CN102329225A - Method for purifying roburic acid - Google Patents
Method for purifying roburic acid Download PDFInfo
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- CN102329225A CN102329225A CN201110210032A CN201110210032A CN102329225A CN 102329225 A CN102329225 A CN 102329225A CN 201110210032 A CN201110210032 A CN 201110210032A CN 201110210032 A CN201110210032 A CN 201110210032A CN 102329225 A CN102329225 A CN 102329225A
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Abstract
The invention discloses a method for purifying roburic acid, which comprises the following steps: (1) taking the raw material large leaf gentian root, drying, pulverizing, soaking in an enzyme solution, carrying out water bath enzymolysis, and sufficiently stirring; (2) filtering, extracting the filter residue with 5-10 times of ethanol water solution 2-3 times, merging the extracting solutions, and concentrating under reduced pressure to obtain an extract; (3) passing the extract through macroporous adsorbent resin columns, eluting with ethanol water solution, and collecting the eluate; (4) recycling ethanol from the eluate under reduced pressure, extracting by ethyl acetate, concentrating the extracting liquid, passing the extracting liquid through silica gel columns, carrying out cyclohexane-ethanol gradient elution, carrying out TLC (thin layer chromatography) tracking and monitoring, and collecting the roburic acid fraction; and (5) concentrating the roburic acid fraction under reduced pressure to small volume, standing to crystallize, and carrying out methanol/petroleum ether-acetone ethanol recrystallization. The invention is simple to operate, has the advantages of high product yield and low cost, and is suitable for large-scale production.
Description
Technical field
The invention belongs to the traditional Chinese medicine extraction technical field, relate to a kind of method of from bark of ash, extracting the acid of purifying oak goitre.
Background technology
Oak goitre acid (Roburic acid) has another name called the acid of oak cherry, molecular formula: C
30H
48O
2Molecular weight: 440.707; Molecular structural formula is following:
The acid of oak cherry is from bark of ash, to separate the composition that obtains; Bark of ash is a kind of conventional Chinese medicine material; Beginning is stated from Shennong's Herbal; Effect with wind-damp dispelling, clearing away damp-heat, stopping numbness pain, reducing the asthenic fever is used for that rheumatic arthralgia, apoplexy and hemiplegia, the contraction of muscle arteries and veins, joint are ached, jaundice due to damp-heat, osteopyrexia and fever, infantile malnutrition heating.Regulation bark of ash of 2010 editions pharmacopeia is the gentianaceae plant bark of ash
Gentiana macrophylliaPall., gentiana straminea maxim
Gentiana stramineaMaxim., gentiana crassicaulis Duthie
Gentiana crassicaulisDuthie ex Burk. or radix gentiane dahuvicae
Gentiana dahuricaFisch. dry root.And differentiating that having increased under the item with the acid of oak goitre is the composition discriminating of reference substance, has effectively distinguished the pseudo-article of the bark of ash that contains gentiopicrin.Through retrieval, still there be not the patent and the bibliographical information of the industrial process of preparation oak goitre acid at home.
Summary of the invention
The method of purification that the purpose of this invention is to provide a kind of oak goitre acid.
The objective of the invention is to realize through following technical scheme:
A kind of method of purification of oak goitre acid is characterized in that may further comprise the steps:
(1) enzymolysis: get raw material bark of ash root,, soak with enzyme solution through dry, pulverization process, water enzyme digestion 2-6 hour, and fully stir;
(2) extract: above-mentioned zymolyte is filtered, get filter residue and extract 2-3 time with the aqueous ethanolic solution that 5-10 doubly measures, united extraction liquid, concentrating under reduced pressure gets medicinal extract;
(3) enrichment: medicinal extract is crossed macroporous adsorptive resins, and pure water elution is collected elutriant;
(4) purifying: with above-mentioned elutriant decompression recycling ethanol, use ethyl acetate extraction, extraction liquid concentrates the back and goes up silicagel column (the silica gel granularity is the 100-400 order), hexanaphthene-ethanol gradient elution, and the TLC tracking monitor is collected oak goitre acid flow point;
(5) recrystallization: concentrating under reduced pressure oak goitre acid flow point leaves standstill crystallization to small volume, and methyl alcohol/sherwood oil-acetone refluxes and dissolves, and places crystallization, leaches the dry product that gets.
Enzyme solution described in the step (1) is selected from a kind of in cellulase solution or the polygalacturonase solution, and the pH of enzyme solution is 4.0-4.8, and hydrolysis temperature is 40-50 ℃.
The concentration of aqueous ethanolic solution is 65-90% described in the step (2).
Macroporous resin described in the step (3) is selected from a kind of in D4006, X-5, NKA-2, AB-8, the D101 type.
Pure water elution described in the step (3) is 3-4BV10%-20% ethanolic soln → 4-6BV70%-90% ethanolic soln.
Gradient elution described in the step (4) be hexanaphthene-ethanol by volume from high to low, be followed successively by 10:1,5:1,1:1.
The present invention adopts enzymolysis and extraction, lets effective constituent ooze out more easily, improves extraction yield; The employing macroporous resin separates, and the different concentration ethanol wash-out is removed impurity; Use silicagel column, hexanaphthene-ethanol gradient column chromatography can obtain more purified oak goitre acid, and product purity is higher.
Embodiment
Embodiment 1:
Get gentiana macrophylla medicine and pulverized 50 mesh sieves, take by weighing 1kg, the cellulase aqueous solution (per kilogram crude drug add 140mg cellulase) that adds 8LpH4.4 soaks half a hour, then 40 ℃ of following water enzyme digestions 5 hours, and fully stirs; Filter, get filter residue, extract united extraction liquid 3 times with the 80% alcohol heating reflux extraction of 8 times of amounts 1 hour; Concentrating under reduced pressure gets medicinal extract, and medicinal extract is heated water-dispersion, adds the NKA-2 macroporous adsorptive resins, gets 3BV20% ethanol elution impurity earlier; Get 5BV75% ethanol elution effective constituent again, decompression recycling ethanol gets crude extract, with the ethyl acetate extraction of the long-pending amount of isoploid, continuous extraction 4 times; Combining extraction liquid, extraction liquid concentrates the back and goes up silica gel column chromatography (the silica gel granularity is the 100-200 order), presses 10:1,5:1,1:1 gradient elution, TLC tracking monitor with hexanaphthene-ethanol; Collect oak goitre acid flow point, be evaporated to small volume, leave standstill crystallization, leach coarse-grain; Use sherwood oil-acetone (1:1) to reflux again and dissolve, place crystallization, leach the dry acid of oak goitre, the content 93.5% of getting.
Embodiment 2:
Get gentiana macrophylla medicine and pulverized 60 mesh sieves, take by weighing 1kg, the cellulase aqueous solution (per kilogram crude drug add 140mg cellulase) that adds 8LpH4.5 soaks half a hour, then 50 ℃ of following water enzyme digestions 2 hours, and fully stirs; Filter, get filter residue, extract united extraction liquid 2 times with the 60% alcohol heating reflux extraction of 10 times of amounts 1 hour; Concentrating under reduced pressure gets medicinal extract, and medicinal extract is heated water-dispersion, adds the D4006 macroporous adsorptive resins, gets 3BV10% ethanol elution impurity earlier; Get 5BV80% ethanol elution effective constituent again, decompression recycling ethanol gets crude extract, with the ethyl acetate extraction of the long-pending amount of isoploid, continuous extraction 3 times; Combining extraction liquid, extraction liquid concentrates the back and goes up silica gel column chromatography (the silica gel granularity is the 100-200 order), presses 10:1,5:1,1:1 gradient elution, TLC tracking monitor with hexanaphthene-ethanol; Collect oak goitre acid flow point, be evaporated to small volume, leave standstill crystallization, leach coarse-grain; With the alcohol reflux dissolving, place crystallization again, leach the dry acid of oak goitre, the content 91.4% of getting.
Embodiment 3:
Get gentiana macrophylla medicine and pulverized 100 mesh sieves, take by weighing 1kg, the cellulase aqueous solution (per kilogram crude drug add 140mg cellulase) that adds 8LpH4.0 soaks half a hour, then 45 ℃ of following water enzyme digestions 4 hours, and fully stirs; Filter, get filter residue, extract united extraction liquid 3 times with the 75% alcohol heating reflux extraction of 6 times of amounts 1 hour; Concentrating under reduced pressure gets medicinal extract, and medicinal extract is heated water-dispersion, adds the X-5 macroporous adsorptive resins, gets 4BV20% ethanol elution impurity earlier; Get 5BV85% ethanol elution effective constituent again, decompression recycling ethanol gets crude extract, with the ethyl acetate extraction of the long-pending amount of isoploid, continuous extraction 4 times; Combining extraction liquid, extraction liquid concentrates the back and goes up silica gel column chromatography (the silica gel granularity is the 200-300 order), presses 10:1,5:1,1:1 gradient elution, TLC tracking monitor with hexanaphthene-ethanol; Collect oak goitre acid flow point, be evaporated to small volume, leave standstill crystallization, leach coarse-grain; With the methanol eddy dissolving, place crystallization again, leach the dry acid of oak goitre, the content 92.2% of getting.
Embodiment 4:
Get gentiana macrophylla medicine and pulverized 80 mesh sieves, take by weighing 1kg, the cellulase aqueous solution (per kilogram crude drug add 140mg cellulase) that adds 8LpH4.8 soaks half a hour, then 50 ℃ of following water enzyme digestions 6 hours, and fully stirs; Filter, get filter residue, extract united extraction liquid 2 times with the 90% alcohol heating reflux extraction of 7 times of amounts 1 hour; Concentrating under reduced pressure gets medicinal extract, and medicinal extract is heated water-dispersion, adds the D101 macroporous adsorptive resins, gets 3BV20% ethanol elution impurity earlier; Get 4BV70% ethanol elution effective constituent again, decompression recycling ethanol gets crude extract, with the ethyl acetate extraction of the long-pending amount of isoploid, continuous extraction 5 times; Combining extraction liquid, extraction liquid concentrates the back and goes up silica gel column chromatography (the silica gel granularity is the 300-400 order), presses 10:1,5:1,1:1 gradient elution, TLC tracking monitor with hexanaphthene-ethanol; Collect oak goitre acid flow point, be evaporated to small volume, leave standstill crystallization, leach coarse-grain; Use sherwood oil-acetone (1:1) to reflux again and dissolve, place crystallization, leach the dry acid of oak goitre, the content 92.6% of getting.
Embodiment 5:
Get gentiana macrophylla medicine and pulverized 40 mesh sieves, take by weighing 2kg, the cellulase aqueous solution (per kilogram crude drug add 140mg cellulase) that adds 8LpH4.6 soaks half a hour, then 40 ℃ of following water enzyme digestions 3 hours, and fully stirs; Filter, get filter residue, extract united extraction liquid 2 times with the 85% alcohol heating reflux extraction of 5 times of amounts 1 hour; Concentrating under reduced pressure gets medicinal extract, and medicinal extract is heated water-dispersion, adds the AB-8 macroporous adsorptive resins, gets 4BV20% ethanol elution impurity earlier; Get 6BV90% ethanol elution effective constituent again, decompression recycling ethanol gets crude extract, with the ethyl acetate extraction of the long-pending amount of isoploid, continuous extraction 3 times; Combining extraction liquid, extraction liquid concentrates the back and goes up silica gel column chromatography (the silica gel granularity is the 200-300 order), presses 10:1,5:1,1:1 gradient elution, TLC tracking monitor with hexanaphthene-ethanol; Collect oak goitre acid flow point, be evaporated to small volume, leave standstill crystallization, leach coarse-grain; With the alcohol reflux dissolving, place crystallization again, leach the dry acid of oak goitre, the content 91.7% of getting.
Claims (6)
1. the method for purification of oak goitre acid is characterized in that may further comprise the steps:
(1) enzymolysis: get raw material bark of ash root,, soak with enzyme solution through dry, pulverization process, water enzyme digestion 2-6 hour, and fully stir;
(2) extract: above-mentioned zymolyte is filtered, get filter residue and extract 2-3 time with the aqueous ethanolic solution that 5-10 doubly measures, united extraction liquid, concentrating under reduced pressure gets medicinal extract;
(3) enrichment: medicinal extract is crossed macroporous adsorptive resins, and pure water elution is collected elutriant;
(4) purifying: with above-mentioned elutriant decompression recycling ethanol, use ethyl acetate extraction, extraction liquid concentrates the back and goes up silicagel column (the silica gel granularity is the 100-400 order), hexanaphthene-ethanol gradient elution, and the TLC tracking monitor is collected oak goitre acid flow point;
(5) recrystallization: concentrating under reduced pressure oak goitre acid flow point leaves standstill crystallization to small volume, and methyl alcohol/sherwood oil-acetone refluxes and dissolves, and places crystallization, leaches the dry product that gets.
2. method of purification according to claim 1 is characterized in that: enzyme solution described in the step (1) is selected from a kind of in cellulase solution or the polygalacturonase solution, and the pH of enzyme solution is 4.0-4.8, and hydrolysis temperature is 40-50 ℃.
3. method of purification according to claim 1 is characterized in that: the concentration of aqueous ethanolic solution is 65-90% described in the step (2).
4. method of purification according to claim 1 is characterized in that: macroporous resin described in the step (3) is selected from a kind of in D4006, X-5, NKA-2, AB-8, the D101 type.
5. method of purification according to claim 1 is characterized in that: pure water elution described in the step (3) is 3-4BV10%-20% ethanolic soln → 4-6BV70%-90% ethanolic soln.
6. method of purification according to claim 1 is characterized in that: gradient elution described in the step (4) be hexanaphthene-ethanol by volume from high to low, be followed successively by 10:1,5:1,1:1.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201110210032A CN102329225A (en) | 2011-07-26 | 2011-07-26 | Method for purifying roburic acid |
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|---|---|---|---|
| CN201110210032A CN102329225A (en) | 2011-07-26 | 2011-07-26 | Method for purifying roburic acid |
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| CN102329225A true CN102329225A (en) | 2012-01-25 |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2014237611A (en) * | 2013-06-07 | 2014-12-18 | 花王株式会社 | Ceramide production promoter |
| CN109172633A (en) * | 2018-10-19 | 2019-01-11 | 中国科学院西北高原生物研究所 | A kind of Radix Gentianae Macrophyllae extract and the preparation method and application thereof |
| CN113384590A (en) * | 2021-06-18 | 2021-09-14 | 五邑大学 | Application of quercus acutissima acid in preparation of medicine for treating pancreatic cancer |
| WO2021213484A1 (en) * | 2020-04-24 | 2021-10-28 | 珠海岐微生物科技有限公司 | Application of radix gentianae macrophyllae and monomer compound thereof in killing mites |
-
2011
- 2011-07-26 CN CN201110210032A patent/CN102329225A/en active Pending
Non-Patent Citations (4)
| Title |
|---|
| 《中国中药杂志》 20051031 陈千良等 "陕西产秦艽的化学成分研究" 第1519-1522页 1-6 第30卷, 第19期 * |
| 《中草药》 20100830 危士虎等 "麻花秦艽非环烯醚萜成分的研究" 第1242-1245页 1-6 第41卷, 第8期 * |
| 危士虎等: ""麻花秦艽非环烯醚萜成分的研究"", 《中草药》 * |
| 陈千良等: ""陕西产秦艽的化学成分研究"", 《中国中药杂志》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2014237611A (en) * | 2013-06-07 | 2014-12-18 | 花王株式会社 | Ceramide production promoter |
| CN109172633A (en) * | 2018-10-19 | 2019-01-11 | 中国科学院西北高原生物研究所 | A kind of Radix Gentianae Macrophyllae extract and the preparation method and application thereof |
| WO2021213484A1 (en) * | 2020-04-24 | 2021-10-28 | 珠海岐微生物科技有限公司 | Application of radix gentianae macrophyllae and monomer compound thereof in killing mites |
| CN113384590A (en) * | 2021-06-18 | 2021-09-14 | 五邑大学 | Application of quercus acutissima acid in preparation of medicine for treating pancreatic cancer |
| CN113384590B (en) * | 2021-06-18 | 2022-06-21 | 五邑大学 | Application of quercus acutissima acid in preparation of medicine for treating pancreatic cancer |
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Application publication date: 20120125 |