CN102206138A - Method for separating and purifying two fragrance precursors from tobacco - Google Patents
Method for separating and purifying two fragrance precursors from tobacco Download PDFInfo
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Abstract
The invention belongs to the technical field of separation and purification and relates to a technique for separating and purifying active ingredients in tobacco, in particular to a method for extracting, separating and purifying alpha-4,8,13-duvatriene-1,3-diol and beta-4,8,13-Duvatriene-1,3-diol from fresh tobacco. The separation and purification method disclosed by the method comprises: (1) leaching; (2) liquid-liquid extraction; (3) separating by normal phase silica gel column chromatography; (4) separating by normal phase cyanogen-based column chromatography; and (5) separating by antiphase C18 column chromatography to obtain two fragrance precursors. The purities of the two fragrance precursors obtained by the separation and purification method are both over 99 percent, so that the two fragrance precursors can be used as standard products in related scientific research; and the product recovery rate of the whole separation and purification process, the process operation is simple and convenient, the automation degree is high, and batch production can be realized easily.
Description
Technical field
The invention belongs to the separating and purifying technology field, relate to the separating and purifying technology of effective constituent in the tobacco, be specifically related to a kind of extraction from new fresh tobacco leaf, separation and purifying α-4,8,13-west cypress triolefin-1,3-two pure and mild β-4,8,13-west cypress triolefin-1, the method for 3-glycol.
Background technology
α-4,8,13-west cypress triolefin-1,3-two pure and mild β-4,8,13-west cypress triolefin-1,3-glycol are importantly in the tobacco to cause fragrant precursor, are mainly derived from the surperficial secretory product of new fresh tobacco leaf and fireworks, most of can the degraded after modulation, generating important aroma component solanone and derivative thereof, the fragrance style and the quality of tobacco leaf had decisive influence, is one of crucial chemical substance of carrying out quality of tobacco research.On the other hand, α-4,8,13-west cypress triolefin-1,3-two pure and mild β-4,8,13-west cypress triolefin-1, the 3-glycol has biological activitys such as stronger anti-tumor activity, antibacterial and coordinate plant growth, for nicotine addiction certain restraining effect is arranged also, therefore also receives the concern of more and more biochemists and Pharmaceutical Chemist day by day.Yet, α-4,8,13-west cypress triolefin-1,3-two pure and mild β-4,8,13-west cypress triolefin-1, the separation and purification of 3-glycol is difficulty comparatively, relates to the coupling of multiple technologies such as leaching, extraction, the separation of multistep column chromatography and recrystallization, the technological operation complexity, preparation cost is higher, does not still have standard substance to sell at present both at home and abroad, and related science institute mostly is the patronage of external large-scale tobacco company with pure product, the technology specificity is strong, has greatly limited the development of China's correlative study.Therefore, carry out α in the tobacco-4,8,13-west cypress triolefin-1,3-two pure and mild β-4,8,13-west cypress triolefin-1, the Separation Research of 3-glycol, preparation high purity standard substance have crucial meaning for improving China's related science research level.
Summary of the invention
The purpose of this invention is to provide a kind of extraction from new fresh tobacco leaf, separation and purifying α-4,8,13-west cypress triolefin-1,3-two pure and mild β-4,8,13-west cypress triolefin-1, the method for 3-glycol.
Two kinds of the present invention to cause fragrant precursor be α-4,8,13-west cypress triolefin-1, and 3-two pure and mild β-4,8,13-west cypress triolefin-1, the 3-glycol, chemical structural formula is as follows respectively:
α-4,8 in the separation and purification tobacco of the present invention, 13-west cypress triolefin-1,3-two pure and mild β-4,8,13-west cypress triolefin-1, the method for 3-glycol, its technical process may further comprise the steps as shown in Figure 1:
(1) leaching: adopt the surperficial secretory product that embathes method extracting fresh tobacco leaf, extraction liquid is concentrated into dried, obtains Vandyke brown medicinal extract S1.
Described extraction liquid is methylene dichloride or trichloromethane.
(2) liquid-liquid extraction: adopt liquid-liquid extraction method to obtain containing the extracting solution S2 of western cypress alkyl compound.
Described liquid-liquid extraction method specifically comprises the steps: Vandyke brown medicinal extract S1 is added in the hybrid extraction solvent, ultrasonicly carries out liquid-liquid extraction and separates to medicinal extract all after the dissolving, collects upper phase and lower floor's liquid phase respectively; Adopt the hybrid extraction solvent that upper phase and lower floor's liquid phase are extracted respectively repeatedly then, merge the extracting solution of all lower floor's liquid phases, obtain containing the extracting solution S2 of western cypress alkyl compound.
Further, described hybrid extraction solvent is the mixed solvent of normal hexane, first alcohol and water, and the volume ratio of described normal hexane, first alcohol and water is 100: (50-90): (10-50).
Further, the volume ratio of first alcohol and water is 80: 20 in the described hybrid extraction solvent, and the cumulative volume of described first alcohol and water equates that with the volume of normal hexane the volume ratio of promptly described normal hexane, first alcohol and water is 100: 80: 20.
Described upper phase and lower floor's liquid phase are extracted respectively repeatedly as 3 times at least.
(3) normal phase silica gel column chromatography separates: adopt normal phase silicagel column that the extracting solution S2 that obtains is carried out gradient elution, obtaining essential substance is α-4,8,13-west cypress triolefin-1,3-two pure and mild β-4,8,13-west cypress triolefin-1, the elutriant S3 of 3-glycol, described moving phase is selected for use increases the agent of polar gradient elution gradually.
Described normal phase silica gel column chromatography separation specifically comprises the steps: to adopt normal phase silicagel column, select for use normal hexane and ethyl acetate as the gradient elution agent, the extracting solution S2 that obtains is carried out gradient elution, and the collected volume ratio is 60: 40-40: it is α-4 that 60 wash-out effluent liquid, concentrating under reduced pressure obtain essential substance, 8,13-west cypress triolefin-1,3-two pure and mild β-4,8,13-west cypress triolefin-1, the elutriant S3 of 3-glycol.
Further, during described normal phase silica gel column chromatography separates, the extracting solution S2 that obtains is carried out gradient elution, collected volume is than being (45-50): wash-out effluent liquid (50-55).
Preferably, during described normal phase silica gel column chromatography separates, the extracting solution S2 that obtains is carried out gradient elution, the collected volume ratio is 50: 50 a wash-out effluent liquid.
The gradient elution agent that described gradient elution agent can adopt the volume ratio of normal hexane and ethyl acetate to be followed successively by 100: 0,75: 25,50: 50,25: 75,0: 100.
(4) positive cyano group column chromatography is separated: adopt positive cyano group preparative column that the elutriant S3 that obtains is carried out gradient elution, obtaining essential substance is α-4,8,13-west cypress triolefin-1,3-two pure and mild β-4,8,13-west cypress triolefin-1, the elutriant S4 of 3-glycol, described moving phase is selected for use increases the agent of polar gradient elution gradually.
Described positive cyano group column chromatography separation specifically comprises the steps: to adopt positive cyano group preparative column, the mixed system of selecting normal hexane and ethyl acetate for use is as the gradient elution agent, the elutriant S3 that obtains is carried out gradient elution, and the collected volume ratio is 85: 15-65: it is α-4 that 35 wash-out effluent liquid, concentrating under reduced pressure obtain essential substance, 8,13-west cypress triolefin-1,3-two pure and mild β-4,8,13-west cypress triolefin-1, the elutriant S4 of 3-glycol.
Further, during described positive cyano group column chromatography is separated, the elutriant S3 that obtains is carried out gradient elution, collected volume is than being (80-75): wash-out effluent liquid (20-25).
Preferably, during described positive cyano group column chromatography is separated, the elutriant S3 that obtains is carried out gradient elution, the collected volume ratio is 75: 25 a wash-out effluent liquid.
During described positive cyano group column chromatography was separated, described gradient elution agent can adopt the volume ratio of normal hexane and ethyl acetate to be followed successively by 90: 10,75: 25,50: 50 eluent.
(5) anti-phase C18 column chromatography is separated: adopt anti-phase C18 preparative column that the elutriant S4 that obtains is carried out gradient elution, during wash-out in 210nm place priority two strong absorption peaks appear, collect the elutriant S5 and the elutriant S6 of two strong absorption peak correspondences respectively, elutriant S5 and elutriant S6 are obtained α-4,8 after the concentrating under reduced pressure drying respectively, 13-west cypress triolefin-1,3-two pure and mild β-4,8,13-west cypress triolefin-1,3-glycol.
Described anti-phase C18 column chromatography separation specifically comprises the steps:: use the C18 preparative column, the mixed system of selecting the first alcohol and water for use carries out gradient elution as the gradient elution agent to the elutriant S4 that obtains; Adopt the absorbancy of UV-detector monitoring stream fluid simultaneously at the 210nm place, and record uv-absorbing gradient elution curve; Volume ratio is 90: 10-95: two strong absorption peaks occur in 210nm place priority when 5 methyl alcohol and water elution agent wash-out, collect the elutriant S5 and the elutriant S6 of two absorption peak correspondences respectively, elutriant S5 and elutriant S6 are obtained α-4 after the concentrating under reduced pressure drying respectively, 8,13-west cypress triolefin-1,3-two pure and mild β-4,8,13-west cypress triolefin-1, the 3-glycol.
Preferably, during described anti-phase C18 column chromatography is separated, the elutriant S4 that obtains is carried out gradient elution, when volume ratio is 95: 5 methyl alcohol and water elution agent wash-out, collect 210nm place successively the elutriant S5 and the elutriant S6 of two strong absorption peak correspondences of appearance respectively.
During described anti-phase C18 column chromatography is separated, the eluent that can adopt the volume ratio of first alcohol and water to be followed successively by 75: 25,85: 15,95: 5,100: 0.
In the step (1), described new fresh tobacco leaf is selected from flue-cured tobacco, Turkish tobaccos and the burley tobaccos of any kind.
In the step (1), the described method of embathing is for dipping extraction, soaking extraction or ultrasonic extraction.
Chromatographic separation in step (3), (4), (5) can adopt low pressure, medium-pressure or high pressure preparative liquid chromatography.
In step (3) and (4), the operational condition of described concentrating under reduced pressure is: bath temperature 40-50 ℃, and vacuum tightness 240-300mbar.Preferred bath temperature is 40 ℃.
In the step (5), the operational condition of described concentrating under reduced pressure is: bath temperature 40-50 ℃, and vacuum tightness 60-72mbar.Preferred bath temperature is 45 ℃.
The organic solvent that relates in step (3), (4), (5) can recycle after reclaiming.
Outstanding advantage of the present invention is: can be effectively to α-4,8 in the fresh tobacco leaf surface secretory product, and 13-west cypress triolefin-1,3-two pure and mild β-4,8,13-west cypress triolefin-1, the 3-glycol is realized extracting fully; Final product α-4,8,13-west cypress triolefin-1,3-two pure and mild β-4,8,13-west cypress triolefin-1,3-glycol purity can be used as standard substance and is applied in the related science research all greater than 99%; Whole separation and purification product recovery rate height, technological operation is easy, and the level of automation height is easily realized preparation in enormous quantities.
Description of drawings
Fig. 1: prepare α-4,8 in the new fresh tobacco leaf, 13-west cypress triolefin-1,3-two pure and mild β-4,8,13-west cypress triolefin-1, the technical process of 3-glycol.
Fig. 2: the gas chromatography mass spectrometry figure of fresh tobacco leaf surface secretory product medicinal extract.
Fig. 3: α-4,8,13-west cypress triolefin-1, the gas chromatography mass spectrometry figure of the pure product of 3-glycol.
Fig. 4: β-4,8,13-west cypress triolefin-1, the gas chromatography mass spectrometry figure of the pure product of 3-glycol.
Embodiment
Further set forth the present invention below in conjunction with specific embodiment, should be understood that these embodiment only are used to the present invention is described and are not used in restriction protection scope of the present invention.
Embodiment 1
α in the separation and purification tobacco-4,8,13-west cypress triolefin-1,3-two pure and mild β-4,8,13-west cypress triolefin-1, the method for 3-glycol, its technical process may further comprise the steps as shown in Figure 1:
(1) leaching: in the Yuxi tobacco leaf place of production, Yunnan Province, be object, gather 1000 new fresh tobacco leafs with the K326 cured tobacco leaf.Blade is dipped in the 10L methylene dichloride 3 times continuously, and each 2s embathes the back solution filter paper filtering that anhydrous sodium sulphate is housed.Filtrate is concentrated into dried at 40 ℃ reduce pressure down (10-13kPa), obtain the about 80g of Vandyke brown medicinal extract.Detect through GC/MS, obtain the color atlas of fresh tobacco leaf surface secretory product medicinal extract as shown in Figure 2, as we know from the figure, target compound α-4,8,13-west cypress triolefin-1,3-two pure and mild β-4,8,13-west cypress triolefin-1, the total content of 3-glycol is 67.6%.
(2) liquid-liquid extraction: take by weighing 20g tobacco leaf surface secretory product medicinal extract and place the 250ml triangular flask, add 50ml methanol-water (80: 20) mixing solutions and 50ml normal hexane, add a cover ultrasonic 20min.After sample fully dissolves, solution is transferred in the 250ml separating funnel, thermal agitation 2-3min makes two to be separated, and collects lower floor's water and upper organic phase respectively.Methanol-water used respectively with volume normal hexane and methanol-water (80: 20) mixing solutions mutually with normal hexane mutually extract repeatedly 3 times, merge all methanol-water phase extracting solution filter paper filterings, obtain the about 300ml of filtrate.Detect through GC/MS, in the methanol extracting solution, target compound α-4,8,13-west cypress triolefin-1,3-two pure and mild β-4,8,13-west cypress triolefin-1, the total content of 3-glycol is 73.5%.
(3) normal phase silica gel column chromatography separates: use silicagel column (particle diameter 40-60 μ m, specification 35.0 * 500mm), select for use the n-hexane/ethyl acetate mixed system as eluent, with increase gradually polar volume ratio order (100: 0,75: 25,50: 50,25: 75,0: 100) extracting solution that step (2) is obtained carries out gradient elution, the effluent liquid of collected volume than 50: 50, under 40 ℃ of reduced pressure, (240mbar) is concentrated into about 200ml with Rotary Evaporators.Detect through GC/MS, the methanol extraction liquid after through silica gel silicon chromatographic separation, target compound α-4,8,13-west cypress triolefin-1,3-two pure and mild β-4,8,13-west cypress triolefin-1, the total content of 3-glycol is 83.65%.
(4) positive cyano group column chromatography is separated: use Ultimate XB-CN preparative column (particle diameter 5 μ m, specification 21.2 * 250mm), select for use the n-hexane/ethyl acetate mixed system as eluent, to increase polar volume ratio order (90: 10 gradually, 75: 25,50: 50) sample that step (3) is obtained carries out gradient elution, the effluent liquid of collected volume than 75: 25, under 40 ℃ of reduced pressure, (vacuum tightness 240mbar) is concentrated into about 200ml with Rotary Evaporators.Detect through GC/MS, after separating through the cyano group column chromatography, target compound α-4,8,13-west cypress triolefin-1,3-two pure and mild β-4,8,13-west cypress triolefin-1, the total content of 3-glycol is 95.41%.
(5) anti-phase C18 column chromatography is separated: use Ultimate XB-C18 preparative column (particle diameter 5 μ m, specification 21.2 * 250mm), select for use the methanol mixed system as eluent, with volume ratio (75: 25,85: 15,95: 5,100: 0) order the sample that step (4) obtains is carried out gradient elution, UV-detector monitoring stream fluid writes down uv-absorbing gradient elution curve in the absorbancy at 210nm place.During 95: 5 wash-outs of volume ratio in 210nm place priority two strong absorption peaks appear, collect the effluent liquid of two peak correspondences respectively, under 40 ℃ of reduced pressure, be concentrated into driedly with Rotary Evaporators (vacuum tightness 72mbar), obtain 2.3g α-4 respectively, 8,13-west cypress triolefin-1,3-two pure and mild 760mg β-4,8,13-west cypress triolefin-1, the pure product of 3-glycol.Detect through GC/MS, obtain α-4,8 as shown in Figure 3,13-west cypress triolefin-1, the gas of the pure product of 3-glycol/matter coupling figure and β as shown in Figure 4-4,8,13-west cypress triolefin-1, the gas of the pure product of 3-glycol/matter coupling figure, as we know from the figure, after the separation of C18 column chromatography, gained target compound α-4,8,13-west cypress triolefin-1,3-two pure and mild β-4,8,13-west cypress triolefin-1, the content of 3-glycol is respectively 99.54% and 99.21%; α-4,8,13-west cypress triolefin-1,3-two pure and mild β-4,8,13-west cypress triolefin-1, the 3-glycol is respectively 11.5% and 3.8% with respect to the rate of recovery of tobacco leaf surface secretory product medicinal extract.
GC/MS testing conditions: GC condition: chromatographic column: DB-5MS quartz capillary column; Injector temperature: 250 ℃; Carrier gas: He, 1.0mL/min; Splitting ratio: 10: 1; Sample size: 1.0 μ L.Degree heats up: 120 ℃ keep 5min, are warmed up to 210 ℃ with 20 ℃/min, are warmed up to 220 ℃ with 0.5 ℃/min again, are warmed up to 280 ℃ with 10 ℃/min then, keep 30min.MS condition: 280 ℃ of transmission line temperature; 150 ℃ of level Four bar temperature; 230 ℃ of EI ion source temperatures; Ionizing energy 70eV; Total mass number scope 30-800amu.
Embodiment 2
α in the separation and purification tobacco-4,8,13-west cypress triolefin-1,3-two pure and mild β-4,8,13-west cypress triolefin-1, the method for 3-glycol, its technical process may further comprise the steps as shown in Figure 1:
(1) leaching: in the Yuxi tobacco leaf place of production, Yunnan Province, be object, gather 1000 new fresh tobacco leafs with the K326 cured tobacco leaf.Blade is dipped in the 10L trichloromethane 3 times continuously, and each 2s embathes the back solution filter paper filtering that anhydrous sodium sulphate is housed.Filtrate is concentrated into dried at 40 ℃ reduce pressure down (10-13kPa), obtain the about 80g of Vandyke brown medicinal extract.Detect through GC/MS, target compound α-4,8,13-west cypress triolefin-1,3-two pure and mild β-4,8,13-west cypress triolefin-1, the total content of 3-glycol is 67.6%.
(2) liquid-liquid extraction: take by weighing 20g tobacco leaf surface secretory product medicinal extract and place the 250ml triangular flask, add 50ml methanol-water (50: 50) mixing solutions and 50ml normal hexane, add a cover ultrasonic 20min.After sample fully dissolves, solution is transferred in the 250ml separating funnel, thermal agitation 2-3min makes two to be separated, and collects lower floor's water and upper organic phase respectively.Methanol-water used respectively with volume normal hexane and methanol-water (50: 50) mixing solutions mutually with normal hexane mutually extract repeatedly 3 times, merge all methanol-water phase extracting solution filter paper filterings, obtain the about 300ml of filtrate.Detect through GC/MS, in the methanol extracting solution, target compound α-4,8,13-west cypress triolefin-1,3-two pure and mild β-4,8,13-west cypress triolefin-1, the total content of 3-glycol is 73.5%.(3) normal phase silica gel column chromatography separates: use silicagel column (particle diameter 40-60 μ m, specification 35.0 * 500mm), select for use the n-hexane/ethyl acetate mixed system as eluent, with increase gradually polar volume ratio order (100: 0,75: 25,45: 55,25: 75,0: 100) extracting solution that step (2) is obtained carries out gradient elution, the effluent liquid of collected volume than 45: 55, under 40 ℃ of reduced pressure, (240mbar) is concentrated into about 200ml with Rotary Evaporators.Detect through GC/MS, the methanol extraction liquid after through silica gel silicon chromatographic separation, target compound α-4,8,13-west cypress triolefin-1,3-two pure and mild β-4,8,13-west cypress triolefin-1, the total content of 3-glycol is 83.65%.
(4) positive cyano group column chromatography is separated: use Ultimate XB-CN preparative column (particle diameter 5 μ m, specification 21.2 * 250mm), select for use the n-hexane/ethyl acetate mixed system as eluent, to increase polar volume ratio order (90: 10 gradually, 80: 20,50: 50) sample that step (3) is obtained carries out gradient elution, the effluent liquid of collected volume than 80: 20, under 40 ℃ of reduced pressure, (vacuum tightness 240mbar) is concentrated into about 200ml with Rotary Evaporators.Detect through GC/MS, after separating through the cyano group column chromatography, target compound α-4,8,13-west cypress triolefin-1,3-two pure and mild β-4,8,13-west cypress triolefin-1, the total content of 3-glycol is 95.41%.
(5) anti-phase C18 column chromatography is separated: use Ultimate XB-C18 preparative column (particle diameter 5 μ m, specification 21.2 * 250mm), select for use the methanol mixed system as eluent, with volume ratio (75: 25,85: 15,95: 10,100: 0) order the sample that step (4) obtains is carried out gradient elution, UV-detector monitoring stream fluid writes down uv-absorbing gradient elution curve in the absorbancy at 210nm place.During 95: 10 wash-outs of volume ratio in 210nm place priority two strong absorption peaks appear, collect the effluent liquid of two peak correspondences respectively, under 40 ℃ of reduced pressure, be concentrated into driedly with Rotary Evaporators (vacuum tightness 72mbar), obtain 2.2g α-4 respectively, 8,13-west cypress triolefin-1,3-two pure and mild 700mg β-4,8,13-west cypress triolefin-1, the pure product of 3-glycol.Detect through GC/MS, after separating through the C18 column chromatography, gained target compound α-4,8,13-west cypress triolefin-1,3-two pure and mild β-4,8,13-west cypress triolefin-1, the content of 3-glycol is respectively 99.50% and 99.20%; α-4,8,13-west cypress triolefin-1,3-two pure and mild β-4,8,13-west cypress triolefin-1, the 3-glycol is respectively 11.0% and 3.5% with respect to the rate of recovery of tobacco leaf surface secretory product medicinal extract.
GC/MS testing conditions: GC condition: chromatographic column: DB-5MS quartz capillary column; Injector temperature: 250 ℃; Carrier gas: He, 1.0mL/min; Splitting ratio: 10: 1; Sample size: 1.0 μ L.Degree heats up: 120 ℃ keep 5min, are warmed up to 210 ℃ with 20 ℃/min, are warmed up to 220 ℃ with 0.5 ℃/min again, are warmed up to 280 ℃ with 10 ℃/min then, keep 30min.MS condition: 280 ℃ of transmission line temperature; 150 ℃ of level Four bar temperature; 230 ℃ of EI ion source temperatures; Ionizing energy 70eV; Total mass number scope 30-800amu.
Embodiment 3
In step (2), the volume ratio of normal hexane, first alcohol and water is 100: 90: 10 in the hybrid extraction solvent, and the vacuum tightness of Rotary Evaporators is that the vacuum tightness of Rotary Evaporators in 300mbar and the step (5) is beyond the 60mbar in step (3) and the step (4), and other steps are identical with embodiment 1, the α of gained-4,8,13-west cypress triolefin-1,3-two pure and mild β-4,8,13-west cypress triolefin-1, the 3-glycol is respectively 2.1g and 680mg, and purity is all greater than 99%; α-4,8,13-west cypress triolefin-1,3-two pure and mild β-4,8,13-west cypress triolefin-1, the 3-glycol is respectively 10.5% and 3.4% with respect to the rate of recovery of tobacco leaf surface secretory product medicinal extract.
Claims (13)
1. α-4,8 in the separation and purification tobacco, 13-west cypress triolefin-1,3-two pure and mild β-4,8,13-west cypress triolefin-1, the method for 3-glycol comprises the steps:
(1) leaching: adopt the surperficial secretory product that embathes method extracting fresh tobacco leaf, extraction liquid is concentrated into dried, obtains Vandyke brown medicinal extract S1;
(2) liquid-liquid extraction: adopt liquid-liquid extraction method to obtain containing the extracting solution S2 of western cypress alkyl compound;
(3) normal phase silica gel column chromatography separates: adopt normal phase silicagel column that the extracting solution S2 that obtains is carried out gradient elution, obtaining essential substance is α-4,8,13-west cypress triolefin-1,3-two pure and mild β-4,8,13-west cypress triolefin-1, the elutriant S3 of 3-glycol, described moving phase is selected for use increases the agent of polar gradient elution gradually;
(4) positive cyano group column chromatography is separated: adopt positive cyano group preparative column that the elutriant S3 that obtains is carried out gradient elution, obtaining essential substance is α-4,8,13-west cypress triolefin-1,3-two pure and mild β-4,8,13-west cypress triolefin-1, the elutriant S4 of 3-glycol, described moving phase is selected for use increases the agent of polar gradient elution gradually;
(5) anti-phase C18 column chromatography is separated: adopt anti-phase C18 preparative column that the elutriant S4 that obtains is carried out gradient elution, during wash-out in 210nm place priority two strong absorption peaks appear, collect the elutriant S5 and the elutriant S6 of two strong absorption peak correspondences respectively, elutriant S5 and elutriant S6 are obtained α-4,8 after the concentrating under reduced pressure drying respectively, 13-west cypress triolefin-1,3-two pure and mild β-4,8,13-west cypress triolefin-1,3-glycol.
2. separation purification method as claimed in claim 1 is characterized in that, in the step (1), described extraction liquid is methylene dichloride or trichloromethane.
3. separation purification method as claimed in claim 1, it is characterized in that, in the step (2), described liquid-liquid extraction method specifically comprises the steps: Vandyke brown medicinal extract S1 is added in the hybrid extraction solvent, ultrasonic to the whole dissolvings of medicinal extract, carry out liquid-liquid extraction and separate, collect upper phase and lower floor's liquid phase respectively; Adopt the hybrid extraction solvent that upper phase and lower floor's liquid phase are extracted respectively repeatedly then, merge the extracting solution of all lower floor's liquid phases, obtain containing the extracting solution S2 of western cypress alkyl compound.
4. separation purification method as claimed in claim 3 is characterized in that, described hybrid extraction solvent is the mixed solvent of normal hexane, first alcohol and water, and the volume ratio of described normal hexane, first alcohol and water is 100: (50-90): (10-50).
5. separation purification method as claimed in claim 1, it is characterized in that, in the step (3), described normal phase silica gel column chromatography separation specifically comprises the steps: to adopt normal phase silicagel column, select for use normal hexane and ethyl acetate as the gradient elution agent, the extracting solution S2 that obtains is carried out gradient elution, the collected volume ratio is 60: 40-40: 60 wash-out effluent liquid, it is α-4,8 that concentrating under reduced pressure obtains essential substance, 13-west cypress triolefin-1,3-two pure and mild β-4,8,13-west cypress triolefin-1, the elutriant S3 of 3-glycol.
6. separation purification method as claimed in claim 6 is characterized in that, during described normal phase silica gel column chromatography separates, the extracting solution S2 that obtains is carried out gradient elution, and collected volume is than being (45-50): wash-out effluent liquid (50-55).
7. separation purification method as claimed in claim 7 is characterized in that, during described normal phase silica gel column chromatography separated, the collected volume ratio was 50: 50 a wash-out effluent liquid.
8. separation purification method as claimed in claim 1, it is characterized in that, in the step (4), described positive cyano group column chromatography separation specifically comprises the steps: to adopt positive cyano group preparative column, the mixed system of selecting normal hexane and ethyl acetate for use is as the gradient elution agent, the elutriant S3 that obtains is carried out gradient elution, the collected volume ratio is 85: 15-65: 35 wash-out effluent liquid, it is α-4,8 that concentrating under reduced pressure obtains essential substance, 13-west cypress triolefin-1,3-two pure and mild β-4,8,13-west cypress triolefin-1, the elutriant S4 of 3-glycol.
9. separation purification method as claimed in claim 9 is characterized in that, during described positive cyano group column chromatography is separated, the elutriant S3 that obtains is carried out gradient elution, and collected volume is than being (80-75): wash-out effluent liquid (20-25).
10. separation purification method as claimed in claim 10 is characterized in that, during described positive cyano group column chromatography was separated, the collected volume ratio was 75: 25 a wash-out effluent liquid.
11. separation purification method as claimed in claim 1, it is characterized in that in the step (5), described anti-phase C18 column chromatography separation specifically comprises the steps: to use the C18 preparative column, the mixed system of selecting the first alcohol and water for use carries out gradient elution as the gradient elution agent to the elutriant S4 that obtains; Adopt the absorbancy of UV-detector monitoring stream fluid simultaneously at the 210nm place, and record uv-absorbing gradient elution curve; Volume ratio is 90: 10-95: two strong absorption peaks occur in 210nm place priority when 5 methyl alcohol and water elution agent wash-out, collect the elutriant S5 and the elutriant S6 of two absorption peak correspondences respectively, elutriant S5 and elutriant S6 are obtained α-4 after the concentrating under reduced pressure drying respectively, 8,13-west cypress triolefin-1,3-two pure and mild β-4,8,13-west cypress triolefin-1, the 3-glycol.
12. separation purification method as claimed in claim 10, it is characterized in that, during described anti-phase C18 column chromatography is separated, when adopting volume ratio to be 95: 5 methyl alcohol and water elution agent wash-out, collect 210nm place successively the elutriant S5 and the elutriant S6 of two strong absorption peak correspondences of appearance respectively.
13. separation purification method as claimed in claim 1 is characterized in that, in step (3) and (4), the operational condition of described concentrating under reduced pressure is: bath temperature 40-50 ℃, and vacuum tightness 240-300mbar; In the step (5), the operational condition of described concentrating under reduced pressure is: bath temperature 40-50 ℃, and vacuum tightness 60-72mbar.
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Cited By (11)
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| CN102586020A (en) * | 2012-02-23 | 2012-07-18 | 上海烟草集团有限责任公司 | Separation method of weak acidic aromatic components in cigarette mainstream smoke, and its application |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4701570A (en) * | 1984-11-05 | 1987-10-20 | Japan Tobacco Inc. | Antitumor agent |
| JPS6368524A (en) * | 1986-09-11 | 1988-03-28 | Japan Tobacco Inc | Inhibitor for aldose reductase |
-
2010
- 2010-12-30 CN CN 201010617666 patent/CN102206138B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4701570A (en) * | 1984-11-05 | 1987-10-20 | Japan Tobacco Inc. | Antitumor agent |
| JPS6368524A (en) * | 1986-09-11 | 1988-03-28 | Japan Tobacco Inc | Inhibitor for aldose reductase |
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