CN103012542A - Method for extracting high-purity ursolic acid from paulownia leaves - Google Patents
Method for extracting high-purity ursolic acid from paulownia leaves Download PDFInfo
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- CN103012542A CN103012542A CN2012105575345A CN201210557534A CN103012542A CN 103012542 A CN103012542 A CN 103012542A CN 2012105575345 A CN2012105575345 A CN 2012105575345A CN 201210557534 A CN201210557534 A CN 201210557534A CN 103012542 A CN103012542 A CN 103012542A
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Abstract
The invention discloses a method for extracting high-purity ursolic acid from paulownia leaves. The method comprises the following steps: smashing paulownia leaves, extracting the smashed paulownia leaves with alcohol to obtain an extract; adding water into the extract for boiling; taking the water precipitation at the lower layer, dissolving the water precipitation with alcohol; adjusting pH to 9.0 to 11.0 with aqueous solution of sodium hydroxide at the temperature of 40 to 70 DEG C; filtering to remove precipitation; adjusting the pH to 1.0 to 3.0 with hydrochloric acid; filtering to take the precipitation; taking the precipitation once more; dissolving the precipitation with ethanol, and decolorizing and crystallizing with activated carbon to obtain the ursolic acid. The extracting method is simple, convenient and quick; the used reagent is only limited to ethanol, sodium hydroxide, hydrochloric acid, activated carbon and other auxiliary materials approved by pharmacopoeia rather than organic solvent and macroporous resin; no endogenic impurities are introduced; the obtained ursolic acid is stable in crystal form, small in impurity, and 98%-99.5% in purity, meets the reporting requirement of a new medicament, and can be used as a raw material of injection preparation.
Description
Technical field
The present invention relates to a kind of method of from plant, extracting the medical active single compound, but be specifically related to a kind of method of from Folium paulowniae, extracting the ursolic acid crystal of highly purified injection.
Background technology
Ursolic acid is a kind of triterpene compound that is present in the natural phant, have the various biological effects such as calmness, anti-inflammatory, antibiotic, anti-diabetic, antiulcer agent, lowering blood glucose, ursolic acid also has obvious anti-oxidant function, thereby is widely used as medicine and cosmetic material.Ursolic acid is widely distributed at occurring in nature, has in the various plants such as glossy privet, loquat, hawthorn, paulownia etc.Compare with other plant (such as glossy privet, loquat etc.), although ursolic acid content in Folium paulowniae is moderate, but paulownia are widely distributed, and paulownia mainly as timber with tree, its leaf is with low cost, it is convenient to collect, be suitable for scale operation, so lower than other plant on cost.The method of extracting ursolic acid from plant is more, reflux extraction is arranged, enzymolysis process, ultrasonic extraction, the normal-temperature superhigh pressure extraction method, the soybean dietary fiber method, percolation etc., in all multi-methods, adopt the solvent refluxing simple and convenient extraction, and process stabilizing, be suitable for continuous production, but when refluxing extraction with an organic solvent, often introduce sherwood oil, ethyl acetate, chloroform, the compositions such as methyl alcohol, these materials have certain toxicity, in a single day the residual of these organic solvents arranged in the ursolic acid, when being applied in human body and the animal body as medicine especially injectable drug, then will cause uncertain physiological response, in addition, there is report to use macroporous resin separating-purifying ursolic acid, because of macroporous resin as macromolecular material, the unknowable impurity of wherein introducing is more, the ursolic acid that obtains of producing is not suitable for pharmaceutical prod yet.
For the extraction of ursolic acid, the Patents report is arranged, for example, patent CN1453289 discloses a kind of method of extracting separating ursolic acid from plant, and the method is take Folium paulowniae as raw material.Used extraction solvent is methyl alcohol, and the extract washing with acetone has methyl alcohol, acetone remnants in the gained ursolic acid, ursolic acid is polluted, and is not suitable for pharmaceutical prod.Patent CN101205248 discloses the method for preparing ursolic acid take Folium paulowniae as raw material, and the method macroporous resin extraction ursolic acid is introduced unknown impuritie, also is not suitable for pharmaceutical prod.Patent CN1629180A discloses a kind of method of acid-base process for preparing ursolic acid, and the raw material of the method does not relate to Folium paulowniae widely distributed, cheap and easy to get, and the requirement that the purity of the ursolic acid of gained can not fulfilling medicinal.
Summary of the invention
The present invention is directed to the deficiencies in the prior art, a kind of method of extracting high-purity ursolic acid from Folium paulowniae is provided, the method gained ursolic acid purity is high, stable crystal form, does not introduce exogenous impurity, and is safe, can be directly used in pharmaceutical prod.
Technical solution of the present invention is as follows:
A kind of method of extracting high-purity ursolic acid from Folium paulowniae is characterized in that may further comprise the steps:
(1) Folium paulowniae is pulverized, with alcohol reflux twice, united extraction liquid is recycled to without the alcohol flavor, gets extract;
(2) add entry in extract, be heated to boiling, make extract form suspension, then at room temperature leave standstill, pour out supernatant liquor, it is stand-by to stay layer depositing in water thing;
(3) add ethanol in the depositing in water thing, be heated to moltenly, keeping solution temperature is 40-70 ℃, regulates pH to 9.0-11.0 with aqueous sodium hydroxide solution, removes by filter precipitation, then uses the salt acid for adjusting pH to 1.0-3.0, filters taking precipitate; Transfer pH, hydrochloric acid to transfer the pH step process one time according to above-mentioned dissolve with ethanol, sodium hydroxide again throw out, get sedimentable matter;
(4) with the sedimentable matter dissolve with ethanol, add activated carbon decolorizing, after the decolouring with solution at 15-30 ℃ of lower crystallization, filter, collect crystal, be ursolic acid.
The used Folium paulowniae of the inventive method can be the leaf of Paulownia elongata (P. elongata), royal paulownia (P.tomentosa), fortune paulownia (P.fortunei) or river paulownia (P.fargesii).
Folium paulowniae of the present invention will be pulverized when extracting, and in the Folium paulowniae of pulverizing, should be lower than 5% greater than the shive content of 10mm * 10mm.
In the above-mentioned steps (1), the ethanol volumetric concentration is 85 ~ 95%, and the weight ratio of ethanol and Folium paulowniae is 6:1 ~ 12:1, and preferred, the ethanol volumetric concentration is 95%, and the weight ratio of ethanol and Folium paulowniae is 10:1.
In the above-mentioned steps (1), extract 2h at every turn.
In the above-mentioned steps (2), add entry the soluble impurity in the extract is removed, the add-on of water is 20-25 times of extract quality, is preferably 20 times.
In the above-mentioned steps (3), aqueous sodium hydroxide solution concentration is 0.1 ~ 2mol/L, and concentration of hydrochloric acid is 0.1 ~ 2mol/L, when regulator solution alkalescence, should maintain the temperature between 40-70 ℃, thereby improve the ursolic acid yield, and preferred, keeping solution temperature is 60 ℃.
In the above-mentioned steps (3), the depositing in water thing is 20-25 times of depositing in water amount with the dissolve with ethanol of volumetric concentration 80-85%, the consumption of ethanol, preferred 20 times.
In the above-mentioned steps (3), being 11.0 with sodium hydrate regulator solution pH preferably, is 2.0 with the salt acid for adjusting pH.
In the above-mentioned steps (4), the sedimentable matter dissolve with ethanol of volumetric concentration more than 90%, the temperature of ethanol is 50-80 ℃.
In the above-mentioned steps (4), activated carbon dosage is the 1%-5% of sedimentable matter quality, and the activated carbon decolorizing number of times is 2-5 time.Activated carbon decolorizing can improve the quality of ursolic acid, makes ursolic acid be easier to crystallization, improves the yield of ursolic acid, if the product that do not decolour is difficult to crystallization, wherein activated carbon dosage 2%, and 3 times the effect of decolour is best, product yield height.
In the above-mentioned steps (4), crystallization time is 48-240h.
In the inventive method, used ethanol, hydrochloric acid, sodium hydroxide, gac are medicinal rank, and institute's water is distilled water.
Simple and convenient extraction of the present invention is quick, employed reagent only limits to the pharmacopeia such as ethanol, sodium hydroxide, hydrochloric acid, gac and records the auxiliary material that approval is used, not not with an organic solvent and macroporous resin, introduce without exogenous impurity, gained ursolic acid stable crystal form, impurity is few, purity is more than 98%, reach as high as more than 99.5%, meet the new drug requirements of customs declaration, can be used as the material medicine of injection formulations.It is good that the emulsion injection liquid that adopts the present invention to extract the ursolic acid preparation of gained is injected rear security in animal body, can not cause toxic reaction.
Embodiment
The present invention will be further elaborated below by specific embodiment, should be understood that, following explanation only is in order to explain the present invention, its content not to be limited.If no special instructions, following used ethanol is volumetric concentration.
The hydrochloric acid of using among the following embodiment, sodium hydroxide and gac, ethanol all should meet the pharmacopeia regulation, can buy in market.
The used Folium paulowniae of the inventive method can be any one in Paulownia elongata, royal paulownia, fortune paulownia or the river paulownia, and used Folium paulowniae is fortune paulownia among the following embodiment.
Embodiment 1
1. get the Folium paulowniae that dries, pulverize, the gained fragment is lower than 5% greater than the shive content of 10mm * 10mm, get the Folium paulowniae of pulverizing, add 95% alcohol reflux of 10 times of amounts, extract twice, each two hours, extracting solution is merged, and Recycled ethanol to solution is distinguished the flavor of without alcohol, the extract obtained weight of weighing, add 20 times of amount distilled water, be heated to boiling, make extract form suspension, left standstill 12 hours under the room temperature.
2. get the ursolic acid depositing in water liquid of layering, slowly incline and upper water liquid and discard, with 15 times of amount ethanol heating for dissolving, keeping solution temperature is 60 ℃, transfers to PH11.0 with 1mol/L sodium hydroxide with lower sediment, centrifugal or remove by filter precipitation, supernatant liquor is transferred to PH2.0 with 1mol/L hydrochloric acid, centrifugal or filtration, abandoning supernatant, transfer pH, hydrochloric acid to transfer the pH step to process again the collecting precipitation thing one time according to dissolve with ethanol, sodium hydroxide lower sediment;
3. with 95% dissolve with ethanol of throw out with 50-80 ℃, add the activated carbon decolorizing that is equivalent to weight of precipitate 2%, repeatedly decolour 3 times, this moment, solution was faint yellow.
4. solution is put crystallization under the room temperature, after about 4 days, with solution filter, collected crystallization, be ursolic acid, gained ursolic acid crystal is white, needle-shaped crystals, and stable crystal form carries out three batches of experiments according to the method described above, and acquired results is as shown in the table:
Wherein, the purity determination method adopts pharmacopeia HPLC detection method commonly used: stationary phase is kromasil C18 chromatographic column (4.6*250mm, 5 μ m), and moving phase is acetonitrile-water (90:10), flow velocity 1mL/min, and 25 ℃ of column temperatures detect wavelength 210nm.Detect respectively reference substance and product of the present invention, by product of the present invention and reference substance relatively Reinheitszahl.
Ursolic acid yield method of calculation are: ursolic acid crystallization weight/Folium paulowniae weight * 1,000 ‰.
Can find out that from upper table data it is more to process more ursolic acid of paulownia yield, this may be the reason of Folium paulowniae loss less in leaching process when many, and yield was large when the ursolic acid yield was than the laboratory lab scale when producing in enormous quantities in a word.
Still contain certain ursolic acid behind the crystallization of solution in the mother liquor of gained, further concentrated mother liquor can also obtain ursolic acid by crystallization, and still gained ursolic acid purity is lower than 98% after testing, can not be directly as medicinal, but can be used for the low field of other purity requirements.
Embodiment 2
1. get the Folium paulowniae 10kg that dries rear simple pulverizing, 85% alcohol reflux that adds 12 times of amounts, extract twice, each two hours, extracting solution is merged, Recycled ethanol to solution is distinguished the flavor of without alcohol, the extract obtained weight of weighing adds 25 times of amount distilled water, is heated to boiling, make extract form suspension, left standstill 12 hours under the room temperature.
2. get the ursolic acid depositing in water liquid of layering, slowly incline and upper water liquid and discard, with 12 times of amount ethanol heating for dissolving, keeping solution temperature is 40 ℃, transfers to pH9.0 with 0.1mol/L sodium hydroxide with lower sediment, centrifugal or remove by filter precipitation, supernatant liquor is transferred to pH3.0 with 0.1mol/L hydrochloric acid, centrifugal or filtration, abandoning supernatant, transfer pH, hydrochloric acid to transfer the pH step to process again the collecting precipitation thing one time according to dissolve with ethanol, sodium hydroxide lower sediment.
3. with 90% dissolve with ethanol of throw out with 50-80 ℃, add the activated carbon decolorizing that is equivalent to weight of precipitate 1%, repeatedly decolour 5 times, this moment, solution was faint yellow.
4. solution is put 15-20 ℃ of lower crystallization, behind about 180h, with solution filter, collected crystallization, get white, needle-shaped crystals, resulting ursolic acid crystal purity is high, and detecting purity through HPLC is 99.04%, and yield is 4.87 ‰.
Comparative Examples 1
Adopt the method for patent CN1629180A to extract ursolic acid, method is:
Get commercially available Folium paulowniae 10kg, after the pulverizing, add water 100kg, infiltrate and boiled 1 hour after 30 minutes, filter, abandon liquid to boil water, filter residue adds 60kg water again, boils 1 hour, gets filter residue, oven dry, pulverizing; 1 part of rear Folium paulowniae slag (doing) is boiled in water intaking, adds 10 parts of 95% ethanol of 85-(g/v), and leaching 2 hours refluxes in Chinese medicament extracting device, collect alcohol extract (1) after filtering, residue is used 90-95% ethanol (1:8, g/v) refluxing extraction 2 hours again, gets alcohol extract (2); Merge (1), (2) alcohol extract;
Add 0.3% gac (g/v) in alcohol extract, whip attachment 60 minutes is filtered, is removed residue, gets Folium paulowniae decolouring alcohol extract;
Above-mentioned decolouring alcohol extract is transferred pH to 11 with lmol/LNaOH, hold over night, centrifugal or suction filtration except precipitated impurities, gets alkaline alcohol extract;
Alkaline alcohol extract is transferred pH to 3 with 6mol/LHCl, and through concentrating under reduced pressure, suction filtration gets Folium paulowniae acid alcohol precipitate.
In Folium paulowniae acid alcohol precipitate, add the acid distilled water of 100 times of (v/v) pH3, stir and dissolve and wash away 15 minutes, 100 times of (v/v) pH12 of the follow-up usefulness of the layer alkaline water that anhydrates of inclining dissolves and washes away 15 minutes, uses the neutral distillation water washing 2 times again, gets water-insoluble;
Measure the ursolic acid content in the water-insoluble, by ursolic acid content, be dissolved in the 95%-100% ethanol with 6-8mg/ml concentration, after the heating for dissolving, put 10-20 ℃ of lower nature crystallization, separate out without crystal this moment.This shows that the method for patent CN1629180A is not suitable for extracting ursolic acid from Folium paulowniae.
The ursolic acid safety experiment
The ursolic acid that uses this patent method to extract, injection Yelkin TTS in addition, soybean oil, oleic acid etc. are made injectable emulsion, give the injection of SD rat tail vein by the 50mg/kg body weight, once a day, injection is 15 days continuously, and rat behavior is normal, checks that through dissecting each histoorgan and control rats are without considerable change, ursolic acid and preparation security thereof that proof uses present method to extract are good, can be directly as material medicine.
Claims (10)
1. method of extracting high-purity ursolic acid from Folium paulowniae is characterized in that may further comprise the steps:
(1) Folium paulowniae is pulverized, with alcohol reflux twice, united extraction liquid is recycled to without the alcohol flavor, gets extract;
(2) add entry in extract, be heated to boiling, make extract form suspension, then at room temperature leave standstill, pour out supernatant liquor, it is stand-by to stay layer depositing in water thing;
(3) add ethanol in the depositing in water thing, be heated to moltenly, keeping solution temperature is 40-70 ℃, regulates pH to 9.0-11.0 with aqueous sodium hydroxide solution, removes by filter precipitation, then uses the salt acid for adjusting pH to 1.0-3.0, filters taking precipitate; Transfer pH, hydrochloric acid to transfer the pH step process one time according to above-mentioned dissolve with ethanol, sodium hydroxide again throw out, get sedimentable matter;
(4) with the sedimentable matter dissolve with ethanol, add activated carbon decolorizing, after the decolouring with solution at 15-30 ℃ of lower crystallization, filter, collect crystal, be ursolic acid.
2. method according to claim 1, it is characterized in that: described Folium paulowniae is the leaf of Paulownia elongata, royal paulownia, fortune paulownia or river paulownia.
3. method according to claim 1 is characterized in that: in the Folium paulowniae of pulverizing, be lower than 5% greater than the shive content of 10mm * 10mm.
4. method according to claim 1, it is characterized in that: in the step (1), the ethanol volumetric concentration is 85 ~ 95%, and the weight ratio of ethanol and Folium paulowniae is 6:1 ~ 12:1; Each 2h that extracts.
5. method according to claim 1 is characterized in that: in the step (2), the add-on of water be extract quality 20-25 doubly.
6. method according to claim 1, it is characterized in that: in the step (3), aqueous sodium hydroxide solution concentration is 0.1 ~ 2mol/L, and concentration of hydrochloric acid is 0.1 ~ 2mol/L, and the ethanol volumetric concentration is 80-85%, the consumption of ethanol be the depositing in water amount 20-25 doubly.
7. method according to claim 1 is characterized in that: in the step (4), sedimentable matter is with the dissolve with ethanol of volumetric concentration more than 90%, and the temperature of ethanol is 50-80 ℃.
8. method according to claim 1, it is characterized in that: in the step (4), activated carbon dosage is the 1%-5% of sedimentable matter quality, and the activated carbon decolorizing number of times is 2-5 time.
9. method according to claim 1, it is characterized in that: in the step (4), crystallization time is 48-240h.
10. method according to claim 1, it is characterized in that: used ethanol, hydrochloric acid, sodium hydroxide, gac are medicinal rank, and institute's water is distilled water.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104447939A (en) * | 2014-11-06 | 2015-03-25 | 童必炎 | Method for preparing ursolic acid by using paulownia tomentosa leaves as raw material |
| CN105503994A (en) * | 2015-12-10 | 2016-04-20 | 苏州泽达兴邦医药科技有限公司 | Preparation method of high-purity UA (Ursolic Acid) |
| CN106589044A (en) * | 2016-11-28 | 2017-04-26 | 南昌大学 | Method for separating ursolic acid from apple peel by alkali extraction method |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1453289A (en) * | 2003-05-19 | 2003-11-05 | 西安交通大学 | Process of extracting and separating ursolic acid from plant |
| CN1629180A (en) * | 2003-12-19 | 2005-06-22 | 杨庆尧 | Acid-base process for preparing ursolic acid |
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2012
- 2012-12-20 CN CN2012105575345A patent/CN103012542A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1453289A (en) * | 2003-05-19 | 2003-11-05 | 西安交通大学 | Process of extracting and separating ursolic acid from plant |
| CN1629180A (en) * | 2003-12-19 | 2005-06-22 | 杨庆尧 | Acid-base process for preparing ursolic acid |
Non-Patent Citations (1)
| Title |
|---|
| 韩晶，等: "从毛泡桐叶中提取、分离熊果酸的新工艺研究", 《西北大学学报（自然科学版）》, vol. 33, no. 3, 30 June 2003 (2003-06-30), pages 304 - 306 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104447939A (en) * | 2014-11-06 | 2015-03-25 | 童必炎 | Method for preparing ursolic acid by using paulownia tomentosa leaves as raw material |
| CN105503994A (en) * | 2015-12-10 | 2016-04-20 | 苏州泽达兴邦医药科技有限公司 | Preparation method of high-purity UA (Ursolic Acid) |
| CN106589044A (en) * | 2016-11-28 | 2017-04-26 | 南昌大学 | Method for separating ursolic acid from apple peel by alkali extraction method |
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Application publication date: 20130403 |