CN103102376B - A kind of separation method concurrently separating Vaccarin and Semen Vaccariae total saponins - Google Patents
A kind of separation method concurrently separating Vaccarin and Semen Vaccariae total saponins Download PDFInfo
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- CN103102376B CN103102376B CN201210512777.7A CN201210512777A CN103102376B CN 103102376 B CN103102376 B CN 103102376B CN 201210512777 A CN201210512777 A CN 201210512777A CN 103102376 B CN103102376 B CN 103102376B
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- 210000000582 semen Anatomy 0.000 title claims abstract description 27
- GYIKGLVKALOGDP-DSBKMWIDSA-N Isovitexin 4'-O-glucoside 2''-O-arabinoside Natural products O([C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1c1c(O)c2C(=O)C=C(c3ccc(O[C@H]4[C@H](O)[C@@H](O)[C@H](O)[C@H](CO)O4)cc3)Oc2cc1O)[C@@H]1[C@H](O)[C@H](O)[C@H](O)CO1 GYIKGLVKALOGDP-DSBKMWIDSA-N 0.000 title claims abstract description 20
- GYIKGLVKALOGDP-HLEFUGOVSA-N Vaccarin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OC1=CC=C(C=2OC3=CC(O)=C([C@H]4[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O4)O[C@H]4[C@@H]([C@@H](O)[C@@H](O)CO4)O)C(O)=C3C(=O)C=2)C=C1 GYIKGLVKALOGDP-HLEFUGOVSA-N 0.000 title claims abstract description 20
- GYIKGLVKALOGDP-UHFFFAOYSA-N Vaccarin Natural products OC1C(O)C(O)C(CO)OC1OC1=CC=C(C=2OC3=CC(O)=C(C4C(C(O)C(O)C(CO)O4)OC4C(C(O)C(O)CO4)O)C(O)=C3C(=O)C=2)C=C1 GYIKGLVKALOGDP-UHFFFAOYSA-N 0.000 title claims abstract description 20
- 229930182490 saponin Natural products 0.000 title claims abstract description 15
- 150000007949 saponins Chemical class 0.000 title claims abstract description 15
- 235000017709 saponins Nutrition 0.000 title claims abstract description 15
- 238000000926 separation method Methods 0.000 title abstract description 6
- 239000011347 resin Substances 0.000 claims abstract description 25
- 229920005989 resin Polymers 0.000 claims abstract description 25
- 238000000034 method Methods 0.000 claims abstract description 12
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 11
- 239000000284 extract Substances 0.000 claims abstract description 9
- 239000001397 quillaja saponaria molina bark Substances 0.000 claims abstract description 4
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 84
- 238000010828 elution Methods 0.000 claims description 35
- 238000010992 reflux Methods 0.000 claims description 9
- 239000008367 deionised water Substances 0.000 claims description 6
- 229910021641 deionized water Inorganic materials 0.000 claims description 6
- 239000012567 medical material Substances 0.000 claims description 4
- 230000000694 effects Effects 0.000 abstract description 13
- 239000000843 powder Substances 0.000 abstract description 12
- 239000003814 drug Substances 0.000 abstract description 9
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- 239000003480 eluent Substances 0.000 abstract description 3
- 235000013305 food Nutrition 0.000 abstract description 3
- 210000004925 microvascular endothelial cell Anatomy 0.000 abstract description 3
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- 239000012141 concentrate Substances 0.000 abstract 1
- 238000000605 extraction Methods 0.000 description 9
- 238000006243 chemical reaction Methods 0.000 description 7
- 238000001514 detection method Methods 0.000 description 7
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 6
- 239000000463 material Substances 0.000 description 6
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 5
- 239000002904 solvent Substances 0.000 description 5
- 239000000126 substance Substances 0.000 description 5
- 238000012360 testing method Methods 0.000 description 5
- 238000004128 high performance liquid chromatography Methods 0.000 description 4
- 239000004615 ingredient Substances 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- WFDIJRYMOXRFFG-UHFFFAOYSA-N Acetic anhydride Chemical compound CC(=O)OC(C)=O WFDIJRYMOXRFFG-UHFFFAOYSA-N 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 238000010521 absorption reaction Methods 0.000 description 3
- 230000033115 angiogenesis Effects 0.000 description 3
- 210000004027 cell Anatomy 0.000 description 3
- 201000010099 disease Diseases 0.000 description 3
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 3
- 229930182486 flavonoid glycoside Natural products 0.000 description 3
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- 239000000243 solution Substances 0.000 description 3
- 230000001629 suppression Effects 0.000 description 3
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 2
- 206010028980 Neoplasm Diseases 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
- 239000003463 adsorbent Substances 0.000 description 2
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- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 description 1
- 201000000736 Amenorrhea Diseases 0.000 description 1
- 206010001928 Amenorrhoea Diseases 0.000 description 1
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- 108010069514 Cyclic Peptides Proteins 0.000 description 1
- 102000001189 Cyclic Peptides Human genes 0.000 description 1
- 208000004880 Polyuria Diseases 0.000 description 1
- 239000007983 Tris buffer Substances 0.000 description 1
- 244000178320 Vaccaria pyramidata Species 0.000 description 1
- HZWXJJCSDBQVLF-UHFFFAOYSA-N acetoxysulfonic acid Chemical compound CC(=O)OS(O)(=O)=O HZWXJJCSDBQVLF-UHFFFAOYSA-N 0.000 description 1
- -1 acetyl sulphonyl Chemical group 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 239000004480 active ingredient Substances 0.000 description 1
- 230000037006 agalactosis Effects 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
- 231100000540 amenorrhea Toxicity 0.000 description 1
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- 238000004821 distillation Methods 0.000 description 1
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- 210000003725 endotheliocyte Anatomy 0.000 description 1
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- 235000013336 milk Nutrition 0.000 description 1
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- 210000004080 milk Anatomy 0.000 description 1
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- 150000002772 monosaccharides Chemical class 0.000 description 1
- 210000004165 myocardium Anatomy 0.000 description 1
- 229930014626 natural product Natural products 0.000 description 1
- 230000010046 negative regulation of endothelial cell proliferation Effects 0.000 description 1
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- 230000008961 swelling Effects 0.000 description 1
- 208000011580 syndromic disease Diseases 0.000 description 1
- YNJBWRMUSHSURL-UHFFFAOYSA-N trichloroacetic acid Chemical compound OC(=O)C(Cl)(Cl)Cl YNJBWRMUSHSURL-UHFFFAOYSA-N 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
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- 230000004614 tumor growth Effects 0.000 description 1
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- Steroid Compounds (AREA)
- Medicines Containing Plant Substances (AREA)
Abstract
The present invention provides a kind of method concurrently separating Vaccarin and Semen Vaccariae total saponins, i.e. extracts Semen Vaccariae defat dry powder with water-containing organic solvent, and is filtered by extracting solution, concentrate, dried continuation macroporous resin separates and prepares, and eluent is concentrated, is dried and forms.The present invention prepares in Semen Vaccariae the propagation to human microvascular endothelial cell (mvec) by a kind of method first simultaneously and has two kinds of components of adverse effect, separation efficiency is higher, Vaccarin and the Semen Vaccariae total saponin extracts of isolated can be used for medicine, food, the purposes such as health product and/or cosmetics.
Description
Technical field
The present invention relates to the technical field that Chinese medicine active ingredient is extracted, use alcohol reflux, method one step that HPD-100 macroporous resin separates is same
Time obtain Vaccarin and Semen Vaccariae total saponins in Semen Vaccariae, and evaluate, compare this two classes component to new vessels generation in terms of
Effect.
Background technology
Chinese medicine reflux extraction is with volatile organic solvent extraction material compositions such as ethanol, and leachate is added thermal distillation, and wherein volatile solvent evaporates
It is cooled again after going out, repeats to flow back to leach in container to extract raw material, so go round and begin again, until effective ingredient reflux, extract, method completely.Backflow
Effective ingredient in Chinese medicine can be extracted by extraction well, can avoid again the dissolution of partial impurities as far as possible.By selecting different solvents permissible
Extract required component targetedly, or the concentration changing solvent more extracts effective ingredient, reduces the dissolution of impurity.Use alcohol reflux
During extraction, it is possible to reduce the addition of solvent, it is also possible to reclaim ethanol, reducing cost, this method has become as present stage extraction Chinese medicine and effectively becomes
The conventional method divided.
Macroporous adsorbent resin is a class without cation exchange groups and the preparation that has macroporous structure, has good macroreticular structure and bigger
Specific surface area, Organic substance can be adsorbed from aqueous solution selectively by physical absorption, therefore can be according to the polarity of required extraction of substance, Yi Jiyu
The power of absorption with macroporous adsorbent resin performance selects suitable macroporous resin and corresponding eluting solvent by target components in mixture and non-targeted group
Separate.Numerous studies report shows have three dimensions solid pore structure inside macroporous resin, has physical and chemical stability height, compares table
Area is big, adsorption capacity is big, selectivity is good, adsorption rate is fast, desorption condition is gentle, Regeneration Treatment is convenient, use cycle length, be suitable for composition to be closed
The plurality of advantages such as road circulation, saving expense, have been widely used in environmental protection, food, medicine and other fields, the most extensive in China in recent years
In the extracting and developing, purification work of Chinese herbal medicine effective ingredients.The HPD-100 type macroporous resin that the present invention relates to is non-polar macroporous resin,
It is primarily adapted for use in saponin in natural product to separate with the extraction of flavonoid glycoside.
Semen Vaccariae is the conventional Chinese medicine simply that Chinese Pharmacopoeia one is included, for pinkwort Vaccaria segetalis Vaccaria segetalis (Neck.) Garcke.
Dry mature seed, there is promoting blood circulation to restore menstrual flow, the effects such as stimulating milk secretion is subsided a swelling, inducing diuresis for treating stranguria syndrome, for amenorrhea, stimulate the menstrual flow, agalactia, acute mastitis swells and ache,
The diseases such as the puckery pain of stranguria [1, Chinese Pharmacopoeia Commission. Chinese Pharmacopoeia first, China Medical Science Press, Beijing, p49.].The medicinal part of Semen Vaccariae,
Tool book on Chinese herbal medicine is recorded, polyphyly root, stem, leaf, flower and seed use, current then use seed.Semen Vaccariae mainly contains triterpene saponin, flavonoid glycoside,
Cyclic peptide, lipoid and fatty acid, monosaccharide etc..[2, Li Fan, Liang Jingyu. the progress [J] of Semen Vaccariae. Strait Pharmaceutical Journal .2007,19 (3)].Early-stage Study
Find, Semen Vaccariae exists new vessels is generated the different material with adverse effect, therefore, select suitable separation method by both pair
Angiogenesis has the material of adverse effect and separates and have preferable realistic meaning.So the present invention uses alcohol reflux, HPD-100 type is big
Method one step that hole resin separates obtains this two classes component simultaneously, and it is carried out the qualification of physicochemical property and examines the activity of new vessels nucleus formation
Survey, to follow-up, this two classes component is comprehensively utilized.
New vessels plays a significant role in human normal is grown and numerous disease develops, and has suppression or promotes the thing of angiogenesis function
Matter is respectively provided with good application prospect and the huge market demand in the fields such as medicine, food, health product, cosmetics.Such as, in treatment painstaking effort
During pipe disease, by promoting that in the existing vascular bed of ischemic myocardium endotheliocyte germinates and the formation vasoganglion that sends forth branches, thus can have
The vital sign improving cardiovascular disease of effect.Tumor growth also has to rely on relevant blood vessel to certain phase just can complete its growth, spreads and turns
The malignant activity moved, therefore treating tumor by the tumor neovasculature growth of suppression has become the study hotspot of lot of domestic and foreign pharmacy worker.
And the propagation of vascular endothelial cell is the most important condition promoting new vessels to generate, so the HMEC cell that the present invention selects, i.e. people's microvascular endothelial
Cell, with its body outer cell proliferation rate for bio-evaluation model, evaluating obtained component to the impact that new vessels generates is feasible method
One of.
Summary of the invention
It is an object of the invention to protect a kind of method concurrently separating Vaccarin and Semen Vaccariae total saponins.
Technological parameter involved in the present invention is: by Semen Vaccariae pulverizing medicinal materials, adds 4 times of petroleum ether degreasings twice, each 12 hours, volatilizes molten
After agent, defat medical material adds 10 times of volume 70% alcohol reflux twice, each 2 hours, and gained extracting solution spin concentration after filtering is the most dry,
Add water and be dried to obtain dry powder through reduced vacuum after redissolving.Gained extracts dry powder follow-up employing HPD-100 type macroporous resin and separates, and concrete operations are to treat
After sample upper prop adsorbs 2 hours, carry out gradient elution with 6 times of column volume deionized waters, 10% ethanol, 30% ethanol and 70% ethanol successively, wash
Separation of flow speed is 4ml/min.Collecting eluent rotated concentration and be vacuum dried to obtain dry powder, wherein, 30% ethanol elution component is Semen Vaccariae
Flavonoid glycoside component, 70% ethanol elution component is Semen Vaccariae total saponins component.
Beneficial effects of the present invention:
Technique of the present invention can simultaneously from Semen Vaccariae isolated angiogenesis is had two kinds of materials of adverse effect, separation efficiency compares
Height, has the material large-scale production of adverse effect for both from now on and comprehensive utilization provides technical support new vessels.
Accompanying drawing explanation
Fig. 1 and Fig. 2 relates separately to the detection of Vaccarin relevant in case study on implementation 2 and case study on implementation 3 and related component cytoactive
Measure.Fig. 1 for detect whether 30% ethanol elution component and 70% ethanol elution component contain Vaccarin by high performance liquid chromatography,
(1:HPD-100 70% ethanol elution component detection figure, 2:HPD-100 30% ethanol elution component detection figure, 3: Vaccarin standard
Product examine mapping).Fig. 2 is Vaccarin component and the impact on human microvascular endothelial cell (mvec) in-vitro multiplication rate of the Semen Vaccariae total saponins component.
Detailed description of the invention
Embodiment 1: the reflux extraction method of Semen Vaccariae sample separates with HPD-100 type macroporous resin
100g Semen Vaccariae degreaser drying powder 1000ml 70% alcohol reflux twice, each 2 hours, after cooling, filters, rotates and steam
Sending to do, deionized water redissolves, and is vacuum dried to obtain dry powder, standby.By HPD-100 type macroporous resin wet method dress post after 95% soak with ethanol,
Blade diameter length ratio is 1: 7.After not showing muddiness after being first washed till eluent and 2 times of deionized water mixing with 95% ethanol, continue to be washed with deionized water post, to be washed
Time in de-liquid without ethanol taste, standby.Above-mentioned prepared Semen Vaccariae dry powder is added deionized water dissolving, accurate absorption Semen Vaccariae sample liquid
20ml (0.013g/ml) upper prop (18cm × 2.5cm), after adsorbing two hours, successively with 6 times of column volume distilled water, 10% ethanol, 30% ethanol, 70%
Ethanol carries out gradient elution, and elution flow rate is 4ml/min.By each for macroporous resin alcohol elution fraction rotary evaporated to dryness, add water redissolution, vacuum drying,
Obtain dry powder, standby.Wherein, the extraction ratio of 30% ethanol elution component be 1.32% ((elution fraction dry powder quality/medical material defat dry powder quality) ×
100%), the extraction ratio of 70% ethanol elution component is 2.02% ((elution fraction dry powder quality/medical material defat dry powder quality) × 100%)
Embodiment 2:HPD-100 type macroporous resin 30% ethanol elution component and the qualitative identification of 70% ethanol elution component
Inspecting of 2.1 Vaccarins:
According to the condition of 2010 editions first middle HPLC method detection Vaccarin of Chinese Pharmacopoeia, to HPD-100 type macroporous resin 30%
In ethanol elution component, Vaccarin is inspected, and concrete chromatographic condition is: with methanol as mobile phase A, with 0.3% phosphoric acid solution for flowing
Phase B, detection wavelength is 280nm；Regulation according to the form below carries out gradient elution.
The flowing phase gradient table of table 1 high performance liquid chromatography detection Vaccarin
| Time (minute) | Mobile phase A (%) | Mobile phase B (%) |
| 0～10 | 35 | 65 |
| 10～20 | 35→40 | 65→60 |
| 20～35 | 40→50 | 60→50 |
By above-mentioned HPLC testing conditions, with Vaccarin as reference substance, to HPD-100 type macroporous resin 30% ethanol elution component and
70% ethanol elution component detects, and testing result shows in FIG.
Fig. 1 result shows, the retention time of Vaccarin standard substance is 8.693min, and Vaccarin is in HPD-100 type macropore tree
Fat 30% ethanol elution component is enriched with, is not detected in 70% ethanol elution component.
The qualitative reaction of 2.2 total saponins is chosen following 3 reactions and is carried out:
2.2.1, foam test: take appropriate above-mentioned macroporous resin elution fraction and be dissolved in water, after strong shaking, persistent foam can be produced then for the positive
Reaction.
2.2.2, acetic anhydride-strong sulfuric acid response: take appropriate above-mentioned macroporous resin elution fraction and be dissolved in acetic anhydride, add concentrated sulphuric acid-acetic anhydride (1: 20), produce yellow,
The color change such as red, purple, blue, finally fade then for positive reaction.
2.2.3, chloroform-strong sulfuric acid response: take appropriate above-mentioned macroporous resin elution fraction and be dissolved in chloroform, after adding concentrated sulphuric acid, present at chloroform layer red
Color or blueness, chloroform layer have that green fluorescence occurs then for positive reaction.
Experimental result such as following table.
Table 2 macroporous resin each component saponin qualitative detection result
Note: "-" is negative reaction；"+" is positive reaction.
According to table 2 result it was determined that HPD-100 type macroporous resin 30% ethanol elution component is Vaccarin component, 70% ethanol is washed
De-component is Semen Vaccariae total saponins component.
Embodiment 3: cytoactive evaluation
Take the logarithm trophophase HMEC-1 cell, be inoculated in 96 orifice plates by 8000, every hole cell, be positioned over 37 DEG C, 50ml/LCO2's
Incubator is cultivated 24h, adds HPD-100 type macroporous resin 30% and 70% ethanol elution component 48h action time, with not dosing group as feminine gender
Matched group, often organizes and sets 3 multiple holes simultaneously, and every batch sample is repeated 3 times test.Cultivate after dosing to 72h, go supernatant, every hole to add gently
100g/L trichloroacetic acid 100 μ l fixes, and moves on to 4 DEG C and places 40min again, incline and fall fixative, be washed with deionized water 5 times after standing 5min, empty
Air dry is dry.Every hole adds the bright B of 4g/L acetyl sulphonyl (SRB) 100 μ l room temperature and places 30min, washes 5 times with 10ml/L acetate solution, adds 150 μ l
10mmol/L Tris alkali liquor (pH 10.5) dissolves, and measures every hole A value by MK3 type microplate reader at wavelength A540.Suppression ratio=[(matched group
The A value of A value-medicine group)/(the A value of the A value of matched group-blank group)] × 100%.
To the HPD-100 type macroporous resin 30% ethanol elution component obtained by above-mentioned and 70% ethanol elution composition activity testing result in fig. 2
Display.
Result shows, the 30% ethanol elution component that HPD-100 type macroporous resin separates and collects, i.e. Vaccarin component have in promotion
The effect of epithelial cell proliferation, 70% ethanol elution component, i.e. Semen Vaccariae total saponins component have the effect of inhibition of endothelial cell proliferation.
The present invention is described in conjunction with the embodiments, but after having read the foregoing of the present invention, those skilled in the art can appreciate the fact that open
Embodiment in make many and change and also can obtain same or similar result, and without departing from the design of the present invention, spirit and scope.More specifically,
Obviously some alternative reagent disclosed herein of chemical and physiological related reagent and obtain same or similar result.All similar replacements and
Modify it is evident to the person skilled in the art that be regarded as in the spirit of the present invention, scope and spirit and right, the most all above-mentioned
These equivalent form of values and all improvement to technological parameter and variation all also belong to claims of the present invention limited range.
Claims (1)
1. concurrently separate Vaccarin and a method for Semen Vaccariae total saponins, it is characterized by choose Semen Vaccariae defat medical material, use 70%
10 times of volume reflux, extract, of ethanol twice, each two hours, extract obtained follow-up employing HPD-100 type macroporous resin separated, and uses successively
Deionized water, 10% ethanol, 30% ethanol and 70% ethanol carry out eluting, and elution volume is 6 times of column volumes, elution flow rate 4mL/min, and
Collecting 30% ethanol elution component and 70% ethanol elution component, extract the most i.e. respectively obtains Vaccarin component and Semen Vaccariae is total
Saponin component.
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| CN101333238A (en) * | 2007-06-25 | 2008-12-31 | 中国科学院上海药物研究所 | Triterpene compounds separated from seed of cowherb of Chinese traditional medicine and uses thereof |
| WO2009117828A1 (en) * | 2008-03-26 | 2009-10-01 | National Research Counsil Of Canada | Saponin extract from saponaria spp. and uses thereof |
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