CN104478986A - Purification process for high-purity saponin product - Google Patents
Purification process for high-purity saponin product Download PDFInfo
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- CN104478986A CN104478986A CN201410834598.4A CN201410834598A CN104478986A CN 104478986 A CN104478986 A CN 104478986A CN 201410834598 A CN201410834598 A CN 201410834598A CN 104478986 A CN104478986 A CN 104478986A
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- silica gel
- purity
- purifying
- wash
- digitonin
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07J—STEROIDS
- C07J71/00—Steroids in which the cyclopenta(a)hydrophenanthrene skeleton is condensed with a heterocyclic ring
- C07J71/0005—Oxygen-containing hetero ring
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- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Steroid Compounds (AREA)
Abstract
The invention discloses a technological method for purifying low-purity digitonide to a product with the purity of more than 70%. The technological method comprises the following steps: purifying the low-purity digitonide by utilizing a normal-phase silica gel chromatographic column, and carrying out crystallizing, drying, purifying with a reverse-phase silica gel chromatographic column, crystallizing and drying to obtain digitonide with the target purity. The technological method disclosed by the invention is suitable for large-scale production, the raw material is easy to obtain, the preparation process is simple and environment-friendly, a solvent can be recycled, the cost is low, the purity of the obtained target product reaches more than 70%, and the total yield reaches more than 20%.
Description
Technical field
The present invention relates to the purifying process that a kind of purity is greater than the digitonin of 70%.
Background technology
Digitonin (
such as formula 1) be a kind of glycoside obtained from the leaf and seed of pale reddish brown foxglove, the one of steroidal Saponin/TSM.During hydrolysis, a part digitogenin (digitogenin) as aglycone (aglycone) part can be generated, and have the wood sugar of bimolecular D-Glucose and two molecule one semi-lactosis and a part as the part of sugar.These sugar moieties are attached on the hydroxyl of thricarbon atom of digitogenin.Because, the steroid (such as cholesterol) that it and can have free 3 β-OH forms the DIGITONIN glycoside compound (digitonide) being insoluble in the molecular compound of water with equimolecular ratio, this characteristic can be used to carry out the qualitative of sterol and be separated.As important native protein solubilizing agent, cholesterol determination agent, there is larger economic worth and social value.Current existing extraction, purifying process are repeatable poor, and solvent toxicity greatly, and can only obtain the digitonin of the liquid phase purity being up to 50%.
Formula 1 digitonin structure.
Summary of the invention
The object of the invention is to provide a kind of purifying technique that can be used in the effective high purity digitonin of cholesterol determination, protein solubilization.This is simple for process, is suitable for batch preparation and suitability for industrialized production.The digitonin liquid chromatography purity obtained reaches more than 70%.
The invention provides following purifying technique scheme:
1, after being pulverized by foxglove seed, and ether solvent is placed in diafiltration cylinder, diafiltration degreasing after immersion, and after degreasing, fine powder loads extractor, and after the methanol aqueous solution heat with 30% extracts 3 times, concentrated extracting solution is extremely without alcohol taste;
2, be loaded on D201 resin column by the concentrated solution obtained, use the distillation of 2 times of cylinder accumulated amounts, 25% methyl alcohol of 3 times of cylinder accumulated amounts, 60% methanol-eluted fractions of 4 times of cylinder accumulated amounts successively, collect 60% alcohol eluen, underpressure distillation, except desolventizing, obtains eluate;
3, a small amount of alcoholic solvent of eluate is dissolved, select purification on normal-phase silica gel, silica gel column chromatography upper end poured into by sample, filler blade diameter length ratio is 1: 8 ~ 12, the weight ratio of alcohol eluate and silica gel is 1: 40 ~ 60, with the chloroform-ethanol of different concns-water gradient elution, detect with thin layer chromatography and instruct wash-out.The condition of thin-layer chromatography is: propyl carbinol-36% Acetic Acid-Water (7: 1: 1 to 7: 3: 1), and developer is methyl alcohol: Glacial acetic acid: the vitriol oil: aubepine=170: 20: 10: 1 (v/v/v), and steroidal saponin is green at chromatoplate.Collect the elutriant containing steroid glucoside, merge, concentrated, obtain the enriched substance of beige white powder shape.
Be unformed silica gel, spherical silica gel for the weighting agent of column chromatography for separation in such scheme.Silica gel is of a size of 100 ~ 200 orders or 200 ~ 300 orders.
4, the enriched substance of upper step is dissolved in a small amount of ethanol, pour the post upper end of reversed-phase bonded silica filler into, chromatography column aspect ratio is 1: 10 ~ 15, and the weight ratio of extract and silica gel is 1: 30 ~ 40, with the aqueous methanol gradient wash-out of different concns, detect with thin layer chromatography simultaneously and instruct wash-out.Collect the elutriant containing steroid glucoside, merge, concentrated, recycling design, vacuum drying oven is dry, obtains highly purified digitonin.
Be eight alkyl silane bonded silica gel or octadecylsilane chemically bonded silicas for the weighting agent of column chromatography for separation in such scheme.The methanol-water ratio of wash-out is (1: 2 ~ 4: 1).
5, carry out quantitative analysis by high pressure liquid chromatography (HPLC) to isolate, the finished product purity is greater than 70%.
Embodiment
Below in conjunction with embodiment, the present invention will be further described, and embodiment is for illustration of the present invention instead of for limiting the scope of the invention.
Embodiment 1
After being pulverized by 3kg foxglove seed, and ether solvent is placed in diafiltration cylinder, diafiltration degreasing after immersion, and after degreasing, fine powder loads extractor, and after the methanol aqueous solution heat with 30% extracts 3 times, concentrated extracting solution is extremely without alcohol taste; Be loaded to by the concentrated solution obtained on D201 resin column, use 25% methyl alcohol of the distilled water of 2 times of cylinder accumulated amounts, 3 times of cylinder accumulated amounts, 60% methanol-eluted fractions of 4 times of cylinder accumulated amounts successively, collect 60% alcohol eluen, underpressure distillation, except desolventizing, obtains eluate 40g;
By a small amount of dissolve with ethanol of 40g eluate, select purification on normal-phase silica gel (100 ~ 200 order), silica gel column chromatography upper end poured into by sample, the weight ratio of alcohol eluate and silica gel is 1: 40, use chloroform-ethanol-water (7: 1: 0.5) wash-out 2 times of column volume wash-outs successively, chloroform-ethanol-water (7: 2: 0.5) wash-out 6 times of column volume wash-outs, chloroform-ethanol-water (7: 2.5: 0.5) wash-out 2 times of column volume wash-outs, collect chloroform-ethanol-water (7: 2.5: 0.5) elutriant to concentrate, obtain the enriched substance of 10g beige white powder shape.
The enriched substance of upper step is dissolved in a small amount of ethanol, pour the post upper end of anti-phase eight alkyl silane bonded silica gel fillers into, the weight ratio of extract and silica gel is 1: 30, with 4 times of column volume 10% methanol aqueous solution wash-outs, and 6 times of column volume 25% methanol aqueous solution wash-outs, collect the elutriant of rear 3 times of column volume 25% methanol aqueous solutions, merge, concentrated, recycling design, vacuum drying oven is dry, obtains the digitonin 2g of 73% purity.
Embodiment 2
After being pulverized by 3kg foxglove seed, and ether solvent is placed in diafiltration cylinder, diafiltration degreasing after immersion, and after degreasing, fine powder loads extractor, and after the methanol aqueous solution heat with 30% extracts 3 times, concentrated extracting solution is extremely without alcohol taste; Be loaded to by the concentrated solution obtained on D201 resin column, use 25% methyl alcohol of the distilled water of 2 times of cylinder accumulated amounts, 3 times of cylinder accumulated amounts, 60% methanol-eluted fractions of 4 times of cylinder accumulated amounts successively, collect 60% alcohol eluen, underpressure distillation, except desolventizing, obtains eluate 40g;
By a small amount of dissolve with ethanol of 40g eluate, select purification on normal-phase silica gel (200 ~ 300 order), silica gel column chromatography upper end poured into by sample, the weight ratio of alcohol eluate and silica gel is 1: 50, use chloroform-ethanol-water (7: 2: 0.5) wash-out 3 times of column volume wash-outs successively, chloroform-ethanol-water (7: 3: 0.5) wash-out 3 times of column volume wash-outs, chloroform-ethanol-water (7: 4: 0.5) wash-out 2 times of column volume wash-outs, collect chloroform-ethanol-water (7: 4: 0.5) elutriant to concentrate, obtain the enriched substance of 8g beige white powder shape.
The enriched substance of upper step is dissolved in a small amount of ethanol, pour the post upper end of anti-phase octadecylsilane chemically bonded silica filler into, the weight ratio of extract and silica gel is 1: 30, with 2 times of column volume 30% methanol aqueous solution wash-outs, and 6 times of column volume 50% methanol aqueous solution wash-outs, collect the elutriant of rear 3 times of column volume 50% methanol aqueous solutions, merge, concentrated, recycling design, vacuum drying oven is dry, obtains the digitonin 1.8g of 75% purity.
Claims (3)
1. a purity is greater than the purifying process of the digitonin of 70%, it is characterized in that the low-purity digitonin after pulverizing, degreasing, macroporous resin column separating-purifying, through purification on normal-phase silica gel column chromatography purification, after crystallizing and drying, purify with reversed-phase silica gel column chromatography further, crystallizing and drying obtains the product of more than 70% purity.
2. technique as claimed in claim 1, is characterized in that purification on normal-phase silica gel column chromatography used silica gel is not limited to unformed silica gel, spherical silica gel.Silica gel is of a size of 100 ~ 200 orders or 200 ~ 300 orders, and post blade diameter length ratio is 1: 8 ~ 12, and the weight ratio of alcohol eluate and silica gel is 1: 40 ~ 60.
3. technique as claimed in claim 1, it is characterized in that reverse phase silica gel post used silica gel includes but not limited to eight alkyl silane bonded silica gel or octadecylsilane chemically bonded silicas, the aspect ratio of chromatography column weighting material is 1: 10 ~ 15, and the weight ratio of extract and silica gel is 1: 30 ~ 40.The methanol-water ratio of wash-out is (1: 2 ~ 4: 1).
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410834598.4A CN104478986A (en) | 2014-12-30 | 2014-12-30 | Purification process for high-purity saponin product |
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| Application Number | Priority Date | Filing Date | Title |
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| CN201410834598.4A CN104478986A (en) | 2014-12-30 | 2014-12-30 | Purification process for high-purity saponin product |
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| CN201410834598.4A Pending CN104478986A (en) | 2014-12-30 | 2014-12-30 | Purification process for high-purity saponin product |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH0854100A (en) * | 1994-04-29 | 1996-02-27 | Envirex Inc | Back-flow preventive system for medium bed reactor |
| CN1803827A (en) * | 2006-01-25 | 2006-07-19 | 中国科学院昆明植物研究所 | Method for preparing plant steroid glucoside |
| CN103113452A (en) * | 2011-11-07 | 2013-05-22 | 河北百灵威超精细材料有限公司 | Digitonin extraction and purification technology |
-
2014
- 2014-12-30 CN CN201410834598.4A patent/CN104478986A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH0854100A (en) * | 1994-04-29 | 1996-02-27 | Envirex Inc | Back-flow preventive system for medium bed reactor |
| CN1803827A (en) * | 2006-01-25 | 2006-07-19 | 中国科学院昆明植物研究所 | Method for preparing plant steroid glucoside |
| CN103113452A (en) * | 2011-11-07 | 2013-05-22 | 河北百灵威超精细材料有限公司 | Digitonin extraction and purification technology |
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Application publication date: 20150401 |