CN102240315A - Purification and application of anti-angiogenesis effective part of cowherb seed - Google Patents
Purification and application of anti-angiogenesis effective part of cowherb seed Download PDFInfo
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- CN102240315A CN102240315A CN2011101160343A CN201110116034A CN102240315A CN 102240315 A CN102240315 A CN 102240315A CN 2011101160343 A CN2011101160343 A CN 2011101160343A CN 201110116034 A CN201110116034 A CN 201110116034A CN 102240315 A CN102240315 A CN 102240315A
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Abstract
The invention relates to purification and application of an anti-angiogenesis effective part of cowherb seed. Crude saponins of the anti-angiogenesis effective part in the cowherb seed are extracted, and are purified by a macroporous resin column, eluted by ethanol with different concentrations and subjected to cell activity evaluation to obtain the highest active ingredient, and the highest active ingredient is subjected to silica gel column chromatography and eluted by a chloroform-methanol system to obtain pure saponins. In the purification and application, the cell activity evaluation is used as an index, and the content of the saponins is measured by thin layer-spectrophotometry to obtain a purification process for the saponins of the anti-angiogenesis effective part of the cowherb seed.
Description
Technical field
The present invention relates in the Semen Vaccariae seed, extract thick saponin, be evaluated as index with cytoactive, with thin layer-spectrophotometry saponin content, adopt macroporous adsorbent resin method and the thick saponin of silica gel column chromatography purification refine, obtain greater activity and purer anti-new vessels effective site saponin component.
Background technology
Macroporous resin (macroporous resin) claim full porous resin polymer absorbant again, is a kind of high molecular polymer that is insoluble to acid, alkali and various organic solvents.To be adsorbed as characteristics, Organic substance had concentrated, centrifugation.The aperture of macroporous resin and specific surface area are all bigger, have the three-dimensional pore structure of three dimensions in resin inside, have the physical and chemical stability height, specific surface area is big, adsorption capacity is big, selectivity is good, adsorption rate is fast, desorption condition is gentle, Regeneration Treatment is convenient, life cycle is long, be suitable for plurality of advantages such as formation closed cycle, cost saving.The D101 macroporous adsorbent resin that this research is used has good network structure and very high specific surface area, can be by physical absorption adsorb organic compound matter selectively from aqueous solution, separate the purpose of purifying thereby reach, and have quick, efficient, convenient, sensitive, good selective.At present, the D101 macroporous adsorbent resin is widely used in the separation and purification of Chinese medicine.
Silica gel column chromatography (Silica gel column chromatography) is to separate according to different the obtaining of the absorption affinity of material on silica gel, the material that polarity is bigger is easily by silica gel adsorption generally speaking, the more weak material of polarity is difficult for by silica gel adsorption, and whole chromatography process promptly is absorption, desorbing, absorption again, desorption process again.At present, silica gel column chromatography has been applied to the monomer separation of a lot of Chinese medicines.
Semen Vaccariae [Vaccaria segetalis (neck.) Garcke] is the dry mature seed of dicotyledon Caryophyllaceae Vaccaria segetalis.Promoting the circulation of blood is stimulated the menstrual flow, expedite the emergence of stimulating milk secretion, skin ulcer is held back in detumescence; Women's amenorrhea, galactostasis, difficult labour, stranguria with blood, carbuncle, the hemorrhage grade of incised wound has very big effectiveness.Mainly contain triterpene saponin, flavonoid glycoside, cyclic peptide, lipoid and fatty acid, monosaccharide etc.Studies show that: the Semen Vaccariae water extraction has the effect [1 that suppresses angiogenesis more by force, Hua Hui, Feng Lei, Zhang Xiaoping, Zhang Lianfen, Jin Jian. Semen Vaccariae suppresses the active substance research of angiogenesis. the time precious traditional Chinese medical science traditional Chinese medicines .2009,20 (3): 698-700.], on this research basis, people such as summer star have obtained the Semen Vaccariae water decoction to suppress with purification process such as macroporous adsorbent resin method ethanol elutions the one-component group [2 of human microvascular endothelial cell (mvec) propagation, the summer star, Feng Lei, Zhang Lianfen, Li Ying, Chu Min, Jin Jian. the separation and the purification of the active component of anti-human microvascular endothelial cell (mvec) propagation in the Semen Vaccariae. biotechnology circular .2009,2:93-97.], and Gao Yueying shows that through toxicology and pharmacodynamic study the saponin that Semen Vaccariae obtains through the ethanol extraction n-butanol extraction is the effective site [3 of inhibition of endothelial cell proliferation by the saponin that different routes extract, Gao Yueying, Jiang Qiulong, Feng Lei, Qiu Liying, Jin Jian. the different extract parts of Semen Vaccariae suppress the research of rebirth blood vessel function. Strait Pharmaceutical Journal .2010,22 (12): 34-37.], therefore this research is evaluated as index with cytoactive, thin layer-spectrophotometry saponin content adopts macroporous adsorbent resin method and silica gel column chromatography that the thick saponin of Semen Vaccariae is carried out purification refine.
Summary of the invention
The objective of the invention is to be evaluated as index,, the anti-new vessels effective site of Semen Vaccariae saponin is carried out purification with thin layer-spectrophotometry saponin content with cytoactive.
Technical scheme of the present invention:
The preparation method of described vacsegoside is got the Semen Vaccariae seed, pulverizes, the petroleum ether soak degreasing, dry, add the alcoholic solution reflux, cold filtration, rotary evaporation is to doing, deionized water redissolves, and with dichloromethane, ethyl acetate, water-saturated n-butanol extraction, is spin-dried for successively, add water and redissolve, vacuum drying gets thick saponin.
The thick saponin of described macroporous adsorbent resin method purification, the dress post, it is water-soluble not muddy that 95% ethanol is washed till eluent, and the reuse deionized water is washed till does not have the alcohol flavor, and sample absorption is gone up in the water-soluble back of thick saponin, deionized water is washed till sugar-free, use 30%, 50%, 75%, 95% concentration ethanol eluting more successively, collect each eluent, be spin-dried for, add water and redissolve vacuum drying.
Described cytoactive is estimated each elution fraction, human microvascular endothelial cell (mvec) in-80 ℃ of refrigerators is recovered, normal cultivation is back with 2% culture fluid hunger, be added to behind the 24h in 96 orifice plates and cultivate, dosing is each eluent and thick saponin behind the 48h, continue to cultivate, srb assay dyeing is surveyed the OD value with microplate reader behind the dosing 48h under the 540nm wavelength.
Described silica gel column chromatography purification macroporous resin elution fraction is the eluant gradient elution with the chloroform-methanol system, controls certain flow velocity and every pipe volume.The solution of each collecting pipe is differentiated with the TLC method, is developing solvent with n-butyl alcohol-ethyl acetate-water, and 10% sulphuric acid ethanol is developer, is associated with the component of saponin material.
Described cytoactive is estimated the component that silicagel column obtains, and with said method silicagel column elution fraction and macroporous resin elution fraction, thick saponin is carried out specific activity.
Described thin layer-spectrophotometry saponin content is a reference substance with ginsenoside Re, makes a standard curve, measures the saponin content of thick saponin, macroporous resin eluent, silica gel column chromatography eluent in the range of linearity.
Beneficial effect of the present invention:
The present invention has obtained a purifying process route of the thick saponin of Semen Vaccariae, also having confirmed in the cytoactive evaluation has cytoactive preferably to human microvascular endothelial cell (mvec), the experimentation that helps the later stage, further separating than pure component of obtaining from silica gel column chromatography obtains the effective monomeric compound of cytoactive.
The specific embodiment
Embodiment 1: the extraction process of the thick saponin of Semen Vaccariae
Twice of 600mL75% alcohol reflux of 100g Semen Vaccariae seed, each 2.5h, after the cooling, filter, rotary evaporation is to doing, and deionized water redissolves to 400ml, equal-volume dichloromethane extraction 4 times, the water that obtains equal-volume ethyl acetate extraction 4 times, the water that obtains once more merges the n-butyl alcohol phase with equal-volume water-saturated n-butanol extraction 4 times.Rotary evaporation adds water and redissolves to doing, and vacuum drying gets dry powder, and is standby.
Embodiment 2: thick saponin preliminary purification technology
With the thick saponin of macroporous adsorbent resin method purification, adopt D101 model resin, the chromatographic column model specification is 20 * 300mm, applied sample amount 300mg, be dissolved in the 30ml deionized water, be washed till the Molish reaction with the 400ml deionized water and be negative, respectively collect 300ml with 30%, 50%, 75%, 95% concentration ethanol eluting successively again, rotary evaporation is to doing, adding water redissolves, vacuum drying gets dry powder, and is standby.
Embodiment 3: the purifying process of silica gel column chromatography
50% ethanol elution component of preliminary purification is further purified with silica gel column chromatography.Eluant chloroform-methanol system gradient elution, flow velocity is 5ml/min, every test tube is collected 10ml, wherein chloroform: methanol is 10: 2.5 collection 250ml, 10: 3.5 collection 350ml, collect 350ml at 10: 4, collect 450ml at 10: 5, the TLC method is differentiated saponin, and the 35th test tube begins to occur saponin, until the 126th test tube, merge the solution of 35-126 root test tube.Rotary evaporation adds water and redissolves to doing, and vacuum drying gets dry powder, and is standby.
The developing solvent of TLC method is n-butyl alcohol-ethyl acetate-water (4: 1: 5), and developer is 10% sulphuric acid ethanol, and the ginsenoside Re is a reference substance, 95 ℃ of baking oven colour developing 10min.
Embodiment 4: the cytoactive evaluation
Human microvascular endothelial cell (mvec) (HMEC-1) in-80 ℃ of refrigerators is recovered, cultivate with 20% culture fluid earlier, reinstating 10% culture fluid in second day cultivates, treat behind the cell normal growth to be added to behind the 24h in 96 orifice plates and to cultivate, 8000 cells in every hole with 2% culture fluid hunger, dosing is each elution fraction and thick saponin behind the 48h, continue to cultivate, srb assay dyeing is surveyed the OD value with microplate reader behind the dosing 48h under the 540mn wavelength.The result is as follows: thick saponin I C
50Be 12.62 μ g/ml, 30% ethanol elution component I C
50Be 39.07 μ g/ml, 50% ethanol elution component I C
50Be 6.33 μ g/ml, 75% ethanol elution component I C
50Be 25.37 μ g/ml, silicagel column elution fraction IC
50Be 5.58 μ g/ml.
Embodiment 5: thin layer-spectrophotometry saponin content
Ginsenoside Re's standard solution is mixed with 1.0mg/ml, accurately measure titer 0.1ml, 0.4ml, 0.8ml, 1.2ml, 1.5ml, 1.8ml, behind the water bath method, each adds the dilution of 200 μ l methanol, get 100 μ l point sample on the silica gel G plate, in chromatography cylinder, launch.Launch the back and take out, volatilize solvent, spray developer, develop the color in 95 ℃ of baking ovens.Scrape corresponding reddish violet band, water-bath is extracted, and extracting solution adds 0.2ml5% vanillin-glacial acetic acid and 0.8ml perchloric acid in 80 ℃ of evaporates to dryness.The close plug of mixing, 60 ℃ of water-bath 15min.Be chilled to room temperature and add glacial acetic acid 4.0ml, shake up the back and measure light absorption value down in the 560nm wavelength.With absorbance OD
560Be vertical coordinate, content of ginsenoside C (μ g/ml) is an abscissa, the drawing standard curve.
Preparing thick saponin, 50% ethanol elution component, silicagel column elution fraction concentration all is 1.0mg/ml, and the absorbance OD value of working sample is respectively 0.176,0.274,0.342 as stated above.
The present invention is described in conjunction with most preferred embodiment, yet after having read foregoing of the present invention, those skilled in the art can understand and do many changes also can obtain identical or similar result in disclosed embodiment, and do not exceed design of the present invention, spirit and scope.More particularly, obviously some chemistry obtains identical or similar result with the alternative reagent disclosed herein of physiological related reagent.All similarly replace and modify for a person skilled in the art, obviously think all in spirit of the present invention, scope and design and the claim scope that promptly all above-mentioned these equivalent form of values all fall within claims of the present invention institute restricted portion equally.
Claims (8)
1. with the defat of Semen Vaccariae seed, alcohol reflux obtains total glycosides to claim with petroleum ether, and reuse dichloromethane, ethyl acetate, water-saturated n-butanol extract successively and obtain the thick saponin of anti-new vessels effective site.
Claim with the thick saponin of the described effective site of claim 1 with the macroporous adsorbent resin method through the different concentration ethanol eluting, each eluent is carried out the cytoactive evaluation, and with thin layer-spectrophotometry content.
3. claim is carried out silica gel column chromatography with the highest elution fraction of the described cytoactive of claim 2, with the chloroform-methanol system as the eluant gradient elution, merge required component with TLC method discriminated union, carry out the cytoactive evaluation, and with thin layer-spectrophotometry content.
4. claim becomes similar route after improving on the described route of claim 1 basis, and the thick saponin of anti-new vessels effective site that obtains.
5. claim becomes similar route after improving on the described route of claim 2 basis, and the highest elution fraction of cytoactive that obtains.
6. claim becomes similar route after improving on the described route of claim 3 basis, and the purer effective site saponin that obtains.
7. the active component saponin that obtains by claim 1,2,3 routes of claim is used for generating with anti-new vessels the prevention and the treatment of other relevant disease, and the different product of making all belongs to claim scope of the present invention.
8. the feature of claim 7 is that the disease that is used for has the outgrowth diseases of aberrant angiogenesis such as tumor, diabetes, retinopathy, hemangioma, Niu Pi Ringworm, rheumatoid arthritis, menoxenia; After coronary heart disease, the scheming infarction, behind the cerebral infarction, the disease that suppresses of angiogenesis such as vascular occlusion thromboangiitis.Product form comprises pharmaceutical preparation, nutriment, health product, cosmetics.
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| CN2011101160343A CN102240315A (en) | 2011-05-06 | 2011-05-06 | Purification and application of anti-angiogenesis effective part of cowherb seed |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103102376A (en) * | 2012-12-05 | 2013-05-15 | 江南大学 | Separation method for simultaneously separating cowherb seed flavonoid glycoside and cowherb seed total saponin |
| CN111265448A (en) * | 2020-03-30 | 2020-06-12 | 江南大学 | Cowherb seed extract toning lotion and preparation method thereof |
-
2011
- 2011-05-06 CN CN2011101160343A patent/CN102240315A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103102376A (en) * | 2012-12-05 | 2013-05-15 | 江南大学 | Separation method for simultaneously separating cowherb seed flavonoid glycoside and cowherb seed total saponin |
| CN103102376B (en) * | 2012-12-05 | 2016-08-31 | 江南大学 | A kind of separation method concurrently separating Vaccarin and Semen Vaccariae total saponins |
| CN111265448A (en) * | 2020-03-30 | 2020-06-12 | 江南大学 | Cowherb seed extract toning lotion and preparation method thereof |
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Application publication date: 20111116 |