CN104557835A - Method for extracting eleocharin D from eleocharis tuberosa peels - Google Patents
Method for extracting eleocharin D from eleocharis tuberosa peels Download PDFInfo
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- CN104557835A CN104557835A CN201410834967.XA CN201410834967A CN104557835A CN 104557835 A CN104557835 A CN 104557835A CN 201410834967 A CN201410834967 A CN 201410834967A CN 104557835 A CN104557835 A CN 104557835A
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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
- C07D311/28—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 with aromatic rings attached in position 2 only
- C07D311/32—2,3-Dihydro derivatives, e.g. flavanones
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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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Abstract
The invention discloses a method for extracting eleocharin D from eleocharis tuberosa peels, and belongs to the field of natural organic chemistry. The method is characterized by comprising the following steps: taking acetone aqueous solution as the solvent, extracting a crude extract with ethyl acetate, separating by medium-pressure MCI column chromatography and polyamide column chromatography, using Sephadex LH-20 gel and semi-preparative HPLC (High Performance Liquid Chromatography) for preparation, and performing recrystallization-assisted purification, wherein the purity of obtained product is above 95%. According to the method for extracting eleocharin D from eleocharis tuberosa peels, waste eleocharis tuberosa peels are used as the raw materials to extract and separate eleocharin D, so as to recycle the waste eleocharis tuberosa peels; the separation effect is good; the purity of products is high; the selected chromatographic materials MCI, polyamide and gel can be used repeatedly; the method has the advantages of accessible raw materials, abundant resources and low production cost, and has good application prospects.
Description
Technical field
The invention belongs to field of natural organic chemistry, relate to a kind of extraction and separation method of water chestnut D prime, particularly relate to a kind of method of extraction and isolation water chestnut D prime from water chestnut skin.
Background technology
Water chestnut (Eleocharis tuberosa), also known as water chestnut, belong to the underground bulb of Cyperaceae perennial shallow water herbaceous plant water chestnut, China's most area has cultivation.Wherein, Guangxi water chestnut output accounts for the whole nation 70%, and He Prefecture water chestnut output accounts for Guangxi 70%.According to the literature, water chestnut have antibacterial, antitumor, prevent and treat respiratory tract disease, sharp intestines defaecation and effect such as diuresis row pouring etc.; Can be used for the diseases such as treatment is had sore throat, phlegm heat cough, pyreticosis polydipsia, dysuria, dysentery clinically.Research shows, the active substance of water chestnut is mainly enriched between pericarp and pulp.In the water chestnut course of processing, the water chestnut cortex amount be dropped accounts for 25% of fresh water chestnut quality, and resource is very abundant.We have found water chestnut D prime in the research of water chestnut skin activeconstituents extraction and isolation, belong to flavanone compound.Water chestnut D prime is the derivative of eriodictyol.This compound has the multiple pharmacologically actives such as anti-oxidant, anti-inflammatory, analgesia and regulates cardiovascular systems, neuroprotective system, diuresis, improves the effects such as diabetes, and food is commonly used for the antioxidant of beverage, food and drinks.Water chestnut D prime is present in the pericarp of sedge water chestnut.Water chestnut D prime is separated to obtain new compound from water chestnut skin.
At present, the bibliographical information of extraction and isolation water chestnut D prime from water chestnut skin is had no.Retrieve through Chinese publication, do not have to find the technical scheme identical with present patent application.
Summary of the invention
The object of the invention is to: a kind of method proposing water chestnut skin extraction of chufa D prime.
Water chestnut D prime chemical structural formula is as follows:
The method of water chestnut skin extraction of chufa D prime of the present invention, is characterized in that: application traditional extraction method and MCI, polymeric amide, gel and Semipreparative chromatography technology extraction and isolation water chestnut D prime from water chestnut skin, and its concrete steps are as follows:
(1) the water chestnut skin by fresh is air-dry, pulverizes, for subsequent use; By weight, take 1 part, water chestnut skin powder and add in extractor, add the aqueous acetone solution of 4-10 part 70% volume percent at every turn, at 25 DEG C, soak 24h, soak 3 times; Filter, merging filtrate, is evaporated to paste, obtains extract;
(2) by weight, get the extract 1 part of step (1), be dispersed in 5 parts of water, make suspension liquid, add 5-10 part extraction into ethyl acetate at every turn, extract 3 times, combining extraction liquid, is evaporated to dry, obtains extract;
(3) by weight, get the extract 1 part of step (2), adding methyl alcohol to dissolving completely, mixing sample with 2-4 part polymeric amide, methyl alcohol is evaporated into dry, dress post, connecting MCI post and carry out the separation of middle pressure, take methanol aqueous solution as eluent gradient wash-out, tlc detects, collect and merge the elutriant that moving phase concentration is 70-90% volume percent, be evaporated to dry, obtain crude product A;
(4) by weight, get the crude product A 1 part of step (3), adding methyl alcohol to dissolving completely, adding 2-4 part polymeric amide and mixing sample, by dry for methyl alcohol volatilization, proceeding to polymeric amide chromatographic column to be separated, is the chloroform-methanol wash-out of 5:1-2:1 by volume ratio, with sherwood oil: ethyl acetate: developping agent made by the mixed solution of formic acid=50:49:1, detect by tlc, collecting merging Rf is the elutriant of 0.7, is evaporated to dry, obtains crude product B;
(5) by weight, get the crude product B 1 part of step (4), be dissolved in 4-8 part methyl alcohol, the water methyl alcohol volume such as adding makes suspension liquid, with Sephadex LH-20 gel chromatography column purification, by methanol-eluted fractions, with sherwood oil: ethyl acetate: developping agent made by the mixed solution of formic acid=50:49:1, detect by tlc, collecting merging Rf is the elutriant of 0.7, concentrating under reduced pressure, obtains crude product C;
(6) by weight, get the crude product C 1 part of step (5), be dissolved in 2-5 part methyl alcohol, take 280nm as ultraviolet detection wavelength, with the methanol aqueous solution of 62-68% volume percent for moving phase, efficient liquid phase chromatographic analysis is carried out with the Zorbax SB-C18 post of 4.6mm × 25cm, partly purifying is prepared with the Zorbax SB-C18 post of 9.4mm × 25cm, by the appearance time of 6.48-9.20min, collect threshold values in chromatographic peak and be not less than the elutriant of 20mAU, concentrating under reduced pressure, obtains product.
Described MCI post specification in described step (3) is 70 × 460mm, and packing material is MCI-gel CHP-20P, and column chromatography condition is: post pressure is 80Psi, and flow rate of mobile phase is 50mL/min, and determined wavelength is 245nm; Described moving phase is the methanol aqueous solution of 40-100% volume percent.
Polymeric amide specification described in described step (4) is 200-300 order; The chloroform-methanol of elutriant used to be volume ratio be 5:1-3:1.
Chromatographic column used in described step (6) is Zorbax SB-C18 (9.4mm 25cm) post; Described methanol concentration scope is 62-68% volume percent.
The present invention compared with prior art its beneficial effect is: design science is reasonable, discarded water chestnut skin is utilized to obtain water chestnut D prime for raw material extraction and isolation, good separating effect, product purity is high, and water chestnut skin is turned waste into wealth, and selected chromatographic material MCI, polymeric amide, gel all can Reusabilities, raw material is easy to get, aboundresources, production cost is low, has good application prospect.
Embodiment
Below in conjunction with embodiment, the invention will be further described, but embodiments of the present invention are not limited thereto.
Method of the present invention, is characterized in that: application traditional extraction method and MCI, polymeric amide, gel and Semipreparative chromatography technology extraction and isolation water chestnut flavones monomer from water chestnut skin, and its concrete steps are as follows:
(1) the water chestnut skin by fresh is air-dry, pulverizes, for subsequent use.The water chestnut skin powder taking certain mass (g) adds in extractor, then adds the aqueous acetone solution of 70% volume percent of 4-10 times of volume (mL), at 25 DEG C, soak 24h, soak 3 times, filter, merging filtrate, be evaporated to paste, obtain extract.
(2) be dispersed in by extract (g) in the water of 5 times of volumes (mL) and make suspension liquid, with the extraction into ethyl acetate 3 times of 1-2 times of volume of water, combining extraction liquid, is evaporated to dry, obtains extract.
(3) in extract, methyl alcohol is added to dissolving completely, sample is mixed with the polymeric amide of 2-4 times of extract quality, methyl alcohol is evaporated into dry, dress post, connect MCI post and carry out the separation of middle pressure, post specification is 70mm × 460mm, packing material is the MCI-gel CHP-20P that Mitsubishi chemical company produces, column chromatography condition is: post pressure is 80Psi, flow rate of mobile phase is 50mL/min, determined wavelength is 254nm, with the methanol aqueous solution of 40-100% volume percent for eluent gradient wash-out, eluate concentration is regulated according to detected peaks changing conditions, collect by every part of 500mL, tlc detects, collect and merge the elutriant that moving phase concentration is 70-90% volume percent, concentrating under reduced pressure, obtain crude product A.
(4) in crude product A, methyl alcohol is added to dissolving completely, sample mixed by the polymeric amide adding 2-4 times of quality, by dry for methyl alcohol volatilization, proceeding to polymeric amide chromatographic column to be separated, is the chloroform-methanol wash-out of 5:1-2:1 by volume ratio, and tlc detects, developping agent is sherwood oil: ethyl acetate: formic acid=50:49:1, collecting merging Rf is the elutriant of 0.7, and concentrating under reduced pressure obtains crude product B.
(5) crude product B (g) is dissolved in the methyl alcohol of 4-8 times of volume (mL), the water methyl alcohol volume such as adding makes suspension liquid, with the Sephadex LH-20 gel chromatography column purification that Amersham Pharmacia Biotech company of Sweden produces, by methanol-eluted fractions, tlc detects, and developping agent is sherwood oil: ethyl acetate: formic acid=50:49:1, and collecting merging Rf is the elutriant of 0.7, concentrating under reduced pressure, obtains crude product C.
(6) by weight, get the crude product C 1 part of step (5), be dissolved in 2-5 part methyl alcohol, take 280nm as ultraviolet detection wavelength, with the methanol aqueous solution of 62-68% volume percent for moving phase, efficient liquid phase chromatographic analysis is carried out with the Zorbax SB-C18 post of 4.6mm × 25cm, partly purifying is prepared with the ZorbaxSB-C18 post of 9.4mm × 25cm, by the appearance time of 6.48-9.20min, collect threshold values in chromatographic peak and be not less than the elutriant of 20mAU, concentrating under reduced pressure, obtains product.
Specific experiment example and detected result:
(1) be added in extractor by fresh for 8kg water chestnut skin powder, add the aqueous acetone solution of 32L 70% volume percent, at 25 DEG C, soak 24h, total immersion steeps 3 times, filters, merging filtrate, concentrating under reduced pressure paste, obtains extract 1.05kg.
(2) added in 5.0L water by extract and make turbid solution, with 5.0L extraction into ethyl acetate 3 times, combining extraction liquid, concentrating under reduced pressure, obtains extract 120g.
(3) in extract, 300mL dissolve with methanol is added, sample is mixed with 260g polymeric amide, methyl alcohol is evaporated into dry, dress post, connect MCI post and carry out the separation of middle pressure, be eluent gradient wash-out with the methanol aqueous solution of 40-100% volume percent, collect by every part of 500mL, tlc detects, and collects and merges the elutriant that moving phase concentration is 70-90% volume percent, concentrating under reduced pressure, obtains crude product A 31g.
(4) crude product A is dissolved in 90mL methyl alcohol, add 60g polymeric amide and mix sample, by dry for methyl alcohol volatilization, proceed to polyamide column to be separated, with the chloroform-methanol wash-out that volume ratio is 5:1, tlc detects (developping agent is sherwood oil: ethyl acetate: formic acid=50:49:1), and collecting merging Rf is the elutriant of 0.7, concentrating under reduced pressure, obtains crude product B 3.2g.
(5) crude product B is dissolved in the methyl alcohol of 15mL, add 15mL water and make suspension liquid, with SephadexLH-20 gel chromatography column purification, by methanol-eluted fractions, tlc detects, and developping agent is sherwood oil: ethyl acetate: formic acid=50:49:1, and collecting merging Rf is the elutriant of 0.7, concentrating under reduced pressure, obtains crude product C 0.35g.
(6) be dissolved in the methyl alcohol of 2.0mL by gained crude product C, with the methanol aqueous solution of 62-68% volume percent for moving phase, purify with Semipreparative chromatography, embodiment is as table 1.
Table 1 Semipreparative chromatography purifies and separates embodiment
(7) gained material passes through
1h-NMR,
13c-NMR, IR, HREIMS and UV spectroscopic technique carries out Structural Identification.Spectral data confirms, the material that institute's extraction purification obtains is water chestnut D prime, and chemical structural formula is as follows:
Water chestnut D prime (Eleocharin D) Yellow amorphous powder, chemistry (-)-(S)-8-methyl-3 '-methoxyl group-5,7 by name, 4 '-trihydroxy-flavanone; [α] 16D: – 4.3 (c 1.34, MeOH); IR (KBr) ν
max: 3428,2924,1637,1519,1456,1340,1300,1161,1111cm
-1; UV (MeOH): λ
max(log ε) 291.0 (3.98), 202.5 (4.39) nm;
1h NMR (600MHz, DMSO-d
6) δ: 5.35 (dd, J=12.6,2.6Hz, H-2), 3.26 (dd, J=17.0,12.6Hz, H-3 α), 2.63 (dd, J=17.0,2.6Hz, H-3 β), 5.92 (s, H-6), 7.07 (d, J=1.4Hz, H-2'), 6.77 (d, J=8.0Hz, H-5'), 6.89 (d, J=8.0Hz, H-6'), 1.86 (s, 3H, CH
3-8), 3.77 (s, 3H, CH
3o-3'), 12.45 (br s, HO-5), 9.23 (br s, HO-3');
13c NMR (150MHz, DMSO-d
6) δ: 78.7 (d, C-2), 42.3 (t, C-3), 195.9 (s, C-4), 160.4 (s, C-5), 94.7 (d, C-6), 160.5 (s, C-7), 103.4 (s, C-8), 160.8 (s, C-9), 101.0 (s, C-10), 129.8 (s, C-1'), 111.1 (d, C-2'), 147.6 (s, C-3'), 146.9 (s, C-4'), 115.2 (d, C-5'), 119.7 (d, C-6'), 7.2 (q, CH
3-8), 55.7 (q, CH
3o-3'); SeeTable 2; ESIMS (negative-ion mode) m/z 315 [M – H]
–; HREIMS m/z 316.0933 [M]
+, calcd for C
17h
16o
6, 316.0947.
Claims (3)
1. a method for water chestnut skin extraction of chufa D prime, comprises extraction, extraction; It is characterized in that: its concrete steps are as follows:
(1) the water chestnut skin by fresh is air-dry, pulverizes, for subsequent use; By weight, take 1 part, water chestnut skin powder and add in extractor, add the aqueous acetone solution of 4-10 part 70% volume percent at every turn, at 25 DEG C, soak 24h, soak 3 times; Filter, merging filtrate, is evaporated to paste, obtains extract;
(2) by weight, get the extract 1 part of step (1), be dispersed in 5 parts of water, make suspension liquid, add 5-10 part extraction into ethyl acetate at every turn, extract 3 times, combining extraction liquid, is evaporated to dry, obtains extract;
(3) by weight, get the extract 1 part of step (2), adding methyl alcohol to dissolving completely, mixing sample with 2-4 part polymeric amide, methyl alcohol is evaporated into dry, dress post, connecting MCI post and carry out the separation of middle pressure, take methanol aqueous solution as eluent gradient wash-out, tlc detects, collect and merge the elutriant that moving phase concentration is 70-90% volume percent, be evaporated to dry, obtain crude product A;
(4) by weight, get the crude product A 1 part of step (3), add methyl alcohol to dissolving completely, add 2-4 part polymeric amide and mix sample, by dry for methyl alcohol volatilization, proceed to polymeric amide chromatographic column and be separated, with the chloroform-methanol wash-out that volume ratio is 5:1-2:1, with sherwood oil: ethyl acetate: developping agent made by the mixed solution of formic acid=50:49:1, detect by tlc, collect and merge R
fbe the elutriant of 0.7, be evaporated to dry, obtain crude product B;
(5) by weight, get the crude product B 1 part of step (4), be dissolved in 4-8 part methyl alcohol, the water methyl alcohol volume such as adding makes suspension liquid, with Sephadex LH-20 gel chromatography column purification, by methanol-eluted fractions, with sherwood oil: ethyl acetate: developping agent made by the mixed solution of formic acid=50:49:1, detect by tlc, collect and merge R
fbe the elutriant of 0.7, concentrating under reduced pressure, obtains crude product C;
(6) by weight, get the crude product C 1 part of step (5), be dissolved in 2-5 part methyl alcohol, take 280nm as ultraviolet detection wavelength, with the methanol aqueous solution of 62-68% volume percent for moving phase, efficient liquid phase chromatographic analysis is carried out with the Zorbax SB-C18 post of 4.6mm × 25cm, partly purifying is prepared with the Zorbax SB-C18 post of 9.4mm × 25cm, by the appearance time of 6.48-9.20min, collect threshold values in chromatographic peak and be not less than the elutriant of 20mAU, concentrating under reduced pressure, obtains product.
2. method according to claim 1, is characterized in that, the described MCI post specification in described step (3) is 70 × 460mm, packing material is MCI-gel CHP-20P, column chromatography condition is: post pressure is 80Psi, and flow rate of mobile phase is 50mL/min, and determined wavelength is 245nm; Described moving phase is the methanol aqueous solution of 40-100% volume percent.
3. method according to claim 1, is characterized in that, the polymeric amide specification described in described step (4) is 200-300 order; The chloroform-methanol of elutriant used to be volume ratio be 5:1-3:1.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109497506A (en) * | 2018-12-10 | 2019-03-22 | 贺州学院 | A kind of acrylamide natural inhibitor |
-
2014
- 2014-12-29 CN CN201410834967.XA patent/CN104557835A/en active Pending
Non-Patent Citations (2)
| Title |
|---|
| YANGHE LUO ET AL.: "Isolation, characterisation, and antioxidant activities of flavonoids from chufa (Eleocharis tuberosa) peels", 《FOOD CHEMISTY》 * |
| 李作美等: "荸荠皮中生物活性物质的研究进展", 《中国食物与营养》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109497506A (en) * | 2018-12-10 | 2019-03-22 | 贺州学院 | A kind of acrylamide natural inhibitor |
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