CN102229526A - Method for separating, extracting and purifying isomagnolol in plant, namely Streblus asper - Google Patents
Method for separating, extracting and purifying isomagnolol in plant, namely Streblus asper Download PDFInfo
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Abstract
The invention discloses a method for separating, extracting and purifying isomagnolol in a plant, namely Streblus asper. The method disclosed by the invention comprises the following steps: mincing dried crude drugs or fresh medicinal materials; carrying out reflux extraction or percolation extraction by heating solvents; extracting the obtained pasty extractives through a solvent I and a solvent II; decompressing and concentrating the extracted solvent II; and carrying out chromatography purification to obtain pure isomagnolol. The method disclosed by the invention has the advantages that an extraction source for extracting the isomagnolol is found; the purity of the extracted and separated isomagnolol is high; the isomagnolol right aims to develop a target medicine for resisting hepatitis B virus and bacteria and has important significances on developing high-efficiency and low-toxin medicines such as natural oral medicines, injections, medicines for external use and the like.
Description
Technical field:
The present invention relates to extraction, the separation of traditional Chinese medicine ingredients iso-agnolol, is the method for a kind of extraction from the plant streblus asper, separation and purifying iso-agnolol specifically.
Background technology:
Iso-agnolol is a kind of Lignanoids compounds, and its structural formula is:
Nineteen eighty-three is obtained by separation first from the sassafras bark of Taiwan such as El-Feraly (El-Feraly, et al, ournal ofNatural Products, 1983).It is to many mushrooms and ACAT demonstration good restraining activity such as hepatitis B virus, Ao Lisi staphylococcus, M. smegmatics, saccharomyces cerevisiae, microsporon mentagrophyteses.Up to now, extraction separation obtains iso-agnolol from natural phant, only retrieves one piece in the report document (El-Feraly, et al, Journal of Natural Products, 1983) of extraction separation from the sassafras of Taiwan.
Streblus asper is a Moraceae streblus asper platymiscium, belongs to arbor or shrub, mainly is distributed in provinces such as Guangxi, Guangdong, Yunnan, Hainan.Streblus asper has multiple medicinal curative effect, and its extract can be antibiotic, treatment fever, dysentery, gingivitis, stomatocace etc.
Summary of the invention:
The objective of the invention is to provide the method for a kind of extraction from the natural phant streblus asper, separation and purifying iso-agnolol.
The technical scheme that realizes the object of the invention is:
Main idea of the present invention is to be the method for raw material extraction, separation and purifying iso-agnolol with the root of streblus asper (Streblus asper), skin, stem.Its preparation method comprises the steps:
(1) dry product or the fresh medicinal material with streblus asper shreds;
(2) with the raw medicinal material through shredding in the solvent heating and refluxing extraction method extraction step (1): add solvent in raw material in proportion, heating and refluxing extraction is 2-5 time repeatedly, and each reflux time is 1-4 hour; With the extracting liquid filtering that aforesaid operations obtains, collect filtrate; Being lower than under 70 ℃ of temperature, filtrate is carried out concentrating under reduced pressure, remove solvent, getting proportion is the medicinal extract shape extract A of 1.1-1.4; The add-on of described solvent is: the volume that at every turn adds solvent rises number: add dry product weight kilogram number=6-12 at first: 1, or be that the volume of solvent rises number: fresh medicinal material weight kilogram number=4-8: 1;
(3) extract A to obtaining in the step (2), add the dissolving of entry suspendible earlier, the weight kilogram number of extract A: the volume of water rises number=1: 3-6, then with solvent I extraction 1-4 time, each water and solvent volume ratio=1 mutually: 0.2-2, isolate the solvent layer, with solvent II extraction 1-4 time, each water is 1 with solvent volume ratio mutually to water layer: 0.2-3 again; Merge the solvent II liquid after each time extracts, get solvent liquid B, wherein contain iso-agnolol;
(4) the solvent liquid B to collecting in the step (3) carries out concentrating under reduced pressure being lower than under 70 ℃ of conditions, obtains paste enriched material B1;
(5) the paste enriched material B1 to obtaining in the step (4) carries out the of the same race of one or many or filler chromatography purification not of the same race with chromatography, obtains content and is 〉=98% oily iso-agnolol;
The another kind of method of the present invention's extraction from streblus asper, separation and purifying iso-agnolol is:
(1) gets the raw medicinal material streblus asper, with crude drug dry product or the chopping of fresh medicinal material;
(2) with soaking the raw medicinal material through shredding in the percolation diacolation step (1): in raw medicinal material, add solvent in proportion, at normal temperatures medicinal material is carried out 1-4 diacolation and extract each diacolation 1-3 days; With the extracting liquid filtering that aforesaid operations obtains, collect filtrate; Being lower than under 70 ℃ of temperature, filtrate is carried out concentrating under reduced pressure, remove solvent, getting proportion is the medicinal extract shape extract A of 1.1-1.4; The add-on of described solvent is: the volume that at every turn adds solvent rises number: add dry product weight kilogram number=6-12 at first: 1, or be that the volume of solvent rises number: fresh medicinal material weight kilogram number=4-8: 1;
(3) extract A to obtaining in the step (2), add the dissolving of entry suspendible earlier, the weight kilogram number of extract A: the volume of water rises number=1: 3-6, then with solvent I extraction 14 times, each water and solvent volume ratio=1 mutually: 0.2-2, isolate the solvent layer, with solvent II extraction 1-4 time, each water is 1 with solvent volume ratio mutually to water layer: 0.2-3 again; Merge the solvent II liquid after each time extracts, get solvent liquid B, wherein contain iso-agnolol;
(4) the solvent liquid B to collecting in the step (3) carries out concentrating under reduced pressure being lower than under 70 ℃ of conditions, obtains paste enriched material B1;
(5) the paste enriched material B1 to obtaining in the step (4) carries out the of the same race of one or many or filler chromatography purification not of the same race with chromatography, obtains content and is 〉=98% oily iso-agnolol;
Solvent described in the above-mentioned steps (2) is that volume ratio is the 30-95% aqueous ethanolic solution, or is a kind of in chloroform, methylene dichloride, ethyl acetate, acetone and the methyl alcohol;
Solvent I described in the step (3) is a kind of in sherwood oil, hexane, hexanaphthene and the normal hexane; Sherwood oil preferably;
Solvent II described in the step (3) is a kind of in the Fatty Alcohol(C12-C14 and C12-C18) of ethyl acetate, methylene dichloride, ethylene dichloride, trichloromethane, tetracol phenixin, ether, butylacetate, pentyl acetate and C3-C5; Ethyl acetate preferably;
The filler of chromatography column is a kind of among silica gel, D101 macroporous resin, neutral alumina, polymeric amide, reverse phase silica gel C-18 and the reverse phase silica gel C-8 in the described purification by chromatography of step (5); Silica gel preferably;
The purification process of the chromatography column of the various fillers of the present invention is respectively:
Silica gel column chromatography is a filler with 60-400 order silica gel in the described chromatography, paste enriched material B1 and silica gel ratio of weight and number are 1: 10～100, with sherwood oil: the mixed solvent of ethyl acetate, sherwood oil: the mixed solvent of chloroform and sherwood oil: a kind of in the mixed solvent of ether, be elutriant by volume from 100: 0 to 50: 50, promptly successively with 100: 0,95: 5,90: 10,85: 15,70: 30,50: 50 mixed solution is a gradient eluent, carry out gradient elution, each gradient elution to no sample detects, increase to next gradient again, with the elutriant of collecting that contains iso-agnolol, concentrating under reduced pressure becomes oily liquids under 70 ℃ of temperature being lower than;
D101 macroporous resin chromatography column is a filler with the D101 macroporous resin in the described chromatography, the ratio of weight and number of paste enriched material B1 and filler is 1: 20～100, it is colourless that water is eluted to the wash-out effluent liquid, and then be that 40%, 60%, 80%, 95% aqueous ethanolic solution carries out gradient elution with volume percent successively, be eluted to the colourless gradient that increases again of wash-out effluent liquid at every turn, collection contains iso-agnolol wash-out effluent liquid, wash-out effluent liquid concentrating under reduced pressure is become oily liquids being lower than under 70 ℃ of temperature again;
The neutral alumina chromatography column is a filler with 60-300 order neutral alumina in the described chromatography, the ratio of weight and number of paste enriched material B1 and neutral alumina is 1: 20～100, with sherwood oil: the mixed solvent of ethyl acetate, sherwood oil: the mixed solvent of chloroform and sherwood oil: a kind of in the mixed solvent of ether, be gradient eluent by volume from 100: 0 to 50: 50, promptly successively with 100: 0,95: 5,90: 10,85: 15,70: 30,50: 50 polarity gradient increases, carry out gradient elution, each gradient elution to no sample detects, increase to next gradient again, collection contains the wash-out effluent liquid of iso-agnolol, controlled temperature is lower than 70 ℃, and wash-out effluent liquid concentrating under reduced pressure is become oily liquids;
Polymeric amide chromatography post is a filler with 60-300 order polymeric amide in the described chromatography, the ratio of weight and number of paste enriched material B1 and polymeric amide is 1: 20～100, with sherwood oil: the mixed solvent of ethyl acetate, be gradient eluent by volume from 100: 0 to 50: 50, promptly successively with 100: 0,95: 5,90: 10,85: 15,70: 30, be gradient eluent at 50: 50, or with sherwood oil: the mixed solvent of chloroform, be gradient eluent by volume from 100: 0 to 50: 50, promptly successively with 100: 0,95: 5,90: 10,85: 15,70: 30,50: 50 mixed solution is a gradient eluent, carry out gradient elution, each gradient elution to no sample detects, increase to next gradient again, collection contains the wash-out effluent liquid of iso-agnolol, and controlled temperature is lower than 70 ℃, and wash-out effluent liquid concentrating under reduced pressure is become oily liquids;
The reversed-phase silica gel chromatography post is a filler with 100-300 order reverse phase silica gel C-18 or C-8 in the described chromatography, the ratio of weight and number of paste enriched material B1 and reverse phase silica gel C-18 or C-8 is 1: 20～100, is elutriant with volume ratio from the mixed solvent of 50: 50 to 100: 0 first alcohol and water, collection contains the elutriant of iso-agnolol, or with the methyl alcohol of volume ratio from 50: 50 to 100: 0: water mixed liquid is a gradient eluent, promptly successively with 50: 50,65: 35,80: 20, be gradient eluent at 100: 0, carry out gradient elution, each gradient elution to no sample detects, increase to next gradient again, collect the wash-out effluent liquid that contains iso-agnolol; Controlled temperature is lower than 70 ℃, the wash-out effluent liquid concentrating under reduced pressure of collecting is become oily liquids then.
In described extraction step, the streblus asper medicinal material can be fresh, but dry product preferably can improve extraction yield like this, and medicinal material is chop up better.
In described extraction step, can use or not with an organic solvent, yet iso-agnolol is water insoluble, so preferably with an organic solvent.
Advantage of the present invention is: found an extraction source that extracts iso-agnolol, this method is extracted, isolating iso-agnolol purity height.Iso-agnolol shows the good restraining activity to many mushrooms such as hepatitis B virus, Ao Lisi staphylococcus, M. smegmatics, saccharomyces cerevisiae, microsporon mentagrophytes and ACAT etc., be the high reactivity composition, the target drug of iso-agnolol center exploitation hepatitis B virus resisting and antibiotic usefulness, for the natural port of exploitation high-efficiency low-toxicity take medicine, medicines such as injection, externally applied agent are significant; In addition, streblus asper is classical simply Chinese medicine commonly used, and the property of medicine is clear, aboundresources, extraction separation hepatitis B virus resisting and antimicrobial iso-agnolol separating technology maturation, simple and easy from streblus asper; The chemical structure and the mechanism of action of streblus asper hepatitis B virus resisting and antimicrobial iso-agnolol are clear and definite, and it is very bright to develop hepatitis B virus resisting and antimicrobial new drug future with this, for a new road has been created in the development and use of streblus asper.
Description of drawings:
Fig. 1 is the iso-agnolol that obtains among the embodiment
1The H-NMR collection of illustrative plates;
Fig. 2 is the iso-agnolol that obtains among the embodiment
13The C-NMR collection of illustrative plates.
Embodiment:
Below by embodiment the present invention is further described, but is not to be used for limiting the scope of the invention.
Get root, skin, the stem of 3.2Kg through the streblus asper of natural drying at room temperature, after shredding with 75% aqueous ethanolic solution (ethanol: water, volume ratio) heating and refluxing extraction is 3 times, each reflux time is 2 hours, the add-on of 75% aqueous ethanolic solution is calculated according to the raw material add-on, each volume that adds 75% aqueous ethanolic solution rises the ratio 10: 1 of number and streblus asper dry product weight kilogram number, with the extracting liquid filtering that aforesaid operations obtains, and collection filtrate; Being lower than under 70 ℃ of temperature, filtrate is carried out concentrating under reduced pressure, remove ethanol, proportion is 1.20 medicinal extract shape extract A (380g); In extract A, add the dissolving of suitable quantity of water suspendible then, the weight kilogram number of extract A: the volume of water rises number=1: 5, with petroleum ether extraction suspension 4 times, mutually volume ratio of each water and solvent=1: 2, isolate the solvent layer, water layer is used ethyl acetate extraction 4 times again, and each water is 1: 2 with ethyl acetate volume ratio mutually; Merge the acetic acid ethyl fluid B after each time extracts, concentrating under reduced pressure obtains paste enriched material B1 (175g).Get the normal pressure glass chromatography column, with 60 order silica gel is filler, paste enriched material B1 and silica gel ratio of weight and number were at 1: 50, with sherwood oil: the ethyl acetate volume ratio increases 100: 0,95: 5,90: 10,85: 15,70: 30,50: 50 polarity gradient, carries out gradient elution, and each gradient elution to no sample detects, increase to next gradient again, collection contains the wash-out effluent liquid of iso-agnolol, and controlled temperature is lower than 70 ℃, and wash-out effluent liquid concentrating under reduced pressure is become oily liquids (32g); And then get the normal pressure glass chromatography column, with 200-300 order silica gel is filler, contain the oily enriched material of iso-agnolol and silica gel ratio of weight and number at 1: 100, with sherwood oil: chloroform volume ratio increases 100: 0,95: 5,90: 10,85: 15,70: 30,50: 50 polarity gradient, carry out gradient elution, each gradient elution to no sample detects, increase to next gradient again, collection contains the wash-out effluent liquid of iso-agnolol, controlled temperature is lower than 70 ℃, with wash-out effluent liquid concentrating under reduced pressure, obtain oily liquids iso-agnolol (20.5g).
Embodiment 2
Get root, skin, the stem of 3.0Kg through the streblus asper of natural drying at room temperature, after shredding, use chloroform heating and refluxing extraction 3 times, each reflux time is 2 hours, the add-on of chloroform is calculated according to the raw material add-on, each volume that adds chloroform rises the ratio 12: 1 of number and streblus asper dry product weight kilogram number, with the extracting liquid filtering that aforesaid operations obtains, collect filtrate; Being lower than under 70 ℃ of temperature, filtrate is carried out concentrating under reduced pressure, remove chloroform, proportion is 1.21 medicinal extract shape extract A (360g); In extract A, add the dissolving of suitable quantity of water suspendible then, the weight kilogram number of extract A: the volume of water rises number=1: 5, with petroleum ether extraction suspension 4 times, mutually volume ratio of each water and solvent=1: 2, isolate the solvent layer, water layer is used chloroform extraction 4 times again, and each water is 1: 2 with chloroform volume ratio mutually; Merge the chloroform solution B after each time extracts, concentrating under reduced pressure obtains paste enriched material B1 (175g).Get the normal pressure glass chromatography column during chromatography, with the D101 macroporous resin is filler, the ratio of weight and number of paste enriched material B1 and filler is 1: 50, it is colourless that water is eluted to the wash-out effluent liquid, and then be that 40%, 60%, 80%, 95% aqueous ethanolic solution carries out gradient elution with volume percent successively, be eluted to the colourless gradient that increases again of wash-out effluent liquid at every turn, collect the main iso-agnolol wash-out effluent liquid that contains, wash-out effluent liquid concentrating under reduced pressure is become oily liquids (117g) being lower than under 70 ℃ of temperature; Get the normal pressure glass chromatography column, with 200-300 order silica gel is filler, extract concentrated oily liquids and silica gel ratio of weight and number at 1: 10, with sherwood oil: the ether volume ratio increases 100: 0,95: 5,90: 10,85: 15,70: 30,50: 50 polarity gradient, carries out gradient elution, and each gradient elution to no sample detects, increase to next gradient again, collection contains the wash-out effluent liquid of iso-agnolol, and controlled temperature is lower than 70 ℃, and wash-out effluent liquid concentrating under reduced pressure is become oily liquids (27g); And then get the normal pressure glass chromatography column, with 200-300 order silica gel is filler, contain the oily enriched material of iso-agnolol and polymeric amide ratio of weight and number at 1: 50, with sherwood oil: chloroform volume ratio increases 100: 0,95: 5,90: 10,85: 15,70: 30,50: 50 polarity gradient, carry out gradient elution, each gradient elution to no sample detects, increase to next gradient again, collection contains the wash-out effluent liquid of iso-agnolol, controlled temperature is lower than 70 ℃, with wash-out effluent liquid concentrating under reduced pressure, obtain oily liquids iso-agnolol (20.3g).
Embodiment 3
Get root, skin, the stem of 3.0Kg through the streblus asper of natural drying at room temperature, after shredding, use methyl alcohol heating and refluxing extraction 3 times, each reflux time is 2 hours, the add-on of methyl alcohol is calculated according to the raw material add-on, each volume that adds methyl alcohol rises the ratio 6: 1 of number and streblus asper dry product weight kilogram number, with the extracting liquid filtering that aforesaid operations obtains, collect filtrate; Being lower than under 70 ℃ of temperature, filtrate is carried out concentrating under reduced pressure, remove methyl alcohol, proportion is 1.19 medicinal extract shape extract A (375g); In extract A, add the dissolving of suitable quantity of water suspendible then, the weight kilogram number of extract A: the volume of water rises number=1: 5, with n-hexane extraction suspension 4 times, the volume ratio of each water and normal hexane=1: 2, isolate the normal hexane layer, water layer is used dichloromethane extraction 4 times again, and each water is 1: 2 with methylene dichloride volume ratio mutually; Merge the dichloromethane solution B after each time extracts, concentrating under reduced pressure obtains paste enriched material B1 (196g).Get the normal pressure glass chromatography column, with the neutral alumina is filler, paste enriched material B1 and neutral alumina ratio of weight and number were at 1: 50, with sherwood oil: the ethyl acetate volume ratio increases 100: 0,95: 5,90: 10,85: 15,70: 30,50: 50 polarity gradient, carry out gradient elution, each gradient elution to no sample detects, increase to next gradient again, collection contains the wash-out effluent liquid of iso-agnolol, controlled temperature is lower than 70 ℃, and wash-out effluent liquid concentrating under reduced pressure is become oily liquids (30g); And then get the normal pressure glass chromatography column, with 200-300 order silica gel is filler, contain the oily enriched material of iso-agnolol and silica gel ratio of weight and number at 1: 100, with sherwood oil: chloroform volume ratio increases 100: 0,95: 5,90: 10,85: 15,70: 30,50: 50 polarity gradient, carry out gradient elution, each gradient elution to no sample detects, increase to next gradient again, collection contains the wash-out effluent liquid of iso-agnolol, controlled temperature is lower than 70 ℃, with wash-out effluent liquid concentrating under reduced pressure, obtain oily liquids iso-agnolol (19.8g).
Embodiment 4
Get root, skin, the stem of 3.0Kg through the streblus asper of natural drying at room temperature, soaking diacolation with acetone after shredding extracts 3 times, each immersion diacolation time is 2 days, the add-on of acetone is calculated according to the raw material add-on, each volume that adds acetone rises the ratio 10: 1 of number and streblus asper dry product weight kilogram number, with the extracting liquid filtering that aforesaid operations obtains, collect filtrate; Being lower than under 70 ℃ of temperature, filtrate is carried out concentrating under reduced pressure, remove acetone, proportion is 1.18 medicinal extract shape extract A (302g); In extract A, add the dissolving of suitable quantity of water suspendible then, the weight kilogram number of extract A: the volume of water rises number=1: 5, extract suspension 4 times with hexanaphthene, the volume ratio of each water and hexanaphthene=1: 2, isolate the hexanaphthene layer, water layer is used dichloromethane extraction 4 times again, and each water is 1: 2 with methylene dichloride volume ratio mutually; Merge the dichloromethane solution B after each time extracts, concentrating under reduced pressure obtains paste enriched material B1 (158g).Get the normal pressure glass chromatography column, with silica gel is filler, paste enriched material B1 and silica gel ratio of weight and number were at 1: 50, with sherwood oil: the ethyl acetate volume ratio increases 100: 0,95: 5,90: 10,85: 15,70: 30,50: 50 polarity gradient, carries out gradient elution, and each gradient elution to no sample detects, increase to next gradient again, collection contains the wash-out effluent liquid of iso-agnolol, and controlled temperature is lower than 70 ℃, and wash-out effluent liquid concentrating under reduced pressure is become oily liquids (31g); And then get the normal pressure glass chromatography column, being filler with 300 order reverse phase silica gel C-18, contain the oily enriched material of iso-agnolol and reverse phase silica gel C-18 ratio of weight and number at 1: 50, with volume ratio methyl alcohol: water mixed liquid increases 50: 50,65: 35,80: 20,100: 0 polarity gradient, carry out gradient elution, each gradient elution to no sample detects, increase to next gradient again, collection contains the wash-out effluent liquid of iso-agnolol, controlled temperature is lower than 70 ℃, with wash-out effluent liquid concentrating under reduced pressure, obtain oily liquids iso-agnolol (18.7g).
Embodiment 5
Get root, skin, the stem of 3.0Kg through the streblus asper of natural drying at room temperature, after shredding with 95% aqueous ethanolic solution (ethanol: water, volume ratio) soaking diacolation extracts 3 times, each immersion diacolation time is 2 days, the add-on of 95% aqueous ethanolic solution is calculated according to the raw material add-on, each volume that adds 95% aqueous ethanolic solution rises the ratio 10: 1 of number and streblus asper dry product weight kilogram number, with the extracting liquid filtering that aforesaid operations obtains, and collection filtrate; Being lower than under 70 ℃ of temperature, filtrate is carried out concentrating under reduced pressure, remove ethanol, proportion is 1.18 medicinal extract shape extract A (298g); In extract A, add the dissolving of suitable quantity of water suspendible then, the weight kilogram number of extract A: the volume of water rises number=1: 5, with hexane extraction suspension 4 times, the volume ratio of each water and hexane=1: 2, isolate hexane, water layer is used dichloromethane extraction 4 times again, and each water is 1: 2 with methylene dichloride volume ratio mutually; Merge the dichloromethane solution B after each time extracts, concentrating under reduced pressure obtains paste enriched material B1 (150g).Get the normal pressure glass chromatography column, with silica gel is filler, paste enriched material B1 and silica gel ratio of weight and number were at 1: 50, with sherwood oil: the ethyl acetate volume ratio increases 100: 0,95: 5,90: 10,85: 15,70: 30,50: 50 polarity gradient, carries out gradient elution, and each gradient elution to no sample detects, increase to next gradient again, collection contains the wash-out effluent liquid of iso-agnolol, and controlled temperature is lower than 70 ℃, and wash-out effluent liquid concentrating under reduced pressure is become oily liquids (28g); And then get the normal pressure glass chromatography column, being filler with 300 order reverse phase silica gel C-8, contain the oily enriched material of iso-agnolol and reverse phase silica gel C-8 ratio of weight and number at 1: 50, with volume ratio methyl alcohol: water mixed liquid increases 50: 50,65: 35,80: 20,100: 0 polarity gradient, carry out gradient elution, each gradient elution to no sample detects, increase to next gradient again, collection contains the wash-out effluent liquid of iso-agnolol, controlled temperature is lower than 70 ℃, with wash-out effluent liquid concentrating under reduced pressure, obtain oily liquids iso-agnolol (18.8g).
Embodiment 6
Press the method steps of embodiment 4, different is: bulk drug extracts with soaking the diacolation method after shredding, and at normal temperatures medicinal material is carried out ethyl acetate and soaks extraction.
The top stream part iso-agnolol that obtains after testing, physical data is as follows:
Detect through liquid phase chromatography, the content of iso-agnolol is greater than 98%;
Iso-agnolol, molecular formula C
18H
18O
2, colourless oil liquid;
UV
nm(logε)：289(2.33)，254(2.65)，208(4.54)；
IR(KBr)v
max(cm
-1)：3467，1673(C＝O)，1641，1600，1496，1423；
ESI-MS?m/z(％)：265(100)[M-H]
-；
1H?NMR(500MHz，CD
3COCD
3)δH(ppm)：6.86(1H，d，J＝1.6Hz，H-3)，7.07(1H，d，J＝8.4Hz，H-5)，6.92(1H，dd，J＝8.4，1.6Hz，H-6)，3.36(2H，d，J＝6.8Hz，H-7)，6.03(H，m，H-8)，5.13(2H，m，H-9)，6.96(2H，d，J＝8.5Hz，H-2′，6′)，7.14(1H，dd，J＝8.5Hz，H-3′，5′)，3.29(2H，d，J＝6.7Hz，H-7′)，6.03(1H，m，H-8′)，5.13(2H，m，H-9′)；
13C?NMR(125MHz，CD
3COCD
3)δC(ppm)：147.3(C-1)，143.6(C-2)，120.8(C-3)，132.1(C-4)，125.1(C-5)，117.2(C-6)，39.4(C-7)，137.9(C-8)，115.5(C-9)，156.5(C-1′)，117.3(C-2′，6′)，129.8(C-3′，5′)，134.2(C-4′)，39.3(C-7′)，137.9(C-8′)，115.4(C-9′)。
Above-mentioned data and document (El-Feraly, et al, Journal of Natural Products, 1983) record is consistent.
Claims (11)
1. the method for extraction separation and purifying iso-agnolol from a streblus asper is characterized in that: comprise the steps:
(1) gets the raw medicinal material streblus asper, with crude drug dry product or the chopping of fresh medicinal material;
(2) with the raw medicinal material through shredding in the solvent heating and refluxing extraction method extraction step (1): add solvent in medicinal material in proportion, heating and refluxing extraction is 2-5 time repeatedly, and each reflux time is 1-4 hour; With the extracting liquid filtering that aforesaid operations obtains, collect filtrate; Being lower than under 70 ℃ of temperature, filtrate is carried out concentrating under reduced pressure, remove solvent, getting proportion is the medicinal extract shape extract A of 1.1-1.4; The add-on of described solvent is: the volume that at every turn adds solvent rises number: add dry product weight kilogram number=6-12 at first: 1, or be that the volume of solvent rises number: fresh medicinal material weight kilogram number=4-8: 1;
(3) extract A to obtaining in the step (2), add the dissolving of entry suspendible earlier, the weight kilogram number of extract A: the volume of water rises number=1: 3-6, then with solvent I extraction 1-4 time, each water and solvent volume ratio=1 mutually: 0.2-2, isolate the solvent layer, with solvent II extraction 1-4 time, each water is 1 with solvent volume ratio mutually to water layer: 0.2-3 again; Merge the solvent II liquid after each time extracts, get solvent liquid B, wherein contain iso-agnolol;
(4) the solvent liquid B to collecting in the step (3) carries out concentrating under reduced pressure being lower than under 70 ℃ of conditions, obtains paste enriched material B1;
(5) the paste enriched material B1 to obtaining in the step (4) carries out the of the same race of one or many or filler purification by chromatography not of the same race with chromatography, obtains content and is 〉=98% oily iso-agnolol.
2. the method for extraction separation and purifying iso-agnolol from a streblus asper is characterized in that: comprise the steps:
(1) gets the raw medicinal material streblus asper, with crude drug dry product or the chopping of fresh medicinal material;
(2) with soaking the raw medicinal material through shredding in the percolation diacolation step (1): in raw medicinal material, add solvent in proportion, at normal temperatures medicinal material is carried out 1-4 diacolation and extract each diacolation 1-3 days; With the extracting liquid filtering that aforesaid operations obtains, collect filtrate; Being lower than under 70 ℃ of temperature, filtrate is carried out concentrating under reduced pressure, remove solvent, getting proportion is the medicinal extract shape extract A of 1.1-1.4; The add-on of described solvent is: the volume that at every turn adds solvent rises number: add dry product weight kilogram number=6-12 at first: 1, or be that the volume of solvent rises number: fresh medicinal material weight kilogram number=4-8: 1;
(3) extract A to obtaining in the step (2), add the dissolving of entry suspendible earlier, the weight kilogram number of extract A: the volume of water rises number=1: 3-6, then with solvent I extraction 1-4 time, each water and solvent volume ratio=1 mutually: 0.2-2, isolate the solvent layer, with solvent II extraction 1-4 time, each water is 1 with solvent volume ratio mutually to water layer: 0.2-3 again; Merge the solvent II liquid after each time extracts, get solvent liquid B, wherein contain iso-agnolol;
(4) the solvent liquid B to collecting in the step (3) carries out concentrating under reduced pressure being lower than under 70 ℃ of conditions, obtains paste enriched material B1;
(5) the paste enriched material B1 to obtaining in the step (4) carries out the of the same race of one or many or filler purification by chromatography not of the same race with chromatography, obtains content and is 〉=98% oily iso-agnolol.
3. method according to claim 1 and 2 is characterized in that: the solvent described in the step (2) is that volume ratio is the 30-95% aqueous ethanolic solution, or is a kind of in chloroform, methylene dichloride, ethyl acetate, acetone and the methyl alcohol.
4. method according to claim 1 and 2 is characterized in that: the solvent I described in the step (3) is a kind of in sherwood oil, hexane, hexanaphthene and the normal hexane.
5. method according to claim 1 and 2 is characterized in that: the solvent II described in the step (3) is a kind of in the Fatty Alcohol(C12-C14 and C12-C18) of ethyl acetate, methylene dichloride, ethylene dichloride, trichloromethane, tetracol phenixin, ether, butylacetate, pentyl acetate and C3-C5.
6. method according to claim 1 and 2 is characterized in that: the filler of chromatography column is a kind of among silica gel, D101 macroporous resin, neutral alumina, polymeric amide, reverse phase silica gel C-18 and the reverse phase silica gel C-8 in the described purification by chromatography of step (5).
7. according to claim 1 or 6 described methods, it is characterized in that: silica gel column chromatography is a filler with 60-400 order silica gel in the described chromatography, paste enriched material B1 and silica gel ratio of weight and number are 1: 10～100, with sherwood oil: the mixed solvent of ethyl acetate, sherwood oil: the mixed solvent of chloroform and sherwood oil: a kind of in the mixed solvent of ether, be elutriant by volume from 100: 0 to 50: 50, promptly successively with 100: 0,95: 5,90: 10,85: 15,70: 30,50: 50 mixed solution is a gradient eluent, carry out gradient elution, each gradient elution to no sample detects, increase to next gradient again, with the elutriant of collecting that contains iso-agnolol, concentrating under reduced pressure becomes oily liquids under 70 ℃ of temperature being lower than.
8. according to claim 1 or 6 described methods, it is characterized in that: D101 macroporous resin chromatography column is a filler with the D101 macroporous resin in the described chromatography, the ratio of weight and number of paste enriched material B1 and filler is 1: 20～100, it is colourless that water is eluted to the wash-out effluent liquid, and then be that 40%, 60%, 80%, 95% aqueous ethanolic solution carries out gradient elution with volume percent successively, be eluted to the colourless gradient that increases again of wash-out effluent liquid at every turn, collection contains iso-agnolol wash-out effluent liquid, wash-out effluent liquid concentrating under reduced pressure is become oily liquids being lower than under 70 ℃ of temperature again.
9. according to claim 1 or 6 described methods, it is characterized in that: the neutral alumina chromatography column is a filler with 60-300 order neutral alumina in the described chromatography, the ratio of weight and number of paste enriched material B1 and neutral alumina is 1: 20～100, with sherwood oil: the mixed solvent of ethyl acetate, sherwood oil: the mixed solvent of chloroform and sherwood oil: a kind of in the mixed solvent of ether, be gradient eluent by volume from 100: 0 to 50: 50, promptly successively with 100: 0,95: 5,90: 10,85: 15,70: 30,50: 50 polarity gradient increases, carry out gradient elution, each gradient elution to no sample detects, increase to next gradient again, collection contains the wash-out effluent liquid of iso-agnolol, controlled temperature is lower than 70 ℃, and wash-out effluent liquid concentrating under reduced pressure is become oily liquids.
10. according to claim 1 or 6 described methods, it is characterized in that: polymeric amide chromatography post is a filler with 60-300 order polymeric amide in the described chromatography, the ratio of weight and number of paste enriched material B1 and polymeric amide is 1: 20～100, with sherwood oil: the mixed solvent of ethyl acetate, be gradient eluent by volume from 100: 0 to 50: 50, promptly successively with 100: 0,95: 5,90: 10,85: 15,70: 30, be gradient eluent at 50: 50, or with sherwood oil: the mixed solvent of chloroform, be gradient eluent by volume from 100: 0 to 50: 50, promptly successively with 100: 0,95: 5,90: 10,85: 15,70: 30,50: 50 mixed solution is a gradient eluent, carry out gradient elution, each gradient elution to no sample detects, increase to next gradient again, collection contains the wash-out effluent liquid of iso-agnolol, controlled temperature is lower than 70 ℃, and wash-out effluent liquid concentrating under reduced pressure is become oily liquids.
11. according to claim 1 or 6 described methods, it is characterized in that: the reversed-phase silica gel chromatography post is a filler with 100-300 order reverse phase silica gel C-18 or C-8 in the described chromatography, the ratio of weight and number of paste enriched material B1 and reverse phase silica gel C-18 or C-8 is 1: 20～100, is elutriant with volume ratio from the mixed solvent of 50: 50 to 100: 0 first alcohol and water, collection contains the elutriant of iso-agnolol, or with the methyl alcohol of volume ratio from 50: 50 to 100: 0: water mixed liquid is a gradient eluent, promptly successively with 50: 50,65: 35,80: 20, be gradient eluent at 100: 0, carry out gradient elution, each gradient elution to no sample detects, increase to next gradient again, collect the wash-out effluent liquid that contains iso-agnolol; Controlled temperature is lower than 70 ℃, the wash-out effluent liquid concentrating under reduced pressure of collecting is become oily liquids then.
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| 《Journal of Natural Products》 19830831 Farouk S. El-Feraly., et al Antimicrobial neolignans of Sassafras randaiense roots 493-498 1-11 第46卷, 第4期 * |
| 《中成药》 20100531 梁成钦等 鹊肾树叶化学成分研究 824-827 1-11 第32卷, 第5期 * |
| 《广西师范大学学报: 自然科学版》 20060930 张艳军等 鹊肾树心材化学成分研究 61-63 1-11 第24卷, 第3期 * |
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