CN102229526B - 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): in raw material, add solvent in proportion, repeated heating refluxing extraction 2-5 time, each reflux time is 1-4 hour; With the extracting liquid filtering that aforesaid operations obtains, collect filtrating; Being lower than under 70 ℃ of temperature, filtrating 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 said 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, water layer is again with solvent II extraction 1-4 time, and each water is 1 with solvent volume ratio mutually: 0.2-3; 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 filtrating; Being lower than under 70 ℃ of temperature, filtrating 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 said 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, water layer is again with solvent II extraction 1-4 time, and each water is 1 with solvent volume ratio mutually: 0.2-3; 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, ETHYLE 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 ETHYLE ACETATE, methylene dichloride, ethylene dichloride, trichloromethane, tetracol phenixin, ether, butylacetate, pentyl acetate and C3-C5; ETHYLE 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 said 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 said chromatography; Paste enriched material B1 and silica gel ratio of weight and number are 1: 10～100; With sherwood oil: the mixed solvent of ETHYLE ACETATE, sherwood oil: the mixed solvent of chloroform and sherwood oil: a kind of in the mixed solvent of ether; Being elutriant by volume from 100: 0 to 50: 50, is gradient eluent with 100: 0,95: 5,90: 10,85: 15,70: 30,50: 50 mixed solution successively promptly, carries out gradient elution; Each gradient elution to n.s. 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 said 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; And then to use volume percent successively be that 40%, 60%, 80%, 95% aqueous ethanolic solution carries out gradient elution; Be eluted to the colourless gradient that increases again of wash-out effluent at every turn, collect and to contain iso-agnolol wash-out effluent, wash-out effluent 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 said 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 ETHYLE ACETATE, sherwood oil: the mixed solvent of chloroform and sherwood oil: a kind of in the mixed solvent of ether was gradient eluent by volume from 100: 0 to 50: 50, promptly increased with 100: 0,95: 5,90: 10,85: 15,70: 30,50: 50 polarity gradient successively; Carry out gradient elution; Each gradient elution to n.s. detects, and increases to next gradient again, collects the wash-out effluent that contains iso-agnolol; Controlled temperature is lower than 70 ℃, and wash-out effluent concentrating under reduced pressure is become oily liquids;
Polymeric amide chromatography post is a filler with 60-300 order polymeric amide in the said chromatography, and 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 ETHYLE ACETATE; Being gradient eluent by volume from 100: 0 to 50: 50, was gradient eluent with 100: 0,95: 5,90: 10,85: 15,70: 30,50: 50 successively promptly, or with sherwood oil: the mixed solvent of chloroform; Be gradient eluent by volume from 100: 0 to 50: 50; Be gradient eluent with 100: 0,95: 5,90: 10,85: 15,70: 30,50: 50 mixed solution successively promptly, carry out gradient elution, each gradient elution to n.s. detects; Increase to next gradient again; Collection contains the wash-out effluent of iso-agnolol, and controlled temperature is lower than 70 ℃, and wash-out effluent 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 said 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, collects the elutriant that contains iso-agnolol; Or with the methyl alcohol of volume ratio from 50: 50 to 100: 0: water mixed liquid is a gradient eluent; Be gradient eluent with 50: 50,65: 35,80: 20,100: 0 successively promptly, carry out gradient elution, each gradient elution to n.s. detects; Increase to next gradient again, collect the wash-out effluent that contains iso-agnolol; Controlled temperature is lower than 70 ℃, the wash-out effluent 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 is high.Iso-agnolol shows that to many mushrooms such as hepatitis B virus, Ao Lisi staphylococcus, M. smegmatics, saccharomyces cerevisiae, microsporon mentagrophytes and ACAT etc. good restraining is active; 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, and extraction separation hepatitis B virus resisting and antimicrobial iso-agnolol separating technology are ripe, 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:
Through embodiment the present invention is further described below, but be not to be used for limiting scope of the present invention.
Get root, skin, the stem of 3.2Kg through the streblus asper of natural drying at room temperature; With 75% aqueous ethanolic solution (ethanol: water, volume ratio) heating and refluxing extraction 3 times, the reflux time is 2 hours at every turn through chopping back; 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, collects and filtrates; Being lower than under 70 ℃ of temperature, filtrating 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 is isolated the solvent layer; Water layer is used ethyl acetate extraction 4 times again, and each water is 1: 2 with ETHYLE 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).Getting the normal pressure glass chromatography column, is filler with 60 order silica gel, and paste enriched material B1 and silica gel ratio of weight and number were at 1: 50; With sherwood oil: the ETHYLE 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 n.s. detects; Increase to next gradient again; Collection contains the wash-out effluent of iso-agnolol, and controlled temperature is lower than 70 ℃, and wash-out effluent concentrating under reduced pressure is become oily liquids (32g); And then get the normal pressure glass chromatography column, and be filler with 200-300 order silica gel, the oily enriched material and the silica gel ratio of weight and number that contain iso-agnolol were 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 n.s. detects, and increases to next gradient again; Collection contains the wash-out effluent of iso-agnolol; Controlled temperature is lower than 70 ℃, with wash-out effluent concentrating under reduced pressure, obtains 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 chopping with chloroform heating and refluxing extraction 3 times; Each reflux time is 2 hours, and the add-on of chloroform is calculated according to the raw material add-on, and the volume that at every turn 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 filtrating; Being lower than under 70 ℃ of temperature, filtrating 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 is isolated 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, and the ratio of weight and number of paste enriched material B1 and filler is 1: 50, and it is colourless that water is eluted to the wash-out effluent; And then to use volume percent successively be that 40%, 60%, 80%, 95% aqueous ethanolic solution carries out gradient elution; Be eluted to the colourless gradient that increases again of wash-out effluent at every turn, collect the main iso-agnolol wash-out effluent that contains, wash-out effluent concentrating under reduced pressure is become oily liquids (117g) being lower than under 70 ℃ of temperature; Getting the normal pressure glass chromatography column, is filler with 200-300 order silica gel, extracts 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 n.s. detects; Increase to next gradient again; Collection contains the wash-out effluent of iso-agnolol, and controlled temperature is lower than 70 ℃, and wash-out effluent concentrating under reduced pressure is become oily liquids (27g); And then get the normal pressure glass chromatography column, and be filler with 200-300 order silica gel, the oily enriched material and the polymeric amide ratio of weight and number that contain iso-agnolol were 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 n.s. detects, and increases to next gradient again; Collection contains the wash-out effluent of iso-agnolol; Controlled temperature is lower than 70 ℃, with wash-out effluent concentrating under reduced pressure, obtains 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 chopping with methyl alcohol heating and refluxing extraction 3 times; Each reflux time is 2 hours, and the add-on of methyl alcohol is calculated according to the raw material add-on, and the volume that at every turn 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 filtrating; Being lower than under 70 ℃ of temperature, filtrating 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 is isolated 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, and paste enriched material B1 and neutral alumina ratio of weight and number be at 1: 50, and with sherwood oil: the ETHYLE 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 n.s. detects, and increases to next gradient again, collects the wash-out effluent that contains iso-agnolol; Controlled temperature is lower than 70 ℃, and wash-out effluent concentrating under reduced pressure is become oily liquids (30g); And then get the normal pressure glass chromatography column, and be filler with 200-300 order silica gel, the oily enriched material and the silica gel ratio of weight and number that contain iso-agnolol were 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 n.s. detects, and increases to next gradient again; Collection contains the wash-out effluent of iso-agnolol; Controlled temperature is lower than 70 ℃, with wash-out effluent concentrating under reduced pressure, obtains 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; After chopping, soaking diacolation with acetone extracts 3 times; Each to soak the diacolation time be 2 days, and the add-on of acetone is calculated according to the raw material add-on, and the volume that at every turn 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 filtrating; Being lower than under 70 ℃ of temperature, filtrating 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 is isolated 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).Getting the normal pressure glass chromatography column, is filler with silica gel, and paste enriched material B1 and silica gel ratio of weight and number were at 1: 50; With sherwood oil: the ETHYLE 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 n.s. detects; Increase to next gradient again; Collection contains the wash-out effluent of iso-agnolol, and controlled temperature is lower than 70 ℃, and wash-out effluent concentrating under reduced pressure is become oily liquids (31g); And then get the normal pressure glass chromatography column, to be filler with 300 order reverse phase silica gel C-18, the oily enriched material and the reverse phase silica gel C-18 ratio of weight and number that contain iso-agnolol were 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 n.s. detects, and increases to next gradient again; Collection contains the wash-out effluent of iso-agnolol; Controlled temperature is lower than 70 ℃, with wash-out effluent concentrating under reduced pressure, obtains 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 chopping, soak diacolation with 95% aqueous ethanolic solution (ethanol: water, volume ratio) and extract 3 times, soak the diacolation time is 2 days at every turn; 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, collects and filtrates; Being lower than under 70 ℃ of temperature, filtrating 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 is isolated 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).Getting the normal pressure glass chromatography column, is filler with silica gel, and paste enriched material B1 and silica gel ratio of weight and number were at 1: 50; With sherwood oil: the ETHYLE 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 n.s. detects; Increase to next gradient again; Collection contains the wash-out effluent of iso-agnolol, and controlled temperature is lower than 70 ℃, and wash-out effluent concentrating under reduced pressure is become oily liquids (28g); And then get the normal pressure glass chromatography column, to be filler with 300 order reverse phase silica gel C-8, the oily enriched material and the reverse phase silica gel C-8 ratio of weight and number that contain iso-agnolol were 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 n.s. detects, and increases to next gradient again; Collection contains the wash-out effluent of iso-agnolol; Controlled temperature is lower than 70 ℃, with wash-out effluent concentrating under reduced pressure, obtains 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 the ETHYLE ACETATE soaking and extracting.
The top stream part iso-agnolol that obtains is through detecting, and physical data is following:
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 (8)
1. the method for extraction separation and purifying iso-agnolol from a streblus asper is characterized in that: comprise the steps:
(1) gets root, skin, the stem of 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): in medicinal material, add solvent in proportion, repeated heating refluxing extraction 2-5 time, each reflux time is 1-4 hour; With the extracting liquid filtering that aforesaid operations obtains, collect filtrating; Being lower than under 70 ℃ of temperature, filtrating 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 said 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; Described solvent is that volume ratio is the 30-95% aqueous ethanolic solution, or is a kind of in chloroform, methylene dichloride, ETHYLE ACETATE, acetone and the methyl alcohol;
(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, water layer is again with solvent II extraction 1-4 time, and each water is 1 with solvent volume ratio mutually: 0.2-3; Merge the solvent II liquid after each time extracts, get solvent liquid B, wherein contain iso-agnolol; Described solvent I is a kind of in sherwood oil, hexane and the hexanaphthene; A kind of in the Fatty Alcohol(C12-C14 and C12-C18) that described solvent II is ETHYLE ACETATE, methylene dichloride, ethylene dichloride, trichloromethane, tetracol phenixin, ether, butylacetate, pentyl acetate and C3-C5;
(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 root, skin, the stem of 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 filtrating; Being lower than under 70 ℃ of temperature, filtrating 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 said 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; Described solvent is that volume ratio is the 30-95% aqueous ethanolic solution, or is a kind of in chloroform, methylene dichloride, ETHYLE ACETATE, acetone and the methyl alcohol;
(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, water layer is again with solvent II extraction 1-4 time, and each water is 1 with solvent volume ratio mutually: 0.2-3; Merge the solvent II liquid after each time extracts, get solvent liquid B, wherein contain iso-agnolol; Described solvent I is a kind of in sherwood oil, hexane and the hexanaphthene; A kind of in the Fatty Alcohol(C12-C14 and C12-C18) that described solvent II is ETHYLE ACETATE, methylene dichloride, ethylene dichloride, trichloromethane, tetracol phenixin, ether, butylacetate, pentyl acetate and C3-C5;
(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 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 said purification by chromatography of step (5).
4. method according to claim 3; It is characterized in that: silica gel column chromatography is a filler with 60-400 order silica gel in the said chromatography; Paste enriched material B1 and silica gel ratio of weight and number are 1: 10～100, and with sherwood oil: the mixed solvent of ETHYLE ACETATE, sherwood oil: the mixed solvent of chloroform and sherwood oil: a kind of in the mixed solvent of ether was elutriant by volume from 100: 0 to 50: 50; Be gradient eluent with 100: 0,95: 5,90: 10,85: 15,70: 30,50: 50 mixed solution successively promptly; Carry out gradient elution, each gradient elution to n.s. detects, and increases 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.
5. method according to claim 3; It is characterized in that: D101 macroporous resin chromatography column is a filler with the D101 macroporous resin in the said 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, and then to use volume percent successively be that 40%, 60%, 80%, 95% aqueous ethanolic solution carries out gradient elution, is eluted to the colourless gradient that increases again of wash-out effluent at every turn; Collection contains iso-agnolol wash-out effluent, wash-out effluent concentrating under reduced pressure is become oily liquids being lower than under 70 ℃ of temperature again.
6. method according to claim 3; It is characterized in that: the neutral alumina chromatography column is a filler with 60-300 order neutral alumina in the said chromatography, and 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 ETHYLE 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 increase, carry out gradient elution, each gradient elution to n.s. detects; Increase to next gradient again; Collection contains the wash-out effluent of iso-agnolol, and controlled temperature is lower than 70 ℃, and wash-out effluent concentrating under reduced pressure is become oily liquids.
7. method according to claim 3 is characterized in that: polymeric amide chromatography post is a filler with 60-300 order polymeric amide in the said chromatography, and 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 ETHYLE ACETATE; Being gradient eluent by volume from 100: 0 to 50: 50, was gradient eluent with 100: 0,95: 5,90: 10,85: 15,70: 30,50: 50 successively promptly, or with sherwood oil: the mixed solvent of chloroform; Be gradient eluent by volume from 100: 0 to 50: 50; Be gradient eluent with 100: 0,95: 5,90: 10,85: 15,70: 30,50: 50 mixed solution successively promptly, carry out gradient elution, each gradient elution to n.s. detects; Increase to next gradient again; Collection contains the wash-out effluent of iso-agnolol, and controlled temperature is lower than 70 ℃, and wash-out effluent concentrating under reduced pressure is become oily liquids.
8. method according to claim 3; 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 said 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, collects the elutriant that contains iso-agnolol; Or with the methyl alcohol of volume ratio from 50: 50 to 100: 0: water mixed liquid is a gradient eluent; Be gradient eluent with 50: 50,65: 35,80: 20,100: 0 successively promptly, carry out gradient elution, each gradient elution to n.s. detects; Increase to next gradient again, collect the wash-out effluent that contains iso-agnolol; Controlled temperature is lower than 70 ℃, the wash-out effluent concentrating under reduced pressure of collecting is become oily liquids then.
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