 |
| 5 | 4 | 47.4±12.5 |
| 10 | 4 | 32.1±11.7 |
| 20 | 4 | 26.6±9.2 |
| 40 | 4 | 12.3±5.6 |
| 60 | 4 | 6.2±1.4 |
Compare with matched group: P＜0.001
Embodiment 4: the tumor bearing nude mice in vivo test.
Human colon's cancerous cell line Lo Vo cell is made cell suspension with PBS, and the right side thigh portion that is seeded to big BALB/cAnN-nu/nu nude mice of 8 weeks with 2,000,000 cell/200ul is subcutaneous.When treating the tumor bulk-growth, be divided into two groups at random, 5 every group to 5mm * 5mm size.23-hydroxyl radical white birck acid medication group dosage is 25mg/kg (medicine is to contain the PBS solution dissolving of 10%DMSO), and matched group is given the PBS solution 200ul that contains 10%DMSO, and successive administration 60 days writes down the major diameter and the minor axis of tumor body every day.During the medication, untoward reaction does not appear in mice.After putting to death mice, take out tumor, detect the microvessel density of tumor.The result shows that medication group tumor body dwindles than matched group, and growth is obviously slowed down, and microvessel density is starkly lower than matched group.Illustrate that this medical instrument has the effect that suppresses tumor in the body, and side effect is little.
Embodiment 5: the tumor bearing nude mice in vivo test.
Operational approach is with embodiment 4.23-hydroxyl radical white birck acid medication group dosage is 3mg/kg, and the result shows that medication group tumor body dwindles than matched group, decreased growth, and microvessel density is lower than matched group.Illustrate that this medical instrument has the effect that suppresses tumor in the body, and side effect is little.
Embodiment 6: the tumor bearing nude mice in vivo test.
Operational approach is with embodiment 4.23-hydroxyl radical white birck acid medication group dosage is 50mg/kg, and the result shows that medication group tumor body dwindles than matched group, and growth is significantly slowed down, and microvessel density significantly is lower than matched group.Illustrate that this medical instrument has the effect that suppresses tumor in the body, and side effect is little.
Claims (6)
1, the 23-hydroxyl radical white birck acid suppresses the application in the medicine for the treatment of and/or preventing of angiogenesis in preparation, and it is characterized in that: the effective dose of described 23-hydroxyl radical white birck acid in the medicine that suppresses angiogenesis is 3～50mg/Kg body weight/day.
2, application according to claim 1 is characterized in that: described medicine is to suppress the medicine that spot forms in the wound healing.
3, application according to claim 1 is characterized in that: described medicine is the medicine that treats and/or prevents diabetic retinopathy, retinopathy of prematurity and retinal vein occlusion.
4, application according to claim 1 is characterized in that: described medicine is the medicine that treats and/or prevents degeneration of old plate-like speckle and rheumatic arthritis.
5, application according to claim 1 is characterized in that: described medicine is for treating and/or preventing psoriatic medicine.
6, application according to claim 1 is characterized in that: described medicine is for treating and/or preventing atherosclerotic medicine.
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