CN102108073A - Method for preparing senkyunolide H from extract of Chinese angelica - Google Patents
Method for preparing senkyunolide H from extract of Chinese angelica Download PDFInfo
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- CN102108073A CN102108073A CN2009102006942A CN200910200694A CN102108073A CN 102108073 A CN102108073 A CN 102108073A CN 2009102006942 A CN2009102006942 A CN 2009102006942A CN 200910200694 A CN200910200694 A CN 200910200694A CN 102108073 A CN102108073 A CN 102108073A
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Abstract
The invention discloses a method for preparing senkyunolide H from extract of Chinese angelica, which comprises the following steps: (1) purifying by using macroporous resin; (2) purifying by extraction; and (3) purifying by chromatography. The method can simply and conveniently prepare senkyunolide H, is high in yield and can be used in the industrial production of senkyunolide H.
Description
Technical field
The present invention relates to the preparation method of a kind of senkyunolide H (senkyunolide H), particularly relate to a kind of method that from Radix Angelicae Sinensis [Angelica sinensis (Oliv.) Diel] extract, prepares senkyunolide H.
Background technology
Radix Angelicae Sinensis [Angelica sinensis (Oliv.) Diel] is a samphire, and its medicinal part is a rhizome.Invigorate blood circulation menstruction regulating and pain relieving, the effect that relaxes bowel when being classified as China's traditional Chinese medicine, having to enrich blood.Clinical sallow, the dizzy palpitaition of the deficiency of blood, menoxenia, asthenia cold abdominalgia, the dry constipation of intestines, Feng Shi Paralysis pain, the wound etc. of being usually used in.
Phthalide analog compound is a compounds that is present in the Radix Angelicae Sinensis, is proved to be aspect cardiovascular, blood and the unstriated muscle activity being arranged, and can be considered to one of basic substance of Radix Angelicae Sinensis drug effect.
Senkyunolide H (senkynolide H, formula 1) be in Chinese medical extracts such as Ligusticum wallichii and Radix Angelicae Sinensis, to separate a kind of phthalide analog compound that obtains, have well fat-solublely and water-soluble, can enter blood and cerebrospinal fluid rapidly, have the potentiality of certain developing new drug.Its pharmacological action mainly comprises: it can alleviate ConA and draw the damage of erythrocytic deformability, reduces its aggregation [time precious traditional Chinese medical science traditional Chinese medicines, 2003,14 (12): 738～739]; The interior stream of calcium that suppresses guinea pig myocardium cell and human nerve cell knurl; Lower NO in the ischemia-reperfusion animal model brain stem, and suppress the activity [Clin Exp Pharmacology Physion, 1999,26:845～846] of NO synthase.
At present the preparation technology of senkyunolide H does not still have open report, so the preparation technology's of senkyunolide H research has big meaning for the exploitation of its further medicinal use.
Formula 1
Summary of the invention
The technical problem to be solved in the present invention provides a kind of method for preparing senkyunolide H from Radix Angelicae Sinensis extract, to solve the technical problem of senkyunolide H preparation of industrialization.
For solving the problems of the technologies described above, the method for preparing senkyunolide H from Radix Angelicae Sinensis extract of the present invention comprises step:
(1) macroporous resin purification step
(this concentration of aqueous solution is equivalent to crude drug 0.5gml with the aqueous solution of Radix Angelicae Sinensis extract
-1～1.0gml
-1) [weight ratio of macroporous resin and crude drug (Radix Angelicae Sinensis) is 0.3: 1～0.5: 1, and the absorption flow velocity in the macroporous resin column is 1～2BVh to go up the HPD-100 macroporous resin column
-1, BV refers to column volume], be to discard elutriant behind 0%～10% ethanol elution, 4～6BV with volumetric concentration, be 40%～50% ethanol elution, 4～6BV with volumetric concentration again, collect elutriant, decompression and solvent recovery must be through the medicinal extract B of macroporous resin purification;
(2) abstraction purification step
(this concentration of aqueous solution is equivalent to crude drug 5～10gml with the aqueous solution of step (1) gained medicinal extract B
-1) colourless to ethyl acetate layer with ethyl acetate extraction, get ethyl acetate extract; It is colourless substantially to water layer that the ethyl acetate extract water is come together, and discards ethyl acetate layer, water layer concentrate medicinal extract C;
(3) chromatographic purification step
Step (2) gained medicinal extract C is separated (silica gel is tlc silica gel, and medicinal extract C sample and silica gel weight ratio are 1: 50～1: 100) with silica gel column chromatography, and be to carry out wash-out in 100: 1～50: 1, with elutriant thin-layer silicon offset plate (GF with the chloroform-methanol volume ratio
254) observe down in ultraviolet lamp 254nm behind the point sample, collect the part that contains senkyunolide H, and decompression and solvent recovery, must promptly get the senkyunolide H of purifying through the medicinal extract of silica gel chromatography purifying;
Or with step (2) gained medicinal extract C C
18Reversed-phase silica gel column chromatography separates (medicinal extract C sample and C
18The reverse phase silica gel weight ratio is=1: 30～1: 50), and be to discard elutriant behind wash-out 4～6BV in 20: 80～30: 70 with the methanol-water volume ratio, be 40: 60～50: 50 wash-out 4～6BV with the methanol-water volume ratio again, collect elutriant and decompression and solvent recovery, promptly get the senkyunolide H of purifying;
The used extraction solvent of Radix Angelicae Sinensis extract is that volumetric concentration is 0%～95% ethanolic soln in the described step (1).
Adopt method of the present invention can prepare senkyunolide H comparatively easily, and have higher yield, can be used for the suitability for industrialized production of senkyunolide H.
Embodiment
Below in conjunction with specific embodiment the present invention is described in further details.
The preparation of embodiment 1 senkyunolide H
Angelica sinensis 1kg is ground into powder behind the removal impurity, and volumetric concentration is 95% ethanol 10L refluxing extraction 2 times, and each 2 hours, united extraction liquid reclaimed solvent and gets the about 200g of extractum A.
Extractum A is diluted to about 2000mL with deionized water, and (this concentration of aqueous solution is equivalent to crude drug 0.5gml
-1) HPD-100 macroporous resin column [weight ratio of macroporous resin and crude drug (angelica sinensis) is 0.3: 1, macroporous resin column specification Φ 150*1500mm] on the back, the absorption flow velocity is 2BVh
-1, with discarding elutriant behind the deionized water wash-out 6BV, be 50% ethanol elution 4BV with volumetric concentration again, collect 50% ethanol eluate, decompression and solvent recovery gets the about 30g of medicinal extract B.
(this concentration of aqueous solution is equivalent to crude drug 10gml with the dispersion of 100mL deionized water with medicinal extract B
-1), and with behind the ethyl acetate extraction three times (each 100mL), ethyl acetate layer is bleach, and the combined ethyl acetate position, the reclaim under reduced pressure ethyl acetate is to 100ml; With ethyl acetate extract with deionized water extraction three times (each 100mL) after, water layer is colourless substantially, discards ethyl acetate layer, water layer concentrate the about 3g of medicinal extract C.
Medicinal extract C is separated (silica gel is tlc silica gel, and medicinal extract C sample and silica gel weight ratio are 1: 100, silicagel column specification Φ 100*1000mm) with silica gel column chromatography, and be to carry out wash-out at 50: 1, with elutriant thin-layer silicon offset plate (GF with the chloroform-methanol volume ratio
254) point sample, with chloroform: the methyl alcohol volume ratio is to launch be placed on ultraviolet lamp 254nm at 10: 1 to observe down, collects the part that contains senkyunolide H, and decompression and solvent recovery, promptly gets the about 0.6g of senkyunolide H of purifying.
Final gained senkyunolide H through the known NMR method of routine identify [
13C-NMR (CDCl
3, 400M): 169.4 (C-1), 153.3 (C-3), 148.2 (C-3a), 18.4 (C-4), 25.7 (C-5), 67.3 (C-6), 63.4 (C-7), 125.4 (C-7a), 114.5 (C-8), 28.1 (C-9), 22.3 (C-10), 13.8 (C-11); δ
H4.61 (1H, d, J=3.1Hz, H-7)] [Chem Pharm Bull, 1987,35 (4): 1417～1432], and through high-efficient liquid phase technique [Kromasil-C
18Post (4.6mm*250mm) detects wavelength 278nm, and moving phase: volume percent is Glacial acetic acid-methyl alcohol (volume ratio 55: 45) of 1%, flow velocity 1mLmin
-1, sample size 10 μ l, 25 ℃ of column temperatures, theoretical plate number is not less than 5000 in senkyunolide H] detection level is more than 96%, its yield is 0.06%.
The preparation of embodiment 2 senkyunolide H
Angelica sinensis 1kg is ground into powder behind the removal impurity, and volumetric concentration is 95% ethanol 10L refluxing extraction 2 times, and each 2 hours, united extraction liquid reclaimed solvent and gets the about 200g of extractum A.
Extractum A is diluted to about 2000mL with deionized water, and (this concentration of aqueous solution is equivalent to crude drug 0.5gml
-1) HPD-100 macroporous resin column (weight ratio of macroporous resin and crude drug is 0.3: 1, macroporous resin column specification Φ 150*1500mm) on the back, the absorption flow velocity is 1BVh
-1, with discarding elutriant behind the 10% ethanol elution 4BV, be 40% ethanol elution 6BV with volumetric concentration again, collect 40% ethanol eluate, decompression and solvent recovery gets the about 30g of medicinal extract B.
(this concentration of aqueous solution is equivalent to crude drug 10gml with the dispersion of 100mL deionized water with medicinal extract B
-1), and with behind the ethyl acetate extraction three times (each 100mL), ethyl acetate layer is bleach, and the combined ethyl acetate position, the reclaim under reduced pressure ethyl acetate is to 100ml; With ethyl acetate extract with deionized water extraction three times (each 100mL) after, water layer is colourless substantially, discards ethyl acetate layer, water layer concentrate the about 3g of medicinal extract C.
Medicinal extract C is separated (silica gel is tlc silica gel, and medicinal extract C sample and silica gel weight ratio are 1: 50, silicagel column specification Φ 100*1000mm) with silica gel column chromatography, and be to carry out wash-out at 100: 1, with elutriant thin-layer silicon offset plate (GF with the chloroform-methanol volume ratio
254) point sample, with chloroform: the methyl alcohol volume ratio is to launch be placed on ultraviolet lamp 254nm at 10: 1 to observe down, collects the part that contains senkyunolide H, and decompression and solvent recovery, promptly gets the about 0.7g of senkyunolide H of purifying.
Final gained senkyunolide H identifies [Chem PharmBull, 1987,35 (4): 1417～1432] through the known NMR method of routine, and through high-efficient liquid phase technique [Kromasil-C
18Post (4.6mm*250mm) detects wavelength 278nm, moving phase 1% Glacial acetic acid-methyl alcohol (55: 45), flow velocity 1mLmin
-1, sample size 10 μ l, 25 ℃ of column temperatures, theoretical plate number is not less than 5000 in senkyunolide H] detection level is more than 96%, its yield is 0.07%.
The preparation of embodiment 3 senkyunolide H
Angelica sinensis 1kg is ground into powder behind the removal impurity, and volumetric concentration is 50% ethanol 10L refluxing extraction 2 times, and each 2 hours, united extraction liquid reclaimed solvent and gets the about 200g of extractum A.
Extractum A is diluted to about 1250mL with deionized water, and (this concentration of aqueous solution is equivalent to crude drug 0.8gml
-1) HPD-100 macroporous resin column (weight ratio of macroporous resin and crude drug is 0.4: 1, macroporous resin column specification Φ 150*1500mm) on the back, the absorption flow velocity is 1.5BVh
-1, be to discard elutriant behind the 5% ethanol elution 5BV with volumetric concentration, be 45% ethanol elution 5BV with volumetric concentration again, collect 45% ethanol eluate, decompression and solvent recovery gets the about 30g of medicinal extract B.
(this concentration of aqueous solution is equivalent to crude drug 5gml with the dispersion of 200mL deionized water with medicinal extract B
-1), and with behind the ethyl acetate extraction three times (each 200mL), ethyl acetate layer is bleach, and the combined ethyl acetate position, the reclaim under reduced pressure ethyl acetate is to 100ml; With ethyl acetate extract with deionized water extraction three times (each 100mL) after, water layer is colourless substantially, discards ethyl acetate layer, water layer concentrate the about 3.5g of medicinal extract C.
With the anti-phase C of medicinal extract C
18Silica gel column chromatography separates (medicinal extract C sample and C
18The reverse phase silica gel weight ratio is=1: 50, C
18Reverse phase silica gel post specification Φ 30*300mm), and being wash-out 6BV after to discard elutriant at 20: 80 with the methanol-water volume ratio, is 50: 50 wash-out 4BV with the methanol-water volume ratio again, collects elutriant and decompression and solvent recovery, promptly gets the about 0.9g of senkyunolide H of purifying.
Final gained senkyunolide H identifies [Chem PharmBull, 1987,35 (4): 1417～1432] through the known NMR method of routine, and through high-efficient liquid phase technique [Kromasil-C
18Post (4.6mm*250mm) detects wavelength 278nm, moving phase 1% Glacial acetic acid-methyl alcohol (55: 45), flow velocity 1mLmin
-1, sample size 10 μ l, 25 ℃ of column temperatures, theoretical plate number is not less than 5000 in senkyunolide H] detection level is more than 98%, its yield is 0.09%.
The preparation of embodiment 4 senkyunolide H
Angelica sinensis 1kg is ground into powder behind the removal impurity, deionized water 10L refluxing extraction 2 times, and each 2 hours, united extraction liquid reclaimed solvent and gets the about 200g of extractum A.
Extractum A is diluted to about 1000mL with deionized water, and (this concentration of aqueous solution is equivalent to crude drug 1.0gml
-1) HPD-100 macroporous resin column (weight ratio of macroporous resin and crude drug is 0.5: 1, macroporous resin column specification Φ 150*1500mm) on the back, the absorption flow velocity is 1BVh
-1, be to discard elutriant behind the 10% ethanol elution 4BV with volumetric concentration, be 40% ethanol elution 6BV with volumetric concentration again, collect 40% ethanol eluate, decompression and solvent recovery gets the about 30g of medicinal extract B.
(this concentration of aqueous solution is equivalent to crude drug 6.67gml with the dispersion of 150mL deionized water with medicinal extract B
-1), and with behind the ethyl acetate extraction three times (each 100mL), ethyl acetate layer is bleach, and the combined ethyl acetate position, the reclaim under reduced pressure ethyl acetate is to 100ml; With ethyl acetate extract with deionized water extraction three times (each 100mL) after, water layer is colourless substantially, discards ethyl acetate layer, water layer concentrate the about 3g of medicinal extract C.
With the anti-phase C of medicinal extract C
18Silica gel column chromatography separates (medicinal extract C sample and C
18The reverse phase silica gel weight ratio is=1: 30, C
18Reverse phase silica gel post specification Φ 30*300mm), and being wash-out 4BV after to discard elutriant at 30: 70 with the methanol-water volume ratio, is 40: 60 wash-out 6BV with the methanol-water volume ratio again, collects elutriant and decompression and solvent recovery, promptly gets the about 1.0g of senkyunolide H of purifying.
Final gained senkyunolide H identifies [Chem PharmBull, 1987,35 (4): 1417～1432] through the known NMR method of routine, and through high-efficient liquid phase technique [Kromasil-C
18Post (4.6mm*250mm) detects wavelength 278nm, moving phase 1% Glacial acetic acid-methyl alcohol (55: 45), flow velocity 1mLmin
-1, sample size 10 μ l, 25 ℃ of column temperatures, theoretical plate number is not less than 5000 in senkyunolide H] detection level is more than 98%, its yield is 0.1%.
Above experimental result shows that it is comparatively easy that present method prepares senkyunolide H, and its purity is more than 96%, yield 6/10000ths between the thousandth.Therefore, the present invention can be used for the industrial preparation of senkyunolide H.
Claims (5)
1. from Radix Angelicae Sinensis extract, prepare the method for senkyunolide H, it is characterized in that: comprise step:
(1) macroporous resin purification step
With macroporous resin column on the aqueous solution of Radix Angelicae Sinensis extract, with volumetric concentration is to discard elutriant behind 0%～10% ethanol elution, 4～6BV, is ethanol elution 4～6BV of 40%～50% again with volumetric concentration, collects elutriant, decompression and solvent recovery must be through the medicinal extract B of macroporous resin purification;
(2) abstraction purification step
The aqueous solution of medicinal extract B is colourless to ethyl acetate layer with ethyl acetate extraction, get ethyl acetate extract; It is colourless substantially to water layer that the ethyl acetate extract water is come together, and discards ethyl acetate layer, water layer concentrate medicinal extract C;
(3) chromatographic purification step
C separates with silica gel column chromatography with medicinal extract, and is to carry out wash-out in 100: 1～50: 1 with the chloroform-methanol volume ratio, with elutriant silica-gel plate point sample, collects the elutriant part that contains senkyunolide H, and decompression and solvent recovery promptly gets the senkyunolide H of purifying;
Or with the anti-phase C of medicinal extract C
18Silica gel column chromatography separates, and be to discard elutriant behind wash-out 4～6BV in 20: 80～30: 70 with the methanol-water volume ratio, be 40: 60～50: 50 wash-out 4～6BV with the methanol-water volume ratio again, collect elutriant and decompression and solvent recovery, promptly get the senkyunolide H of purifying.
2. the method for preparing senkyunolide H from Radix Angelicae Sinensis extract as claimed in claim 1 is characterized in that: used extraction solvent is that volumetric concentration is 0%～95% ethanolic soln in the middle Radix Angelicae Sinensis extract of described step (1); The concentration of aqueous solution of Radix Angelicae Sinensis extract is equivalent to crude drug 0.5gml
-1～1.0gml
-1
3. the method that from Radix Angelicae Sinensis extract, prepares senkyunolide H as claimed in claim 1, it is characterized in that: the macroporous resin in the described step (1) is the HPD-100 macroporous resin, wherein, the weight ratio of macroporous resin and crude drug is 0.3: 1～0.5: 1, and the absorption flow velocity in the macroporous resin column is 1～2BVh
-1
4. the method for preparing senkyunolide H from Radix Angelicae Sinensis extract as claimed in claim 1 is characterized in that: the concentration of aqueous solution of medicinal extract B is equivalent to crude drug 5gml in the described step (2)
-1～10gml
-1
5. the method that from Radix Angelicae Sinensis extract, prepares senkyunolide H as claimed in claim 1, it is characterized in that: in described step (3) the medicinal extract C silica gel chromatography purification step, silica gel is tlc silica gel, and medicinal extract C sample and silica gel weight ratio are 1: 50～1: 100;
Medicinal extract C uses in the reverse-phase chromatography purification step, medicinal extract C sample and C
18The reverse phase silica gel weight ratio is=1: 30～1: 50.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104003963A (en) * | 2014-05-30 | 2014-08-27 | 长沙高新技术产业开发区博海生物科技有限公司 | Separation and preparation method of ligustilide |
-
2009
- 2009-12-24 CN CN2009102006942A patent/CN102108073A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104003963A (en) * | 2014-05-30 | 2014-08-27 | 长沙高新技术产业开发区博海生物科技有限公司 | Separation and preparation method of ligustilide |
| CN104003963B (en) * | 2014-05-30 | 2016-08-17 | 长沙博海生物科技有限公司 | A kind of method for separating and preparing of cnidium lactone |
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Application publication date: 20110629 |