CN101456854A - Medicine novel use of procyanidine oligomer and multimer - Google Patents
Medicine novel use of procyanidine oligomer and multimer Download PDFInfo
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- CN101456854A CN101456854A CNA2009100100545A CN200910010054A CN101456854A CN 101456854 A CN101456854 A CN 101456854A CN A2009100100545 A CNA2009100100545 A CN A2009100100545A CN 200910010054 A CN200910010054 A CN 200910010054A CN 101456854 A CN101456854 A CN 101456854A
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Abstract
The invention provides novel application of proanthocyanidin oligomer F2 and a polymer F3 in preparing anti-tumor health care food and medicine, in particular novel application in preparing health care food and medicine for treating glioma. The application researches inhibiting function of F2 on brain tumor, blood system tumor, alimentary canal tumor, respiratory system tumor and urinary genital system tumor. Firstly, F2 and F3 are discovered to have the inhibiting functions on glioma cell strains U-87 and U251, humanized leukemia HL-60 cell strains, humanized liver cancer Hep3B cell strains, humanized colon cancer Colon-205 cell strains, humanized prostate cancer Du-145 cell strains and humanized A549 lung cancer cell strains, and the inhibiting function is improved along with progressive increase of dosage. In addition, the F2 can also remarkably inhibit chemotaxis of human malignant glioma U-87 cells induced by formyl peptide and expression of formyl peptide receptor (FPR) in the cells. The receptor has important function on mediating migration, survival, growth and vasculogenesis of the human malignant glioma cells.
Description
Technical field:
The invention belongs to medical technical field, the new medical use that relates to pycnogenols oligomer (F2) and high polymer (F3), specifically relate to its tumour, hematological system tumor, digestive tract tumor, tumor in respiratory system and urogenital neoplasm in preparation control brain, the purposes of especially preventing and treating people's glioblastoma protective foods and pharmaceutical preparations.
Background technology:
Pycnogenols (proanthocyanidins or procyanidins are called for short PC) is the general name of an extensive big class polyphenolic substance that exists in the plant.People have carried out big quantity research to the pycnogenols that extracts from numerous different plants for many years.Antioxidant as efficiency natural, it also has other numerous good biopharmacology activity, now be subjected to people's favor day by day in fields such as nutritive health-care, medical and health and cosmetics, have multiple important biomolecule activity, can be used for preventing and treating cardiovascular disorder, anti-diabetic, antitumor, anti-ageing, improve immunosuppression etc.
Malignant tumour is a kind of common disease, and the human existence quality in serious threat, is called as first killer of human health.Neurospongioma is the modal malignant tumour of central nervous system, the first place that occupies the intracranial tumors sickness rate.It is except that having the necrotic lesion centrostigma, excelling at leveraging microenvironment and support also have rich blood vessel self survival, the characteristics of growing rapidly, and invasion and attack and characteristics such as transfer ability is strong utilize traditional operative treatment to be difficult to thorough excision.Patient's survival rate of suffering from glioblastoma is extremely low.In addition, the tumour of many other systems all is the malignant diseases of healing than refractory as hematological system tumor, digestive tract tumor, tumor in respiratory system and urogenital neoplasm etc.
(formyl peptide receptor is a kind of g protein coupled receptor FPR) to formyl peptide receptor, is distributed widely in human multiple tissue and organ, can participate in multiple pathophysiological processes such as mediated leucocytes chemotactic, immune response and malignant tumour.In recent years, the progress of inside/outside source property FPR receptors ligand is rapid.Its exogenous agonist comprises chemotactic substance formyl peptides (fMLF) and synthetic polypeptide class WKYMVM, the WKYMVm of " classics ".The endogenous agonist comprises the interior formylated small peptide of partial peptide section, plastosome, the glucocorticosteroid adjusting albumen annexin I in the HIV-1 capsid protein and is present in the Cathepsin G of neutrophil leucocyte.Its antagonist comprises ectogenic synthetic polypeptide tBoc, derives from the cyclosporine H (CsH) of fungi, the polypeptide spinorphin (LVVYPWT) of purifying and streptococcus aureus excretory CHIPS from the ox backbone.Recent study finds that FPR energy selective expression does not express at normal astroglia cell and the lower glioma surface of grade malignancy in people's human malignant glioma cell line U-87 and most former generation IV level glioblastoma and III level astrocytoma.This receptor can take place by migration, survival, growth and the blood vessel with the FPR agonist in host source (fMLF that is discharged by the glioma cell of necrosis) effect Mediated Human glioblastoma cell, can also participate in the tumorigenicity of nude mice.Therefore FPR can be used as the molecular target of a development of new anticol matter tumor medicine.
Summary of the invention:
The purpose of this invention is to provide a kind of Semen Vitis viniferae extract pycnogenols oligomer (F2) and high polymer (F3) purposes at preparation control tumor health food and pharmaceutical preparations.
The present invention is achieved by the following scheme:
The preparation technology of Semen Vitis viniferae extract pycnogenols oligomer (F2) and pycnogenols high polymer (F3) is as follows among the present invention: with the hypervelocity pulverizer Semen Vitis viniferae is ground to form the powder that particle diameter is not higher than 1mm.(80:20 V/V) carries out just carrying, and (75:25 v/v) extracts and obtains the phenols crude extract with acetone-water again with methanol-water immediately.After removing organic solvent, this crude extract is separated with chromatographic column Lichroprep RP-18 (200 X 25mm, particle diameter=25-40 μ m), obtain F2, F3.Its process mainly comprises: the distilled water wash-out of at first using PH=7.0 obtains Semen Vitis viniferae extract oligomer F2 with eluent ethyl acetate again to remove phenolic acid.Semen Vitis viniferae extract high polymer F3 adopts methanol-eluted fractions.F2, F3 are being lower than 30 ℃ of following dryings and are making lyophilized powder.Its structural formula is as follows, and wherein the polymerization degree of F2 is 2-15, and the polymerization degree of F3 is 10-33.
1, the invention provides the protective foods of F2, F3 tumour in preparation control brain and the purposes of pharmaceutical preparations.
2, F2, F3 are in the protective foods of preparation control hematological system tumor and the purposes of pharmaceutical preparations.
3, F2, F3 are in the protective foods of preparation control gi system tumour and the purposes of pharmaceutical preparations.
4, F2, F3 are in the protective foods of preparation control respiratory system tumour and the purposes of pharmaceutical preparations.
5, F2, F3 are in the protective foods of preparation control urogenital neoplasm and the purposes of pharmaceutical preparations.
The effective dose of 6 suggestions is 30-100 μ g/ml.
Description of drawings:
Fig. 1 is the influence of F2 to the U-87 cell chemotaxis
Compare with blank group ###p＜0.001,
* *P＜0.001 dosing group and model fMLF group are relatively; The shade bar graph is shown and adds chemoattractant formyl peptides group, and open column shape figure represents the blank solvent group
Fig. 2 is the influence of F3 to the U-87 cell chemotaxis
Compare with blank group ###p＜0.001,
* *P＜0.001 dosing group and model fMLF group are relatively; The shade bar graph is shown and adds chemoattractant formyl peptides group, and open column shape figure represents the blank solvent group
Fig. 3 is the influence that F2 mobilizes for formyl peptides inductive U-87 cell calcium; The solid line group is represented blank group, and the dotted line group is represented F2 (5 μ g/ml) group, and long and short dash line is represented F2 (10 μ g/ml) group
Fig. 4 is that F2 influences for formyl peptides inductive U-87 cell ERK1/2 activatory
Fig. 5 is the influence that F2 expresses U-87 cell FPR, and white lines are represented the expression of FPR
Embodiment:
Embodiment 1, be that target cell carries out F2, F3 for these cell inhibiting effects experiment with humanized's glioma U-87 and U251 cell strain, humanized's leukemia HL-60 cell strain, humanized's liver cancer Hep3B cell strain, humanized's colorectal carcinoma Colon-205 cell strain, humanized's prostate cancer Du-145 cell strain and humanized A549 lung cancer cell line
1.1 material
People's glioblastoma cell U-87 and U251, humanized's leukemia HL-60 cell strain, humanized's liver cancer Hep3B cell strain, humanized's colorectal carcinoma Colon-205 cell strain, humanized's prostate cancer Du-145 cell strain and humanized A549 lung cancer cell line: provide by ATCC.DMEM dry powder: available from Invitrogen/Gibco.Foetal calf serum: available from TBD company.Pancreatin: available from the magnificent Sheng Ke in Beijing Bioisystech Co., Ltd.Tetramethyl-azo tetrazolium bromide (MTT): available from available from the magnificent Sheng Ke in Beijing Bioisystech Co., Ltd.F2, F3 are provided by Portugal national resources laboratory professor Sun Baoshan.
1.2 method
Humanized's glioma U-87 that will provide by ATCC and U251 cell strain, humanized's leukemia HL-60 cell strain, humanized's liver cancer Hep3B cell strain, humanized's colorectal carcinoma Colon-205 cell strain, humanized's prostate cancer Du-145 cell strain and humanized A549 lung cancer cell line with trysinization after, be suspended in the nutrient solution that contains 10% foetal calf serum, blow and beat into cell suspension gently, microscopically cell counting count board living cell counting.In 96 aseptic well culture plates, every hole adds tumour cell suspension 100 μ l with tumor cell inoculation.It is 5% CO that cell is placed 37 ℃, volume percent
2In the incubator after adherent the spending the night, abandoning supernatant adds the F2 solution of different concns then.After medicine acts on certain hour respectively, shift supernatant liquor, add the serum-free medium 100 μ l of 0.5mg/ml tetrazolium salts (MTT) in every hole, 37 ℃, volume percent are 5% CO
2Continue in the incubator to cultivate 4 hours, abandoning supernatant, every hole adds 100 μ l DMSO, measures each hole absorbancy, is calculated as follows average inhibiting rate then.The result adopts Mean ± SEM to represent, data statistics adopts ANOVA that mean between group is relatively carried out the homoscedasticity analysis, and utilization Dunnett ' s t-test organize between relatively.
Calculation formula: (control group OD value-experimental group OD value)/control group OD value X100
1.3 result
The result is as shown in table 1, F2, F3 are inhibited to glioma cell line U-87 and U251 cell strain, humanized's leukemia HL-60 cell strain, humanized's liver cancer Hep3B cell strain, humanized's colorectal carcinoma Colon-205 cell strain, humanized's prostate cancer Du-145 cell strain and humanized A549 lung cancer cell line, and increasing progressively and improve with dosage.This result shows that F2, F3 are a kind of very potential novel, effective antitumour new drugs, has widened the medical applications of Semen Vitis viniferae extract pycnogenols oligomer (F2) and high polymer (F3) thereof.
Table 1, F2, F3 test the inhibition of tumour cell
2.1 material
48 hole chemotactic plates: available from Neuro Probe company.Polycarbonate membrane: available from Neuro Probe company.I type mouse tail collagen: available from genome company.Formyl peptides (fMLF): available from Sigma company.F2, F3 are provided by Portugal national resources laboratory professor Sun Baoshan.
2.2 method
Chemotactic cell method is adopted in the detection of cell chemotaxis, and the hole is that following hole is the 10nM formyl peptides respectively in advance through the F2 of non-cytotoxicity dosage, the U-87 cell suspension that F3 is hatched 1h on the chemotactic cell.Other adds negative control group (following hole is blank damping fluid, and last hole is blank cell suspension), positive controls (following hole is the 10nM formyl peptides, and last hole is blank cell suspension).The polycarbonate membrane with 8 μ M apertures is separated (using the mouse tail glue primordial covering of 50 μ g/mL in advance) between cell up and down, adjusts cell density to 0.8 * 10
6Cells/mL takes out film hatch 4h in incubator after, the cell number that on average moves in the every hole of counting down in light microscopic through fixing, dyeing back.And calculate the ratio of chemotactic cell count in cell count that chemotactic index (CI) is a fMLF group chemotactic and the damping fluid control group.The result adopts Mean ± SEM to represent, data statistics adopts ANOVA that mean between group is relatively carried out the homoscedasticity analysis, and utilization Dunnett ' st-test organize between relatively.
2.3 result
The result as shown in Figure 1, F2 (2.5 μ g/mL, 5 μ g/mL and 10 μ g/mL) acts on the chemotactic that U87 cell 1h can significantly suppress fMLF inductive U-87 cell in advance, and has dose-dependently.Because F2 (2.5 μ g/mL, 5 μ g/mL) acts on the random migration that U-87 cell 1h does not influence U87 in advance, so F2 (2.5 μ g/mL, 5 μ g/mL) does not work by influencing its random migration to the restraining effect of the chemotactic of fMLP inductive U87 cell.Similar, F3 (2.5 μ g/mL, 5 μ g/mL and 10 μ g/mL) acts on the chemotactic that U-87 cell 1h can significantly suppress fMLF inductive U-87 cell in advance, and has dose-dependently.Because F3 (2.5 μ g/mL, 5 μ g/mL) acts on the random migration that U-87 cell 1h does not influence U-87 in advance, so F3 (2.5 μ g/mL, 5 μ g/mL) does not work by influencing its random migration to the restraining effect of the chemotactic of fMLF inductive U-87 cell.The contrast experiment of the anticol matter tumor activity of embodiment 3, total pycnogenols and F2, F3
3.1 material
Be subjected to reagent: F2, F3 and total pycnogenols are all taught from Portugal national resources laboratory Sun Baoshan.
3.2 method
Mtt assay and chemotactic experiment (as previously mentioned).
3.3 result
The result is as shown in table 2, and anti-glioma propagation and the anti-fMLF inductive glioma cell chemotactic activity of F2, F3 all significantly are better than total pycnogenols.
The anticol matter tumor activity contrast of table 2, F2, F3 and total pycnogenols
Embodiment 4, be that target cell carries out the influence experiment that F2 mobilizes for formyl peptides inductive U-87 cell calcium with humanized's glioma U-87 cell strain
4.1 material
Fura-2 probe: available from Biotium company.Microplate reader: available from genome company.Other are the same.
4.2 method
The mensuration of intracellular calcium concentration.The trysinization collecting cell, adjusting cell density is 2 * 10
7Cells/ml, with the nutrient solution suspension cell that contains 10%FBS, adding final concentration is the Fura-2 of 10 μ M, the room temperature lucifuge is hatched 45min.With the cell of damping fluid wash load three times, resuspended and to adjust cell density be 5 * 106cells/ml, be inoculated in 96 hole blackboards, 200 μ l/ holes measure 340 with microplate reader, the fluorescence intensity at 380nm place.Establish following group in the experiment: cell autofluorescence group, high calcium damping fluid group, zero calcium damping fluid group.Condition determination is excitation wavelength 340nm and 380nm, excites grating 5nm; Emission wavelength 505nm, emission grating 10nm.Observe the variation of fluorescence intensity under the different treatment condition, be calculated as follows [Ca2+] i:[Ca2+ in the cell] i=Kd * (Fmin/Fmax) * (R-Rmin)/(Rmax-R).
4.3 result
The result is as shown in Figure 2: (10nM 100nM) all can significantly start the calcium mobilization of U-87 cell to fMLF.The calcium mobilization that 10nM fMLF (Fig.3A) and 100nM fMLF (Fig.3B) bring out all can significantly desensitize after acting on U-87 cell 5min in advance with F2 (5 μ g/ml, 10 μ g/ml) respectively.Therefore F2 can suppress the U-87 cell calcium mobilization that fMLF brings out.This has illustrated that from the signal path angle part F2 can suppress the reason of fMLF inductive U-87 cell chemotaxis.
Embodiment 5, be that target cell carries out F2 for the influence experiment of formyl peptides inductive U-87 cell ERK1/2 activatory with humanized's glioma U-87 cell strain
5.1 material
ERK1/2 and phosphorylation antibody thereof: all available from Cell Signaling company.ECL: available from green skies company.The goat antirabbit two anti-goat anti-mouse two that reach resist: all available from treasured letter bio tech ltd.Other are the same.
5.2 method
Western blot method.F2 with non-cytotoxicity concentration acts on U-87 cell 2h in advance, then with 100nMfMLF effect U-87 cell 2min, extracts cell protein and experimentizes.
5.3 result
The result is as shown in Figure 3: ERK is as the signal of interest molecule of cell chemotaxis, and F2 interaction energy in advance significantly suppresses fMLF inductive U-87 cell ERK phosphorylation, and part is illustrated the mechanism that F2 can suppress fMLF inductive U87 cell chemotaxis.
Embodiment 6, be that target cell carries out the influence experiment that F2 expresses for its formyl peptide receptor with humanized's glioma U-87 cell strain
6.1 material
FPR antibody: available from Santa Cruze company.Two of FITC mark resists: available from Sigma company.Other are the same.
6.2 method
Immunofluorescence dyeing.Behind 24 orifice plate 12h, PBS washs once, fixes with Paraformaldehyde 96 with cell inoculation.Seal with normal rabbit serum then.With resisting colour developing with two of FITC mark behind the FPR antibody incubation, DAPI dyes nuclear with the DNA dyestuff.
6.3 result
The result is as shown in Figure 4: 2.5-10 μ g/ml F2 handled the U87 cell 12-48 hour, can reduce the FPR protein expression.Wherein 2.5 μ g/ml F2 can reduce the FPR protein expression in the time of 48 hours, and at 12 and 24 hours FPR albumen were not had effect; 5 μ g/ml and 10 μ g/ml F2 can reduce the FPR protein expression at 12 hours.
Claims (5)
1, pycnogenols oligomer (F2) and high polymer (F3), its structural formula is as follows, and wherein the polymerization degree of F2 is 2-15, and the polymerization degree of F3 is 10-33.
2, pycnogenols oligomer (F2) and high polymer (F3) are in the protective foods of the anti-curing oncoma of preparation and the purposes in the medicine.
3, purposes according to claim 2 is characterized in that: described tumour comprises brain tumor, hematological system tumor, gi system tumour, respiratory system tumour, urogenital neoplasm.
4, purposes according to claim 3 is characterized in that: the surviving rate of F2, F3 energy time and dose-dependent inhibition people glioblastoma U-87; Can significantly suppress formyl peptides inductive U-87 cell migration; Can significantly reduce the expression of FPR in the U-87 cell.
5, pycnogenols oligomer according to claim 1 (F2) and high polymer (F3) is characterized in that: it can be mixed and made into acceptable tablet, capsule, injection liquid, electuary clinically with pharmaceutically acceptable carrier.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103833718A (en) * | 2013-04-24 | 2014-06-04 | 新乡医学院 | Preparation method and application of proanthocyanidin derivative |
| CN104774909A (en) * | 2014-10-17 | 2015-07-15 | 江苏大学 | Method for analysis of proanthocyanidins induced liver cancer cell autophagic death and application |
| WO2022205137A1 (en) * | 2021-03-31 | 2022-10-06 | 贝尔克斯生技股份有限公司 | Polymeric proanthocyanidin composition and application thereof |
-
2009
- 2009-01-09 CN CNA2009100100545A patent/CN101456854A/en active Pending
Non-Patent Citations (3)
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| 刘丽萍等: "葡萄籽原青花素的功能及提取工艺", 《食品与药品》 * |
| 张凤娇、杨静玉、牟艳华等: "葡萄籽提取物F2对人恶性胶质瘤U-87细胞的抑制增殖作用及对其甲酰肽受( FPR)功能的影响", 《沈阳药科大学学报》 * |
| 王立辉、杨静玉、申英基: "葡萄籽提取物F3对U87细胞抗肿瘤、趋化作用及机制探讨", 《沈阳药科大学学报》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103833718A (en) * | 2013-04-24 | 2014-06-04 | 新乡医学院 | Preparation method and application of proanthocyanidin derivative |
| CN104774909A (en) * | 2014-10-17 | 2015-07-15 | 江苏大学 | Method for analysis of proanthocyanidins induced liver cancer cell autophagic death and application |
| WO2022205137A1 (en) * | 2021-03-31 | 2022-10-06 | 贝尔克斯生技股份有限公司 | Polymeric proanthocyanidin composition and application thereof |
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Application publication date: 20090617 |