CN106349325B - The method that separating ursolic acid is extracted from olive growing leaves - Google Patents
The method that separating ursolic acid is extracted from olive growing leaves Download PDFInfo
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- CN106349325B CN106349325B CN201610728727.0A CN201610728727A CN106349325B CN 106349325 B CN106349325 B CN 106349325B CN 201610728727 A CN201610728727 A CN 201610728727A CN 106349325 B CN106349325 B CN 106349325B
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- C07—ORGANIC CHEMISTRY
- C07J—STEROIDS
- C07J63/00—Steroids in which the cyclopenta(a)hydrophenanthrene skeleton has been modified by expansion of only one ring by one or two atoms
- C07J63/008—Expansion of ring D by one atom, e.g. D homo steroids
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Abstract
The invention discloses a kind of method that ursolic acid is extracted from olive growing leaves, comprise the following steps：1) olive growing leaves are crushed, is extracted with the ethanol solution that volumetric concentration is 70~90%, extract solution is filtered, takes decompression filtrate recycling ethanol, obtains crude extract；2) it is that 50~65% ethanol solutions dissolve again crude extract to be added into volumetric concentration, obtains mixed solution；3) by the large pore resin absorption columns of AB on mixed solution 8, first it is eluted with water to colourless, then eluted using 40~50 DEG C, 0.1~0.15mol/L sodium bicarbonate solution, eluted again with the sodium bicarbonate solution of 75~78 DEG C, 0.6~0.8mol/L, third time eluent is collected, adjusts pH to 3~5, filtering, filter residue and drying is taken, is produced.The ursolic acid purity that the present invention extracts is high, and yield is big.
Description
Technical field
The invention belongs to technical field of biological extraction, and in particular to a kind of side that separating ursolic acid is extracted from olive growing leaves
Method.
Background technology
Olive (Olea europare L.) is world-renowned woody oil tree species, and the cultivation for having more than 4000 years is gone through
History.Among the people medication of the decoction or preserved material of olive growing leaves in mediterranean country frequently as diabetes, there is the effect of certain.Olive
Leaf extract is used as dietary supplements to strengthen immunologic function in America and Europe.Modern data shows, contains in olive growing leaves
The active ingredients such as flavonoids, triterpenes, carbohydrate, row iridoids.Triterpene compound is mainly by 6 isoprene structures
Unit is formed, and most of is the terpenoid containing 30 carbon atoms.Triterpenes components mainly include olive in olive growing leaves
Acid, crataegolic acid and ursolic acid, and these three structures of matter are similar, generally use Column chromatography techniques are separated, but repeatedly
Silica gel column chromatography separates and recrystallization causes the sample size that finally gives seldom.Again due to Column chromatography techniques generally existing processing week
Phase is grown, and continuous operation property is not strong, is not easy the industrial production process for being used for bioactive substance as separating medium.
The content of the invention
Present invention solves the technical problem that it is to provide a kind of method that ursolic acid is extracted from olive growing leaves, this method letter
Single, the cycle is short, and obtained ursolic acid purity is high, and yield is big.
Technical scheme provided by the invention is the method that ursolic acid is extracted from olive growing leaves, is comprised the following steps：
1) after olive growing leaves are crushed, extracted with the ethanol solution that volumetric concentration is 70~90%, extract solution is filtered, taken
Decompression filtrate recycling ethanol, obtain crude extract；
2) it is that 50~65% ethanol solutions dissolve again crude extract to be added into volumetric concentration, obtains mixed solution；
3) by AB-8 large pore resin absorption columns on mixed solution, be first eluted with water to colourless, then using 40~50 DEG C,
0.1~0.15mol/L sodium bicarbonate solution is eluted, then with 75~78 DEG C, 0.6~0.8mol/L sodium bicarbonate solution
Eluted, collect third time eluent, adjusted pH to 3~5, filtering, take filter residue and drying, produce.
In step 1), volumetric concentration is used to extract olive growing leaves for 70~90% ethanol solution, can abundant extract oil
Oleanolic acid, crataegolic acid and ursolic acid in olive leaf.The solid-liquid ratio of olive growing leaves and ethanol solution is 1g：10~20ml.
Described to be extracted as heating and refluxing extraction, extraction time is 1~3 time, and each extraction time is 1~3h.
In step 2), use volumetric concentration to dissolve crude extract for 50~65% ethanol solution, can remove pole in crude extract
The less impurity of property, play the effect of preliminary purification.It is 0.06~0.10g/ml to add ethanol solution to crude extract concentration, now
The adsorption effect of upper prop is optimal.
In step 3), the macroreticular resins of AB-8 models is low pole, and average pore size is 13~14nm, specific surface area 480
~520m2/g.Because oleanolic acid, crataegolic acid, ursolic acid are low pole material, and there is certain hydrophobicity, generate hydrogen
The ability of key is weaker, thus is easily adsorbed by nonpolar and low pole resin.Secondly, during macroporous absorbent resin adsorbent, quilt
Absorbed component is to be diffused into resin internal surface of hole by the aperture of resin and adsorbed, when resin aperture be less than absorption it is effective into
During fractionated molecule, active ingredient molecule can not enter inside resin, can only adsorb in resin outer surface, when resin aperture is more than effectively
During component molecules, active ingredient can be adsorbed also can be adsorbed on inner surface in resin outer surface, to crataegolic acid, oleanolic acid and
For ursolic acid, its molecular weight is respectively 472,456 and 456, therefore preferable using the adsorption effect of AB-8 macroreticular resins.Resin
The blade diameter length ratio of post is 1:3~6 are advisable, with 1:4 is optimal.
Oleanolic acid, crataegolic acid and ursolic acid structure are very much like, and wherein ursolic acid and oleanolic acid are even more isomerism
Body, common column chromatography are difficult to be isolated to come, but the present invention uses 40~50 DEG C, 0.1~0.15mol/L bicarbonate
Sodium solution can be larger with polarity in resin hawthorn acid reaction generation soluble sodium salt, and then elute, and polarity is less than normal
Oleanolic acid and the carboxyl of ursolic acid and the polarizability of hydrone it is poor, it is impossible to ionize out H+, can not enter with sodium acid carbonate
Row reaction generation sodium salt, then stay in resin column.The elution speed of sodium bicarbonate solution is 1~1.5BV/h, its dosage is 1~
During 1.5BV, elution effect is preferable.
Eluted using the sodium bicarbonate solution of 75~78 DEG C, 0.6~0.8mol/L, can now elute ursolic acid,
And oleanolic acid is not eluted, because oleanolic acid and crataegolic acid structure are isomer, therefore, using faster elution speed
2~2.5BV/h is spent, less amount of eluent is 0.5~1BV, can at utmost elute ursolic acid, and oleanolic acid is not washed
It is de-.
Method of the invention is simple, the cycle is short, and the ursolic acid purity of extraction is high, and yield is big.
Embodiment
The present invention is further elaborated for specific examples below, but not as a limitation of the invention.
Embodiment 1
1) olive growing leaves are crushed to 100 mesh, by solid-liquid ratio are 1g with the ethanol solution that volumetric concentration is 70%：10ml's
Ratio mixes, and heating and refluxing extraction 3h, extract solution is filtered, takes decompression filtrate recycling ethanol, obtains crude extract；
2) it is that 50% ethanol solution to crude extract concentration is 0.06g/ml by crude extract addition volumetric concentration, stirs evenly, obtain
Mixed solution；
3) by AB-8 large pore resin absorption columns on mixed solution, resin column blade diameter length ratio is 1:3, first it is eluted with water to colourless,
Then 40 DEG C of addition, 0.1mol/L sodium bicarbonate solution are eluted, and the elution speed of sodium bicarbonate solution is 1BV/h, its
Dosage is 1BV, then is eluted with the sodium bicarbonate solution of 75 DEG C, 0.6mol/L, elution speed 2BV/h, and its dosage is
0.5BV, third time eluent (i.e. the sodium acid carbonate eluent of 0.6mol/L) is collected, adjusts pH to 3, filtering, take filter residue and drying,
Produce.
It is measured according to high performance liquid chromatography, its purity is 99.1%, yield 90.5%.
Embodiment 2
1) olive growing leaves are crushed to 180 mesh, by solid-liquid ratio are 1g with the ethanol solution that volumetric concentration is 90%：20ml's
Ratio mixes, and heating and refluxing extraction 3 times, each extraction time is 3h, and extract solution is filtered, takes decompression filtrate recycling ethanol, is obtained
To crude extract；
2) it is that 65% ethanol solution to crude extract concentration is 0.10g/ml by crude extract addition volumetric concentration, stirs evenly, obtain
Mixed solution；
3) by AB-8 large pore resin absorption columns on mixed solution, resin column blade diameter length ratio is 1:6, first it is eluted with water to colourless,
Then 50 DEG C of addition, 0.15mol/L sodium bicarbonate solution are eluted, and the elution speed of sodium bicarbonate solution is 1.5BV/h,
Its dosage is 1.5BV, then is eluted with the sodium bicarbonate solution of 78 DEG C, 0.8mol/L, elution speed 2.5BV/h, and it is used
Measure as 1BV, collect third time eluent (i.e. the sodium acid carbonate eluent of 0.8mol/L), adjust pH to 5, filter, take filter residue to do
It is dry, produce.
It is measured according to high performance liquid chromatography, its purity is 99.0%, yield 90.6%.
Embodiment 3
1) olive growing leaves are crushed to 150 mesh, by solid-liquid ratio are 1g with the ethanol solution that volumetric concentration is 90%：20ml's
Ratio mixes, and heating and refluxing extraction 2 times, each extraction time is 2h, and extract solution is filtered, takes decompression filtrate recycling ethanol, is obtained
To crude extract；
2) it is that 55% ethanol solution to crude extract concentration is 0.08g/ml by crude extract addition volumetric concentration, stirs evenly, obtain
Mixed solution；
3) by AB-8 large pore resin absorption columns on mixed solution, resin column blade diameter length ratio is 1:4, first it is eluted with water to colourless,
Then 45 DEG C of addition, 0.12mol/L sodium bicarbonate solution are eluted, and the elution speed of sodium bicarbonate solution is 1.2BV/h,
Its dosage is 1.2BV, then is eluted with the sodium bicarbonate solution of 76 DEG C, 0.7mol/L, elution speed 2.5BV/h, and it is used
Measure as 0.5BV, collect third time eluent (0.7mol/L sodium acid carbonate eluent), adjust pH to 4, filtering, take filter residue to do
It is dry, produce.
It is measured according to high performance liquid chromatography, its purity is 99.5%, yield 93.5%.
Embodiment 4
1) olive growing leaves are crushed to 200 mesh, by solid-liquid ratio are 1g with the ethanol solution that volumetric concentration is 70%：20ml's
Ratio mixes, and heating and refluxing extraction 3 times, each extraction time is 1h, and extract solution is filtered, takes decompression filtrate recycling ethanol, is obtained
To crude extract；
2) it is that 65% ethanol solution to crude extract concentration is 0.06g/ml by crude extract addition volumetric concentration, stirs evenly, obtain
Mixed solution；
3) by AB-8 large pore resin absorption columns on mixed solution, resin column blade diameter length ratio is 1:3, first it is eluted with water to colourless,
Then 50 DEG C of addition, 0.1mol/L sodium bicarbonate solution are eluted, and the elution speed of sodium bicarbonate solution is 1.5BV/h,
Its dosage is 1BV, then is eluted with the sodium bicarbonate solution of 75 DEG C, 0.8mol/L, elution speed 2BV/h, and its dosage is
1BV, third time eluent (i.e. the sodium acid carbonate eluent of 0.8mol/L) is collected, adjust pH to 3~5, filtering, take filter residue to do
It is dry, produce.
It is measured according to high performance liquid chromatography, its purity is 99.0%, yield 91.2%.
Claims (6)
1. the method for ursolic acid is extracted from olive growing leaves, it is characterised in that：Comprise the following steps：
1) after olive growing leaves are crushed, extracted with the ethanol solution that volumetric concentration is 70~90%, extract solution is filtered, takes filtrate
Ethanol is recovered under reduced pressure, obtains crude extract；
2) it is that 50~65% ethanol solutions dissolve again crude extract to be added into volumetric concentration, obtains mixed solution；
3) by AB-8 large pore resin absorption columns on mixed solution, be first eluted with water to colourless, then using 40~50 DEG C, 0.1~
0.15mol/L sodium bicarbonate solution is eluted, then is carried out with the sodium bicarbonate solution of 75~78 DEG C, 0.6~0.8mol/L
Elution, third time eluent is collected, adjust pH to 3~5, filtering, take filter residue and drying, produce.
2. the method according to claim 1 that ursolic acid is extracted from olive growing leaves, it is characterised in that：In step 1), oil
The solid-liquid ratio of olive leaf and ethanol solution is 1g：10~20ml.
3. the method according to claim 1 that ursolic acid is extracted from olive growing leaves, it is characterised in that：In step 1), institute
State extraction time 1~3 time, each extraction time is 1~3h.
4. the method according to claim 1 that ursolic acid is extracted from olive growing leaves, it is characterised in that：In step 2), add
It is 0.06~0.10g/ml to enter ethanol solution to crude extract concentration.
5. the method according to claim 1 that ursolic acid is extracted from olive growing leaves, it is characterised in that：It is dense in step 3)
The elution speed for spending the sodium bicarbonate solution for 0.1~0.15mol/L is 1~1.5BV/h, and its dosage is 1~1.5BV.
6. the method according to claim 1 that ursolic acid is extracted from olive growing leaves, it is characterised in that：It is dense in step 3)
The elution speed for spending the sodium bicarbonate solution for 0.6~0.8mol/L is 2~2.5BV/h, and its dosage is 0.5~1BV.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102370713A (en) * | 2010-08-26 | 2012-03-14 | 苏州宝泽堂医药科技有限公司 | Preparation method for momordin |
| CN103169771A (en) * | 2011-12-23 | 2013-06-26 | 中国科学院兰州化学物理研究所 | Method for extracting maslinic acid and oleanolic acid containing mixture from olea europaea L. pomace |
| CN104876998A (en) * | 2015-05-28 | 2015-09-02 | 天津大学 | Method for adopting pH gradient elution method to synchronously separate triterpenoid saponins and licorice flavonoid |
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- 2016-08-25 CN CN201610728727.0A patent/CN106349325B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102370713A (en) * | 2010-08-26 | 2012-03-14 | 苏州宝泽堂医药科技有限公司 | Preparation method for momordin |
| CN103169771A (en) * | 2011-12-23 | 2013-06-26 | 中国科学院兰州化学物理研究所 | Method for extracting maslinic acid and oleanolic acid containing mixture from olea europaea L. pomace |
| CN104876998A (en) * | 2015-05-28 | 2015-09-02 | 天津大学 | Method for adopting pH gradient elution method to synchronously separate triterpenoid saponins and licorice flavonoid |
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