CN1374081A - Application of safrole oxide in the growth and death of vascular endothelial cell - Google Patents
Application of safrole oxide in the growth and death of vascular endothelial cell Download PDFInfo
- Publication number
- CN1374081A CN1374081A CN 02110135 CN02110135A CN1374081A CN 1374081 A CN1374081 A CN 1374081A CN 02110135 CN02110135 CN 02110135 CN 02110135 A CN02110135 A CN 02110135A CN 1374081 A CN1374081 A CN 1374081A
- Authority
- CN
- China
- Prior art keywords
- vascular endothelial
- endothelial cell
- safrole
- safrole oxide
- oxide
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Abstract
The present invention discloses the application of safrole oxide in the growth and death of vascular endothelial cell. The said matter in the amount of 5-25 mg/L can promote the spread and growth of vascular endothelial cell and inhibit cell wall falling-off of vasculr endothelial cell and DNA fragmentation; while that in the amount of 50-100 mg/L has the functions of promoting cell wall falling-off of vascular endothelial cell and DNA fragmentation. Thus, the present invention opens one way of inducing vascular endothelial cell to treat tumor and promoting growth of vascular endothelial cell to treat vessel atrophy.
Description
(1) technical field
The present invention relates to the purposes of safrole oxide, relate in particular to the application of safrole oxide in vascular endothelial cell growth and apoptosis.
(2) background technology safrole oxide structural formula is:
Molecular formula: C
10H
10O
3
Molecular weight: 178, character: light yellow liquid, boiling point b.p.118 ℃/4mmHg, index of refraction n
201.533.
Safrole oxide was synthesized out before more than 40 years.About the report of its application, the beginning sees the seventies in 20th century, and mostly its purposes is this chemical compound is used for as substrate the spectrum analysis of oxide hydrolytic enzyme; Find that safrole (faint carcinogen) produces safrole oxide in animal body during metabolism the eighties in 20th century again.Qiao Te (Qato) in 1995 and 1996 and Gen Sina (Guenthner) have carried out studying to the associativity of safrole oxide and DNA and (have seen " Toxicol Lett " 1995,75 (1-3): 201-207 and " Drug Metab Dispos " 1996,24 (9): 1020-7), find that this chemical compound can combine with DNA external, and by combine the formation adduct with IUDR acid among the DNA, but do not detect the formation of this adduct between safrole oxide and the liver dna in animal body, further studies have shown that the covalent bond that is combined into of safrole oxide and DNA, except that mainly with guanosine nucleotide combines, it also can combine with other 3 kinds of bases of DNA, and its mechanism is relevant with methylene dioxy phenyl and epoxy construction that safrole oxide contains the tool physiologically active.But, above-mentioned all items are to safrole oxide research report, just to toxicity, the pharmacological action of safrole oxide itself, study with the situation that combines of DNA, and utilize safrole oxide in vascular endothelial cell growth and apoptotic process, to carry out pharmacological research, retrieval is looked into newly through authoritative institution, does not appear in the newspapers as yet both at home and abroad at present.
(3) summary of the invention
The object of the present invention is to provide the new purposes of safrole oxide, promptly safrole oxide influences the application and the method thereof of vascular endothelial cell growth and apoptosis.
The application of the safrole oxide that the present invention relates in vascular endothelial cell growth.
Wherein, the promotion vascular endothelial cell is sprawled and is grown, and the safrole oxide consumption that the inhibition vascular endothelial cell takes off wall and dna fragmentationization is 5～25mg/L.
The application of the safrole oxide that the invention still further relates in apoptosis of vascular endothelial cell.
Wherein, promote that the safrole oxide consumption that takes off wall and dna fragmentationization of vascular endothelial cell is 50～100mg/L.
Wherein, the vascular endothelial cell cycle is blocked in G
2The safrole oxide consumption of-M phase is 100mg/L.
In order to understand essence of the present invention better, pharmacological evaluation and the result with safrole oxide illustrates its application in vascular endothelial cell growth and apoptosis below.
The preparation of safrole oxide: (27 grams of the m-chloro-benzoic acid peroxide with 70%, 0.11 mole) be dissolved in 500 milliliters of chloroforms or the benzene, add safrole (mol ratio of safrole and m-chloro-benzoic acid peroxide is 1: 1.0～1: 1.5), stirred 2-24 hour at 0-50 ℃, behind 5%～9% alkali liquid washing three times, wash again three times, use anhydrous magnesium sulfate drying, filter the back concentrating under reduced pressure, residue carries out distilling under reduced pressure or separates with silica gel column chromatography that (developing solvent is a petrol ether/ethyl acetate=1: 1～3: 1, volume ratio), obtain pure safrole oxide, and standby after nuclear magnetic resonance, NMR (seeing accompanying drawing 1) and infrared spectrum (seeing accompanying drawing 2) checking.Its chemosynthesis reaction formula is as follows:
The preparation of vascular endothelial cell: cultivate vascular endothelial cell with conventional method, it is good and vascular endothelial cell that be in exponential phase is standby to choose growth conditions.
Adopt the method for Celluar and Molecular Biology, carry out following experiment: the research safrole oxide is to the growth of the inductive vascular endothelial cell of removal fibroblast growth factor (FGF) and the influence of apoptosis
1, inverted phase contrast microscope observation of cell morphological change, be the formation of apoptotic body: each is organized vascular endothelial cell and adds the safrole oxide processing after 12 hours and 24 hours with conventional method, the formation of the morphological change of direct observation apoptosis of vascular endothelial cell and apoptotic body under the light microscopic.Found that: the vascular endothelial cell of safrole oxide 5-25mg/L concentration processing removal FGF 24 hours, cell is sprawled and is grown and promoted, cell takes off wall and dna fragmentationization is suppressed, safrole oxide 10mg/L concentration cell cycle distribute not to have obviously influence, and what safrole oxide 50-100mg/L concentration promoted vascular endothelial cell takes off wall and dna fragmentationization (seeing accompanying drawing 3).
2, the condensing and karyorrhexis situation of fluorescence microscope nucleus: after each group vascular endothelial cell added safrole oxide and handle with conventional method, carrying out glutaraldehyde fixes, fix 12 hours for 25 ℃, dyeed 20 minutes with Hoechst 33258 in phosphate buffer solution flushing back, when under fluorescence microscope, observing apoptosis of vascular endothelial cell, condensing and the karyorrhexis situation of nucleus DNA, result show experimental group nucleus DNA condensing and cracked obviously (seeing accompanying drawing 4).
3, detect the activity of cell succinate dehydrogenase with mtt assay, to judge the vascular endothelial cell growth situation: vascular endothelial cell is inoculated in 96 well culture plates, the MTT solution 0.02ml that after safrole oxide is handled, adds 5mg/ml, cultivated 4 hours in 37 ℃ of incubators, take out each hole solution, add 0.2ml DMSO, vibrated 10 minutes, measure optical density (OD value) with enzyme-linked immunosorbent assay instrument, get the meansigma methods of each parallel hole OD value, calculate cell relative survival rate: survivaling cell %=(experimental group OD value/matched group OD value) * 100% (is blank group zeroing with not celliferous culture fluid) according to following formula.The influence of safrole oxide cell growth the results are shown in following table: medicine (mg/L) MTT/A570 survival rate (%) matched group 0.151 ± 0.049 39.40 ± 1.91 safrole oxide 5 0.168 ± 0.017
b44.71 ± 0.82
b
10??????0.362±0.037
c?????????94.73±1.81
c
25??????0.239±0.028
c?????????63.12±1.03
c
50????????0.083±0.012
c??????21.09±0.38
c
75????????0.041±0.023
c??????10.46±0.82
c
100???????0.023±0.008
c??????5.16±0.42
c
Annotate: upward safrole oxide was handled n=5 24 hours in the table.X ± s.
aP＜0.01,
bP＜0.05,
cP＜0.01 is compared with matched group.
4, flow cytometry (FCM) detects cell cycle and apoptosis: with the fixing vascular endothelial cell of respectively organizing after safrole oxide is handled of conventional method, after PI dyeing, with flow cytometer (FACSCAN type, U.S. BO company) measures cell cycle distribution and apoptosis rate, found that safrole oxide 100mg/L blocks cell cycle in G
2-M the phase, see the following form: medicine (mg/L) G
0-G
1(%) G
2-M (%) S (%) apoptosis rate % matched group 61.52 ± 1.43 21.91 ± 1.52 16.63 ± 0.92 3.24 ± 0.23 safrole oxides 10 58.50 ± 1.00
a23.28 ± 1.16
a18.25 ± 0.78
a3.16 ± 0.35
a
100??47.23±1.51
c?51.79±1.76
c?1.00±0.47
c?20.21±1.02
c
Annotate: upward safrole oxide was handled n=3 12 hours in the table.X ± s
aP＞0.05,
bP＜0.01 is compared with matched group.
5, agarose gel electrophoresis detects dna break: collect with the vascular endothelial cell after the processing of 100mg/L amount safrole oxide, extract its DNA, through the laggard row agarose gel electrophoresis of RNA enzymic digestion, after bromination second pyridine dyeing, detect the dna break situation on ultraviolet light gel imaging instrument, the result is shown with dna break (seeing accompanying drawing 5).
6, experimental data statistical procedures: experimental data is represented with mean+/-standard error, checks through t: P＜0.05 expression has notable difference.
By above-mentioned experiment and result thereof, can draw as drawing a conclusion:
Safrole oxide is inhibition apoptosis of vascular endothelial cell when 5-25mg/L concentration, promote this cell growth, and when 50-100mg/L concentration, promote apoptosis of vascular endothelial cell, safrole oxide is the material impact medicine of vascular endothelial cell growth and apoptosis, to aspect adjusting angiogenesis and the degeneration very big purposes arranged, have very big development prospect by induced tumor apoptosis of vascular endothelial cell treatment tumor with aspect promotion vascular endothelial cell growth treatment blood vessel atrophy decline.
By animal model experiment, promptly adopt rat artery ring serum-free culture to form the blood capillary method, to measure safrole oxide and suppress angiogenesis function, this conclusion has obtained further proof.
Experiment is divided into 4 groups at random: serum-free medium MCDB131 is matched group I; MCDB131+B
16Melanoma is a control Group II; The MCDB131+ safrole oxide is experimental group I; MCDB131+B
16Melanoma+safrole oxide is experimental group II.Every group of 3 culture dishs, totally 9 agar circles.Add epsilon-amino acetic acid in culture fluid, added 300mg/ml in initial 3 days, add 50mg/ml later every day, changes once the next day of culture fluid.Cultivated 15 days, observe and count the microvessel quantity of growing gradually in the artery segment incision every day under inverted microscope.
Experimental result sees the following form:
| The 3rd day microvessel quantity | The 6th day microvessel quantity | The 9th day microvessel quantity | The 12nd day microvessel quantity | The 15th day microvessel quantity | |
| Matched group I | Begin to occur | ????38 | ????75 | ????105 | ????106 |
| Control Group II | Begin to occur | ????60 | ????115 | ????135 | ????132 |
| Experimental group I | ?- | ????- | ????- | ????3～5 | ????5～8 |
| Experimental group II | ?- | Begin to occur | ????8 | ????28 | ????30 |
Found by interpretation: the blood capillary growth population is much higher than two experimental grouies around two matched group medium-sized artery rings.Particularly inoculated B
16Melanomatous control Group II is because B
16Melanoma has the effect of bringing out neovascularization, and the microvascular time is advanced to the 3rd day so control Group II is grown, and reaches about 135 to the 15th day blood capillary sum.Inoculated B equally
16Melanoma has added the experimental group II of safrole oxide simultaneously, and the zero-time that blood capillary is occurred is postponed till the 6th day, and the 15th day new vessels sum only is about 30.Confirm that safrole oxide has tangible promotion apoptosis of vascular endothelial cell and to B
16Angiogenic growth factor (tumor angionesis factor, inhibitory action TAF) that melanoma produces.Do not inoculating B
16The effect that melanomatous experimental group I, safrole oxide promote apoptosis of vascular endothelial cell and inhibition neovascularity to generate is more remarkable.
(4) description of drawings
Fig. 1 is the safrole oxide nuclear magnetic resonance map
Fig. 2 is a safrole oxide infrared spectrum collection of illustrative plates
Fig. 3 is normal control shown in the microscopically and the vascular endothelial cell after safrole oxide is handled 24 hours
Wherein: A is the normal control group, and B is an experimental group
Fig. 4 be under the fluorescence microscope shown in normal control and the condensing and karyorrhexis of handling through safrole oxide of vascular endothelial cell center
Wherein: A is the normal control group, and B is an experimental group
Fig. 5 is the dna break in the vascular endothelial cell shown in the agarose gel electrophoresis
Wherein: a is a normal control, b is the dna break in the vascular endothelial cell after 100mg/L amount safrole oxide is handled, c is the dna break in the vascular endothelial cell after the methoxy derivatives of 30mg/L amount safrole oxide is handled, and d is the dna break in the vascular endothelial cell after the ethyoxyl derivant of 30mg/L amount safrole oxide is handled
(5) specific embodiment
Synthesizing of embodiment 1 safrole oxide
With content is that 70% m-chloro-benzoic acid peroxide (27 grams, 0.11 mole) is dissolved in 500 milliliters of chloroforms, adds safrole (16.2 grams, 0.10 mole), 25 ℃ were stirred 20 hours down, wash three times (each 80 milliliters) with 5% aqueous sodium hydroxide washes after, wash again three times (each 80 milliliters), use anhydrous magnesium sulfate drying, filter the back concentrating under reduced pressure and steam chloroform, residue carries out distilling under reduced pressure, b.p.118 ℃/4mmHg, obtain pure safrole oxide 14 grams, yield is 80%.
Embodiment 2 is mixed with 5mg/L, 10mg/L, 15mg/L, 20mg/L, 25mg/L, 30mg/L, 40mg/L, 50mg/L, 60mg/L, 70mg/L, 80mg/L, 90mg/L, 100mg/L isoconcentration with safrole oxide, add respectively in a usual manner in the cultured vascular endothelial, handled 24 hours, the variation of a cell of observation in per 4 hours under inverted microscope, the result sees: the ability that the drug level of 5～25mg/L is attached on the culture plate vascular endothelial cell strengthens, the cell spreading ability strengthens, and it is vigorous to grow; When the drug level of 50～100mg/L is handled vascular endothelial cell, constantly breaking away from the culture plate bottom surface with the increase cell of drug level floats in the culture fluid, cell forms apoptotic body gradually then, typical apoptosis promptly takes place, 10% cell survival was only arranged after 24 hours, and blank group survival rate at this moment is 40%.Safrole oxide is described when 5～25mg/L concentration, has to promote vascular endothelial cell to sprawl and grow the inhibition apoptosis of vascular endothelial cell effect; And when 50～100mg/L concentration, the effect of the apoptosis of vascular endothelial cell of promotion is arranged.
Claims (5)
1, the application of safrole oxide in vascular endothelial cell growth.
2, the application described in claim 1, the promotion vascular endothelial cell is sprawled and is grown, and the safrole oxide consumption that the inhibition vascular endothelial cell takes off wall and dna fragmentationization is 5～25mg/L.
3, the application of safrole oxide in apoptosis of vascular endothelial cell.
4, the application described in claim 3 promotes that the safrole oxide consumption that takes off wall and dna fragmentationization of vascular endothelial cell is 50～100mg/L.
5, the application described in claim 3 blocks the vascular endothelial cell cycle in G
2The safrole oxide consumption of-M phase is 100mg/L.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB021101353A CN1164270C (en) | 2002-03-26 | 2002-03-26 | Application of safrole oxide in the growth and death of vascular endothelial cell |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB021101353A CN1164270C (en) | 2002-03-26 | 2002-03-26 | Application of safrole oxide in the growth and death of vascular endothelial cell |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1374081A true CN1374081A (en) | 2002-10-16 |
| CN1164270C CN1164270C (en) | 2004-09-01 |
Family
ID=4740944
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNB021101353A Expired - Fee Related CN1164270C (en) | 2002-03-26 | 2002-03-26 | Application of safrole oxide in the growth and death of vascular endothelial cell |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1164270C (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100445373C (en) * | 2006-12-04 | 2008-12-24 | 山东大学 | Application of safrole oxide in endothelial cell and nerve cell culturing system |
| CN102253191A (en) * | 2011-06-28 | 2011-11-23 | 南昌大学 | Method for quantitatively measuring cell spreading rates |
-
2002
- 2002-03-26 CN CNB021101353A patent/CN1164270C/en not_active Expired - Fee Related
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100445373C (en) * | 2006-12-04 | 2008-12-24 | 山东大学 | Application of safrole oxide in endothelial cell and nerve cell culturing system |
| CN102253191A (en) * | 2011-06-28 | 2011-11-23 | 南昌大学 | Method for quantitatively measuring cell spreading rates |
Also Published As
| Publication number | Publication date |
|---|---|
| CN1164270C (en) | 2004-09-01 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN106085868B (en) | One plant of Aspergillus and its application | |
| CN111154658B (en) | Marine fungus, novel skeleton heteroterpene derivative prepared from marine fungus, and preparation method and application of novel skeleton heteroterpene derivative | |
| CN113801032B (en) | Long-chain fatty acid glycerol alcohol compound Rubracin B, preparation method and application thereof | |
| Stierle et al. | Phomopsolides and related compounds from the alga-associated fungus, Penicillium clavigerum | |
| CN113773217B (en) | Long-chain fatty acid glycerol alcohol compound Rubracin C, preparation method and application thereof | |
| CN109988217B (en) | Betulol derivative and preparation method and application thereof | |
| CN1164270C (en) | Application of safrole oxide in the growth and death of vascular endothelial cell | |
| CN101575327B (en) | Antimalarial active isocumarans compound and composition, preparation method and application thereof | |
| CN111407764B (en) | Application of heteroterpene derivative in preparation of medicine for resisting non-alcoholic steatohepatitis and hepatic fibrosis | |
| CN112538434A (en) | Sea anemone epiphytic fungus SYSU-MS5127, and fermentation compound and application thereof | |
| EP0769064B1 (en) | A method for mass production of taxol by semi-continuous culture | |
| CN114920717A (en) | Pimarane diterpenoid compound and preparation method and application thereof | |
| CN100387232C (en) | Application of 3-benzyl-5-(2-nitrophenoxymethyl)-gamma-butyrolactone in preparing medicine for inhibiting vascular endothelial senility and apoptosis | |
| CN1172927C (en) | Alkoxy derivative of safrole oxide 2-alkoxy-piperonyl ethanol and its prepn and application | |
| CN111904965B (en) | Application of betulinic acid derivative in preparation of anti-nephropathy drugs | |
| CN102319233A (en) | The application of pterostilbene in inhibition apoptosis of vascular endothelial cell | |
| CN113773216A (en) | Long-chain fatty acid glycerol alcohol compound Rubracin A, preparation method and application thereof | |
| CN101856355A (en) | Therapeutical effect of atabrine and substitutes thereof on hepatitis B | |
| CN106344558A (en) | Lipid-lowering drug comprising wild chrysanthemum extract and preparation method of wild chrysanthemum extract | |
| CN113861157B (en) | Iridoid compound with antiviral activity and preparation method and application thereof | |
| CN100445373C (en) | Application of safrole oxide in endothelial cell and nerve cell culturing system | |
| CN100488932C (en) | Diphenylether compound, its preparation and use | |
| CN1275959C (en) | Pterocarya stenoptera extract, its preparation and use | |
| CN114933976B (en) | Monilinia mountain Monilis and application thereof | |
| CN108478558B (en) | Wartwort lactone C and E are used as the purposes of miR-29a inhibitor and apoptosis of mesenchymal stem cell inhibitor |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| C19 | Lapse of patent right due to non-payment of the annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |