CN102372759A - Purification method for alisol A - Google Patents
Purification method for alisol A Download PDFInfo
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- CN102372759A CN102372759A CN2010102630680A CN201010263068A CN102372759A CN 102372759 A CN102372759 A CN 102372759A CN 2010102630680 A CN2010102630680 A CN 2010102630680A CN 201010263068 A CN201010263068 A CN 201010263068A CN 102372759 A CN102372759 A CN 102372759A
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- alisol
- ethanolic soln
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- macroporous resin
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Abstract
The invention provides a purification method for alisol A, which has the advantages of little pollution and low cost. The technical method includes the following steps: water plantain as a medicinal material is ground and then added with 70 to 90 percent of ethanol for reflux extraction, extract is added with active carbon for decolorization, the ethanol is recovered, concentrate is then added with macroporous resin for adsorption under the condition of 60 DEG C to 80 DEG C, water and ethanol solution are used for gradient elution, precipitate is obtained by concentration, concentrate is washed and crystallized, crystallization is then separated by a high-speed counter-current chromatograph, a heptanes-methylene dichloride-acetonitrile solvent system is chosen for use, and thereby a product is prepared. The product purity and yield of the alisol A produced by the purification method are high.
Description
Technical field:
The present invention relates to a kind of method of purification of alisol A, especially relate to a kind of method with macroporous resin enrichment and adverse current chromatogram separation means purifying alisol A.
Background technology:
Alisol A, triterpene substance.
Molecular formula: C
30H
50O
5
Physico-chemical property: amorphous body, [α] 23D+99.6 ° (chloroform).Be dissolved in ETHYLE ACETATE, acetone, chloroform.Be slightly soluble in methyl alcohol, ethanol, water insoluble and sherwood oil.
Experiment showed, and measure cholesterol level in treatment group and the control group blood after ten days when alisol A is fed male rat simultaneously with the food that contains 1% SUV, 2% Sodium cholic acid and 0.2% choline chloride 60, discovery can obviously reduce cholesterol levels in the blood.Alisol A can be difficult for producing resistance property, and toxic side effect be low, good market prospect as treatment hepatitis.
(high-speed countercurrent chromatography is the liquid luquid partition chromatography stripping technique of a kind of continuous high-efficient of growing up the eighties in 20th century HSCCC) to high speed adverse current chromatogram, and it need not any solid-state upholder or carrier.It utilizes two phase solvent system in the spiral tube of high speed rotating, to set up a kind of special one-way fluid dynamic equilibrium, and one as stationary phase, another is as moving phase when wherein, in the process of wash-out continuously, can keep a large amount of stationary phase.
Owing to do not need solid support; The separation of material according to its two mutually in partition ratio difference and realize; Thereby avoided because of sample loss that irreversible adsorption causes, inactivation, sex change etc.; Sample can all be reclaimed, and the sample of recovery more can reflect the characteristic that it is original, is particularly suitable for the separation of natural bioactive ingredients.And, make the preparation amount of sample improve greatly owing to can fully contact between separated material and the liquid stationary phase, be that a kind of ideal prepares separation means.
It is with respect to traditional solid-liquid column chromatography technology, have applied widely, flexible operation, efficient, fast, advantage such as preparation amount is big, expense is low.The HSCCC technology is developing into a kind of novel separating and purifying technology that receives much concern at present; Be widely used in fields such as biological medicine, natural product, foods and cosmetics, in the natural product industry, be considered to a kind of effective new separation technology especially; The separation and purification of small molecules class material in being suitable for.
At present, mostly the method for extracting alisol A is that organic reagent extracts or supercritical extraction, and macroporous resin separates or the silicagel column separation again.Gained alisol A or content is lower, otherwise cost is higher, preparation amount is little.
Like patent (application number 200810197317.3) " a kind of process for extracting and purifying triterpenoids from oriental waterplantain rhizome "; This patent disclosed method is a methanol extraction; Extracting solution concentrates the back ethyl acetate extraction, and macroporous resin column absorption gets total triterpene compound on the extract dissolve with ethanol.
Patent (application number 03124066.6) " rhizoma alismatis steroidal extract and preparation method thereof, quality controlling means " for another example, this patent disclosed method are to adopt supercritical CO
2The fluid extraction rhizoma alismatis makes the rhizoma alismatis total-sterol extract.
Also have patent " pharmaceutical composition of treatment hepatitis B ", this patent disclosed method is an alcohol reflux, the liquid concentrator chloroform extraction, and silicagel column separates, and recrystallization gets the needle crystal alisol A.
Document " the triterpenes The Chemical Constituents is rushed down in the grassy marsh ", the method that provides is an extraction using alcohol, sherwood oil, ethyl acetate extraction, again silicagel column separate the various monomers of alisol.
The disclosed purification process that is macroporous resin to alisol of document " experimental study of the total terpene alcohol of Amberlyst process purifying rhizoma alismatis constituents ".
Summary of the invention:
The present invention wants the technical solution problem to provide a kind of continuous production, handles the method for purification of the alisol A big, that content is high.
The objective of the invention is to realize through following technical scheme:
A kind of method of purification of alisol A is characterized in that comprising following steps:
1) extract decolouring: get rhizoma alismatis pulverizing medicinal materials 40-80 order, add 8-15 and doubly measure the 70-90% ethanolic soln, refluxing extraction 1-3 hour, 2-3 time, united extraction liquid added activated carbon decolorizing, filter destainer;
2) macroporous resin adsorption: reclaim ethanol to said extracted liquid, add suitable quantity of water again and disperse, keep 60-80 ℃ of temperature to add macroporous resin column absorption, water alcohol gradient elution is collected effective constituent elutriant decompression recycling ethanol, places deposition;
3) crystallization: above-mentioned throw out leaches, and adds 5-8 and doubly measures 35-40% ethanolic soln backflow washing, and cooling normal temperature filters insolubles, refluxes dissolving crystallized with absolute ethyl alcohol;
4) high speed adverse current chromatogram separates: leach above-mentioned crystallisate, select the solvent systems of heptane-methylene dichloride-acetonitrile for use, adopt high-speed counter-current chromatograph to separate, make alisol A.
Gac is an injection class medicinal carbon in the said step 1), and consumption is solution amount 0.3-0.5%.
Said step 2) a kind of among the optional D101 of macroporous resin model, AB-8, HZ816 or the HPD-100, eluent is 10-20BV water → 5-7BV20-40% ethanolic soln → 5-8BV60-90% ethanolic soln.
Solvent ratios is heptane (8-13) in the said step 4): methylene dichloride (2-5): acetonitrile (5-9).
Advantage of the present invention is:
1) macroporous resin enrichment and crystallization purifying are adopted in rough handling, because alisol A content is low, like the difficult proportioning of direct high speed adverse current chromatogram purifies and separates solvent, the solvent repeated multiple times is prone to emulsification.
2) it is big to adopt high speed adverse current chromatogram to separate preparation amount, and the cycle is short, and cost is lower, and product purity is high, and sepn process appearance
To combine embodiment to further specify the present invention below, but the scope that the present invention requires to protect is not limited to following embodiment:
Embodiment:
Embodiment 1:
Rhizoma alismatis pulverizing medicinal materials 40 orders are got 10 kilograms, drop in the 200L pilot scale extractor, add 80L70% ethanolic soln refluxing extraction 1 hour; Extract supernatant, add similarity condition again and extract 2 times, united extraction liquid adds 700g injection class medical active carbon decoloring, leaches gac; Decompression recycling ethanol adds suitable quantity of water again and disperses, and is incubated 80 ℃ and adds the absorption of 4LD101 macroporous resin column, flow velocity 4L/H; Absorption is used 40L water → 20L40% ethanolic soln → 32L60% ethanolic soln wash-out after finishing successively, collects 60% ethanol eluate decompression recycling ethanol, places deposition; Leach, get throw out 300g and add 1.5L40% ethanolic soln backflow washing, cooling normal temperature; Filter insolubles, it is dissolving crystallized to reflux with absolute ethyl alcohol, obtains 61g powdery crystallisate.Get heptane (13): methylene dichloride (2): acetonitrile (9) places separating funnel to mix, and static layering is separated phase up and down; Below being stationary phase mutually, below is moving phase mutually, and it is for use in being dissolved in down mutually in right amount to get above-mentioned powdery crystallisate; Open the preparation of countercurrent chromatography appearance, go into moving phase, when moving phase flows out with the 3ml/min flow pump; The beginning continuous sample introduction, the head and the tail wash-out, rotating speed is controlled to be 700r/min; Regularly collect the alisol A flow point through detector, concentrate drying gets alisol A 13g, high phase Liquid Detection content 98% (area normalization method).
Embodiment 2:
Rhizoma alismatis pulverizing medicinal materials 80 orders are got 10 kilograms, drop in the 200L pilot scale extractor, add 150L90% ethanolic soln refluxing extraction 3 hours; Extract supernatant, add similarity condition again and extract 1 time, united extraction liquid adds 900g injection class medical active carbon decoloring, leaches gac; Decompression recycling ethanol adds suitable quantity of water again and disperses, and is incubated 60 ℃ and adds the absorption of 4L AB-8 macroporous resin column, flow velocity 3L/H; Absorption is used 80L water → 28L20% ethanolic soln → 20L90% ethanolic soln wash-out after finishing successively, collects 90% ethanol eluate decompression recycling ethanol, places deposition; Leach, get throw out 270g and add 2.5L35% ethanolic soln backflow washing, cooling normal temperature; Filter insolubles, it is dissolving crystallized to reflux with absolute ethyl alcohol, obtains 52g powdery crystallisate.Get heptane (8): methylene dichloride (5): acetonitrile (9) places separating funnel to mix, and static layering is separated phase up and down; Below being stationary phase mutually, below is moving phase mutually, and it is for use in being dissolved in down mutually in right amount to get above-mentioned powdery crystallisate; Open the preparation of countercurrent chromatography appearance, go into moving phase, when moving phase flows out with the 2ml/min flow pump; The beginning continuous sample introduction, the head and the tail wash-out, rotating speed is controlled to be 1100r/min; Regularly collect the alisol A flow point through detector, concentrate drying gets alisol A 12g, high phase Liquid Detection content 98.5% (area normalization method).
Embodiment 3:
Rhizoma alismatis pulverizing medicinal materials 60 orders are got 10 kilograms, drop in the 200L pilot scale extractor, add 100L80% ethanolic soln refluxing extraction 2 hours; Extract supernatant, add similarity condition again and extract 2 times, united extraction liquid adds 1.2kg injection class medical active carbon decoloring, leaches gac; Decompression recycling ethanol adds suitable quantity of water again and disperses, and is incubated 80 ℃ and adds the absorption of 4L HZ816 macroporous resin column, flow velocity 5L/H; Absorption is used 60L water → 24L30% ethanolic soln → 26L75% ethanolic soln wash-out after finishing successively, collects 75% ethanol eluate decompression recycling ethanol, places deposition; Leach, get throw out 310g and add 2L40% ethanolic soln backflow washing, cooling normal temperature; Filter insolubles, it is dissolving crystallized to reflux with absolute ethyl alcohol, obtains 48g powdery crystallisate.Get heptane (9): methylene dichloride (4): acetonitrile (7) places separating funnel to mix, and static layering is separated phase up and down; Below being stationary phase mutually, below is moving phase mutually, and it is for use in being dissolved in down mutually in right amount to get above-mentioned powdery crystallisate; Open the preparation of countercurrent chromatography appearance, go into moving phase, when moving phase flows out with the 1.5ml/min flow pump; The beginning continuous sample introduction, the head and the tail wash-out, rotating speed is controlled to be 850r/min; Regularly collect the alisol A flow point through detector, concentrate drying gets alisol A 15g, high phase Liquid Detection content 99% (area normalization method).
Embodiment 4:
Rhizoma alismatis pulverizing medicinal materials 60 orders are got 10 kilograms, drop in the 200L pilot scale extractor, add 14L80% ethanolic soln refluxing extraction 2 hours; Extract supernatant, add similarity condition again and extract 1 time, united extraction liquid adds 1kg injection class medical active carbon decoloring, leaches gac; Decompression recycling ethanol adds suitable quantity of water again and disperses, and is incubated 80 ℃ and adds the absorption of 4L HPD-100 macroporous resin column, flow velocity 2L/H; Absorption is used 40L water → 20L40% ethanolic soln → 30L75% ethanolic soln wash-out after finishing successively, collects 75% ethanol eluate decompression recycling ethanol, places deposition; Leach, get throw out 310g and add 2.4L30% ethanolic soln backflow washing, cooling normal temperature; Filter insolubles, it is dissolving crystallized to reflux with absolute ethyl alcohol, obtains 45g powdery crystallisate.Get heptane (13): methylene dichloride (2): acetonitrile (9) places separating funnel to mix, and static layering is separated phase up and down; Below being stationary phase mutually, below is moving phase mutually, and it is for use in being dissolved in down mutually in right amount to get above-mentioned powdery crystallisate; Open the preparation of countercurrent chromatography appearance, go into moving phase, when moving phase flows out with the 2ml/min flow pump; The beginning continuous sample introduction, the head and the tail wash-out, rotating speed is controlled to be 900r/min; Regularly collect the alisol A flow point through detector, concentrate drying gets alisol A 14g, high phase Liquid Detection content 97.5% (area normalization method).
Claims (4)
1. the method for purification of an alisol A is characterized in that comprising following steps:
1) extract decolouring: get rhizoma alismatis pulverizing medicinal materials 40-80 order, add 8-15 and doubly measure the 70-90% ethanolic soln, refluxing extraction 1-3 hour, 2-3 time, united extraction liquid added activated carbon decolorizing, filter destainer;
2) macroporous resin adsorption: reclaim ethanol to said extracted liquid, add suitable quantity of water again and disperse, keep 60-80 ℃ of temperature to add macroporous resin column absorption, water alcohol gradient elution is collected effective constituent elutriant decompression recycling ethanol, places deposition;
3) crystallization: above-mentioned throw out leaches, and adds 5-8 and doubly measures 35-40% ethanolic soln backflow washing, and cooling normal temperature filters insolubles, refluxes dissolving crystallized with absolute ethyl alcohol;
4) high speed adverse current chromatogram separates: leach above-mentioned crystallisate, select the solvent systems of heptane-methylene dichloride-acetonitrile for use, adopt high-speed counter-current chromatograph to separate, make alisol A.
2. the method for purification of alisol A according to claim 1 is characterized in that gac is an injection class medicinal carbon in the said step 1), and consumption is solution amount 0.3-0.5%.
3. the method for purification of alisol A according to claim 1; It is characterized in that said step 2) in a kind of among the optional D101 of macroporous resin model, AB-8, HZ816 or the HPD-100, eluent is 10-20BV water → 5-7BV20-40% ethanolic soln → 5-8BV60-90% ethanolic soln.
4. the method for purification of alisol A according to claim 1 is characterized in that solvent ratios is heptane (8-13) in the said step 4): methylene dichloride (2-5): acetonitrile (5-9).
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| CN2010102630680A CN102372759A (en) | 2010-08-26 | 2010-08-26 | Purification method for alisol A |
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| CN2010102630680A CN102372759A (en) | 2010-08-26 | 2010-08-26 | Purification method for alisol A |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104497091A (en) * | 2014-12-24 | 2015-04-08 | 陕西嘉禾植物化工有限责任公司 | Method for extracting 24-Acetyl alisol A from rhizoma alismatis |
| CN112876529A (en) * | 2021-01-25 | 2021-06-01 | 福建中医药大学 | Purification preparation method of 23-acetyl alisol C and application thereof in preparing anti-osteoporosis medicine |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101658598A (en) * | 2009-09-16 | 2010-03-03 | 福建中医学院 | Method for extracting and enriching alisma total triterpenic ketone alcohol components from alisma |
| CN101724009A (en) * | 2008-10-23 | 2010-06-09 | 华中科技大学 | Process for extracting and purifying triterpenoids from oriental waterplantain rhizome |
-
2010
- 2010-08-26 CN CN2010102630680A patent/CN102372759A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101724009A (en) * | 2008-10-23 | 2010-06-09 | 华中科技大学 | Process for extracting and purifying triterpenoids from oriental waterplantain rhizome |
| CN101658598A (en) * | 2009-09-16 | 2010-03-03 | 福建中医学院 | Method for extracting and enriching alisma total triterpenic ketone alcohol components from alisma |
Non-Patent Citations (1)
| Title |
|---|
| 林碧芬 等: "高速逆流色谱技术及其在天然菇类分离中的应用", 《福建省畜收兽医学会2009年学术年会论文集》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104497091A (en) * | 2014-12-24 | 2015-04-08 | 陕西嘉禾植物化工有限责任公司 | Method for extracting 24-Acetyl alisol A from rhizoma alismatis |
| CN112876529A (en) * | 2021-01-25 | 2021-06-01 | 福建中医药大学 | Purification preparation method of 23-acetyl alisol C and application thereof in preparing anti-osteoporosis medicine |
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Application publication date: 20120314 |