CN107344930A - A kind of method that young fustic is extracted from Rhus succedanea - Google Patents
A kind of method that young fustic is extracted from Rhus succedanea Download PDFInfo
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- CN107344930A CN107344930A CN201710582652.4A CN201710582652A CN107344930A CN 107344930 A CN107344930 A CN 107344930A CN 201710582652 A CN201710582652 A CN 201710582652A CN 107344930 A CN107344930 A CN 107344930A
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- ethanol water
- young fustic
- ethanol
- fustic
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
- C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
- C07D311/04—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring
- C07D311/22—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4
- C07D311/26—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4 with aromatic rings attached in position 2 or 3
- C07D311/40—Separation, e.g. from natural material; Purification
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- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
The method that the present invention provides extraction purification young fustic in a kind of Rhus succedanea, is converted and is discharged using cellulase and fermentation of bacillus subtilis, then extract, filter through alkali process and alcohol-water flow, be concentrated under reduced pressure to obtain crude extract；Concentrate, crystallization and recrystallization are obtained through macroreticular resin post separation again, filtering, is dried in vacuo to obtain young fustic sterling.The present invention is taken by enzyme process and alkali carries, substantially increase the recovery rate of young fustic, reduce influence of the protein to extraction in Rhus succedanea seed, combined and separated by column chromatography and crystalline phase, substantially increase the purity of young fustic, the operating procedure of purifying is simplified, reduces the production cost of young fustic.
Description
Technical field
The invention belongs to natural products technical field, relate to a kind of preparation method of young fustic, more specifically
The method that relate to the extraction purification young fustic from Rhus succedanea branch.
Background technology
Rhus succedanea (Rhus punjabensis var.sinica Rehd.) is Anacardiaceae Rhus defoliation small arbor,
Its root, stem, leaf, skin, flower and fruit are used as medicine, and do not need with other drug matchings, be used alone effect it is just fine, there is wind-dispelling
Dampness elimination, convergence removing toxic substances, promoting production of body fluid and nourishing the lung, removing fire in the lung and resolving phlegm and other effects.The insect gall formed on the plant sprout leaf is commonly called as Chinese gall, can make
For the important raw material of industry.Young fustic (3,7,3,4- tetrahydroxy flavanone) is with multiple biological activities and medical value
Flavonoid substances.
Young fustic is used as the disease medicaments such as anti-cancer, preventing and treating angiocarpy in states such as Europe, the United States, day, Austria and New Zealand in recent years
Main pharmacodynamics composition and safe and non-toxic novel oxidized enzyme inhibitor be widely applied.So that its demand is got over both at home and abroad
Come bigger, but now rely only on the single development of resources young fustic such as spina gleditsiae and pine and cypress both at home and abroad, market far can not be met
To the demand of young fustic, therefore find that new young fustic content is high, be suitable for again the plant resources of industrialized production into
Study hotspot.Wuling Shan Mountain section Rhus succedanea resource is extremely abundant, but only develops on its sprout leaf the Chinese gall formed, its yield
High fruit is also in state of running its course.
Young fustic is mainly extracted from the spina gleditsiae of legume Chinese honey locust and pine and cypress both at home and abroad at present, and from Rhus succedanea
The research of extraction young fustic has not been reported both at home and abroad.The extracting method of flavone compound mainly has water extraction, added at present
The methods of sour heavy, the ethanol extraction of circumfluence distillation, alkali carries, microwave radiation exaraction, ultrasonic assistant extraction, supercritical extract.Enzyme
Solution is that vegetalitas Chinese medicine effective component extracts very effective technology, because enzyme preparation can effective degrading plant cell
Wall, make to be present in the dissolution to greatest extent of intracellular effective component, while reaction conditions are gentle, product not mutability, ensure that
The property of extract and the stabilization of structure.The raising that recovery rate also can be larger simultaneously, and extraction time can be greatly shortened, it is green
Efficient plant extract technology, the polarity of extract can be improved using the enzyme preparation of correlation, so as to reduce making for organic solvent
With reducing cost.
The content of the invention
For the problems such as Rhus succedanea seed contains substantial amounts of cellulose and protein, young fustic polarity is small, a kind of enzyme is proposed
The method that legal system takes young fustic, and use column chromatography and crystallization isolate the young fustic product of high-purity.
The microorganism fungus kind that the present invention uses is bacillus subtilis (Bacillus subtilis) from the micro- life of China
Thing culture presevation administration committee common micro-organisms center, numbering are respectively CGMCC No.1.769.
The method that the present invention provides extraction purification young fustic in a kind of Rhus succedanea, specific steps include as follows：
(1) Rhus succedanea seed is crushed, the final concentration of 50~200U/kg of cellulase is added, adds 5~10% withered grass gemma
Bacillus liquid ferments 2~3 days at 25~30 DEG C, obtains zymotic fluid.
(2) pH value is adjusted room temperature 4~6h of placement, to add 70%~80% ethanol water to 8~9 with sodium hydroxide zymotic fluid
Solution, it is heated to 85~90 DEG C and carries out 4~8h of refluxing extraction.
(3) by extract solution ceramic membrane filter once, filtrate I is obtained, and ethanol is recovered under reduced pressure under the conditions of 50~65 DEG C, is concentrated
To paste, vacuum drying chamber drying is put to constant-quality, obtains crude extract.
(4) 75%~85% ethanol water of the weight such as crude extract use is dissolved, upper macroporous resin column, with twice of post
For volume to 20% ethanol water of three times column volume, 60%~70% ethanol with four times of column volumes to six times of column volumes is water-soluble
Liquid is eluted, and obtains eluent, and ethanol is recovered under reduced pressure under the conditions of 50~65 DEG C, is concentrated into the 1/5~1/10 of original volume, is obtained
Concentrate.
(5) after concentrate being inserted into -80 DEG C of refrigerator freezings icings, taking-up is put into vacuum drier and vacuumizes crystallization, ceramics
Membrane filtration once, obtains filter residue I, then wherein adds the dissolving of 45% ethanol water, inserts in 0~5 DEG C of refrigerator and is tied again again
Crystalline substance, once, gained filter residue carries out being dried in vacuo to obtain young fustic sterling at 25~35 DEG C ceramic membrane filter.
Above-mentioned bacillus subtilis bacterium solution can be fermented by conventional bacillus subtilis cultural method to be obtained, cellulase
It can buy and obtain from market, enzymatic activity is in more than 2000U/g.
The aperture of above-mentioned ceramic membrane filter is 0.22 μm.
The compound method of above-mentioned 70% ethanol water is that 80mL ethanol mixes with 20mL water.
In a specific embodiment, sodium hydroxide refers to 0.01~0.05mol/L sodium hydroxides described in step (2)
The aqueous solution.
In a specific embodiment, the amount L of 70%~80% ethanol water described in step (2) and Rhus succedanea seed
Weight kg ratios be 2~5：1.
In a specific embodiment, macroreticular resin described in step (4) is the resins such as XDA-8, LsA-21.
In a specific embodiment, the amount L of 45% ethanol water described in step (5) and filter residue I weight kg
Than for 0.5~2：1.
Technique effect
1st, taken by enzyme process and alkali carries, substantially increase the recovery rate of young fustic, reduce protein in Rhus succedanea seed
Influence to extraction.
2nd, combined and separated by column chromatography and crystalline phase, substantially increased the purity of young fustic, simplify purifying
Operating procedure, reduce the production cost of young fustic.
Embodiment
The essentiality content of the present invention is further illustrated with the embodiment of the present invention below, but this is not limited with this
Invention.
Embodiment 1
Rhus succedanea seed 100kg is crushed, the final concentration of 150U/kg of cellulose enzyme powder is added, 10% is added after stirring
Bacillus subtilis bacterium solution is fermented 2 days at 28 DEG C, obtains zymotic fluid.Zymotic fluid is adjusted into pH value to 8 with 0.03mol/L sodium hydroxides,
Room temperature places 4h, adds 80% ethanol water 400L, 85 DEG C of progress refluxing extraction 5h is heated to, by extract solution ceramic membrane filter
Once, 350L filtrates I are obtained, and ethanol is recovered under reduced pressure under the conditions of 55 DEG C, are concentrated into paste, put vacuum drying chamber drying to perseverance
Determine quality, obtain crude extract 6.43kg.Crude extract is dissolved with 85% ethanol water 6.43kg, upper LsA-21 macroreticular resins
Post, with 20% ethanol water of three times column volume, eluted with 65% ethanol water of six times of column volumes, obtain 40L and wash
De- liquid, is recovered under reduced pressure ethanol under the conditions of 55 DEG C, is concentrated into 5L, and after concentrate is inserted into -80 DEG C of refrigerator freezings icings, taking-up is put
Enter vacuum drier and vacuumize crystallization, ceramic membrane filter once, obtains 3.21kg filter residues I, then to add 45% ethanol thereto water-soluble
Liquid 4L dissolves, and inserts in 0~5 DEG C of refrigerator and is recrystallized again, and once, gained filter residue carries out vacuum at 35 DEG C to ceramic membrane filter
Dry 1.74kg young fustic sterlings.The HPLC testing conditions of young fustic：Chromatographic column:C18(4.6 × 250mm, 5 μm)；Stream
The dynamic phosphoric acid (19 of phase acetonitrile -0.4%:81)；UV-detector detects；Flow velocity：1.0mill/min；Column temperature：30℃；Sample size:10
μL.The content of young fustic is 97.45% in obtained sample.
Embodiment 2
Rhus succedanea seed 200kg is crushed, the final concentration of 200U/kg of cellulose enzyme powder is added, 10% is added after stirring
Bacillus subtilis bacterium solution is fermented 3 days at 30 DEG C, obtains zymotic fluid.Zymotic fluid is adjusted into pH value extremely with 0.05mol/L sodium hydroxides
8.5, room temperature places 6h, adds 75% ethanol water 700L, is heated to 85 DEG C of progress refluxing extraction 6h, obtains extract solution.It will carry
Take liquid ceramic membrane filter once, obtain about 600L filtrates I, and ethanol is recovered under reduced pressure under the conditions of 55 DEG C, be concentrated into paste, put true
Empty drying box drying obtains crude extract 13.68kg to constant-quality.Crude extract is molten with 80% ethanol water 13.68kg
Solution, upper XDA-8 macroporous resin columns, with 20% ethanol water of twice of column volume, 70% ethanol with six times of column volumes is water-soluble
Liquid is eluted, and obtains 90L eluents, and ethanol is recovered under reduced pressure under the conditions of 50 DEG C, is concentrated into 10L, obtains concentrate.Concentrate is put
After entering -80 DEG C of refrigerator freezings icings, taking-up is put into vacuum drier and vacuumizes crystallization, and ceramic membrane filter once, obtains 6.75kg filters
Slag I, then 45% ethanol water 8L dissolvings are added thereto, insert in 0~5 DEG C of refrigerator and recrystallized again, ceramic membrane mistake
Once, gained filter residue carries out being dried in vacuo to obtain 3.95kg young fustic sterlings at 30 DEG C for filter.Obtained sample presses the inspection of embodiment 1
Survey method is measured, and the content of young fustic is 97.23%.
Embodiment 3
Rhus succedanea seed 400kg is crushed, the final concentration of 180U/kg of cellulose enzyme powder is added, 10% is added after stirring
Bacillus subtilis bacterium solution is fermented 3 days at 28 DEG C, obtains zymotic fluid.Zymotic fluid is adjusted into pH value extremely with 0.04mol/L sodium hydroxides
8.5, room temperature places 6h, adds 70% ethanol water 1500L, is heated to 85 DEG C of progress refluxing extraction 5h, obtains extract solution.It will carry
Take liquid ceramic membrane filter once, obtain 1400L filtrates I, and ethanol is recovered under reduced pressure under the conditions of 55 DEG C, be concentrated into paste, put true
Empty drying box drying obtains crude extract 28.01kg to constant-quality.Crude extract is molten with 75% ethanol water 28.01kg
Solution, upper XDA-8 macroporous resin columns, with 20% ethanol water of three times column volume, 65% ethanol with six times of column volumes is water-soluble
Liquid is eluted, and obtains 160L eluents, and ethanol is recovered under reduced pressure under the conditions of 50 DEG C, is concentrated into 20L, obtains concentrate.By concentrate
After inserting -80 DEG C of refrigerator freezings icings, taking-up is put into vacuum drier and vacuumizes crystallization, and ceramic membrane filter once, obtains 14.28kg
Filter residue I, then 45% ethanol water 20L dissolvings are added thereto, insert in 0~5 DEG C of refrigerator and recrystallized again, ceramic membrane
Once, gained filter residue carries out being dried in vacuo to obtain 7.84kg young fustic sterlings at 30 DEG C for filtering.Obtained sample is by embodiment 1
Detection method is measured, and the content of young fustic is 97.62%.
Claims (4)
1. the method that the present invention provides extraction purification young fustic in a kind of Rhus succedanea, specific steps include as follows：
(1) Rhus succedanea seed is crushed, the final concentration of 100~400U/kg of cellulase is added, adds 5~10% bacillus subtilis
Bacterium solution is fermented 2~3 days at 25~30 DEG C, obtains zymotic fluid.
(2) pH value is adjusted room temperature 4~6h of placement, to be added to 8~9 with 0.01~0.05mol/L sodium hydrate aqueous solutions zymotic fluid
70%~80% ethanol water, it is heated to 85~90 DEG C and carries out 4~8h of refluxing extraction.
(3) by extract solution ceramic membrane filter once, filtrate I is obtained, and ethanol is recovered under reduced pressure under the conditions of 50~65 DEG C, is concentrated into thick
Paste, vacuum drying chamber drying is put to constant-quality, obtains crude extract.
(4) 75%~85% ethanol water of the weight such as crude extract use is dissolved, upper macroporous resin column, with twice of column volume
To 20% ethanol water of three times column volume, entered with 60%~70% ethanol water of four times of column volumes to six times of column volumes
Row elution, obtains eluent, ethanol is recovered under reduced pressure under the conditions of 50~65 DEG C, be concentrated into the 1/5~1/10 of original volume, must concentrate
Liquid.
(5) after concentrate being inserted into -80 DEG C of refrigerator freezings icings, taking-up is put into vacuum drier and vacuumizes crystallization, ceramic membrane mistake
Filter once, obtains filter residue I, then wherein adds the dissolving of 45% ethanol water, inserts in 0~5 DEG C of refrigerator and is recrystallized again, is made pottery
Once, gained filter residue carries out being dried in vacuo to obtain young fustic sterling at 25~35 DEG C porcelain membrane filtration.
2. method according to claim 1, the amount L of 70%~80% ethanol water and Rhus succedanea seed in the step (2)
Weight kg ratios are 2~5：1.
3. method according to claim 1, macroreticular resin is the resins such as XDA-8, LsA-21 in the step (4).
4. method according to claim 1, the amount L of 45% ethanol water is with the weight kg ratios of filter residue I in the step (5)
0.5~2：1.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710582652.4A CN107344930A (en) | 2017-07-17 | 2017-07-17 | A kind of method that young fustic is extracted from Rhus succedanea |
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| Application Number | Priority Date | Filing Date | Title |
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| CN201710582652.4A CN107344930A (en) | 2017-07-17 | 2017-07-17 | A kind of method that young fustic is extracted from Rhus succedanea |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113754626A (en) * | 2021-07-12 | 2021-12-07 | 雅安职业技术学院 | Method for preparing fisetin by enzyme method |
| CN116023358A (en) * | 2023-01-10 | 2023-04-28 | 四川太时生物科技集团有限公司 | Method for extracting genistein, fisetin and thioflavin from cotinus coggygria |
-
2017
- 2017-07-17 CN CN201710582652.4A patent/CN107344930A/en active Pending
Non-Patent Citations (1)
| Title |
|---|
| 董爱文等: "酶辅助提取协同大孔树脂纯化红麸杨果实黄颜木素", 《林产化学与工业》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113754626A (en) * | 2021-07-12 | 2021-12-07 | 雅安职业技术学院 | Method for preparing fisetin by enzyme method |
| CN113754626B (en) * | 2021-07-12 | 2024-03-15 | 雅安职业技术学院 | Method for preparing fisetin by enzyme method |
| CN116023358A (en) * | 2023-01-10 | 2023-04-28 | 四川太时生物科技集团有限公司 | Method for extracting genistein, fisetin and thioflavin from cotinus coggygria |
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Application publication date: 20171114 |