CN102206138B - 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 tobacco, be specifically related to a kind of extraction from new fresh tobacco leaf, separation and purification of alpha-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 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, aromatic 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 the biological activitys such as stronger anti-tumor activity, antibacterial and coordinate plant growth, for nicotine addiction, certain restraining effect is arranged also, therefore also day by day receives the concern of more and more biochemists and Pharmaceutical Chemist.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 the multiple technologies such as leaching, extraction, the separation of multistep column chromatography and recrystallization, complex operation, preparation cost is higher, does not still have at present standard substance to sell both at home and abroad, and related science institute mostly is the patronage of external large-scale tobacco company with sterling, the technology specificity is strong, has greatly limited the development of China's correlative study.Therefore, carry out α in tobacco-4,8, the western cypress triolefin-1 of 13-, 3-two pure and mild β-4,8, the western cypress triolefin-1 of 13-, the Separation Research of 3-glycol, preparation high purity standard substance are of great significance for improving China's related science research level tool.
Summary of the invention
The purpose of this invention is to provide a kind of extraction from new fresh tobacco leaf, separation and purification of alpha-4,8, the western cypress triolefin-1 of 13-, 3-two pure and mild β-4,8, the western cypress triolefin-1 of 13-, 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 separation and purification tobacco of the present invention, the western cypress triolefin-1 of 13-, 3-two pure and mild β-4,8, the western cypress triolefin-1 of 13-, the method for 3-glycol, its technical process comprises the following 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 all after dissolving to medicinal extract, collects respectively upper phase and lower floor's liquid phase; Then adopt the hybrid extraction solvent that upper phase and lower floor's liquid phase are extracted respectively repeatedly, 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, in described hybrid extraction solvent, the volume ratio of first alcohol and water is 80: 20, and the cumulative volume of described first alcohol and water equates with the volume of normal hexane, and namely the volume ratio of 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 to carry out gradient elution to the extracting solution S2 that obtains, obtaining essential substance is α-4,8,13-west cypress triolefin-1,3-two pure and mild β-4,8, the western cypress triolefin-1 of 13-, the elutriant S3 of 3-glycol, described moving phase is selected increases the agent of the gradient elution of polarity gradually.
Described normal phase silica gel column chromatography separation specifically comprises the steps: to adopt normal phase silicagel column, select 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 the wash-out effluent liquid of 50: 50.
Described gradient elution agent can adopt the volume ratio of normal hexane and ethyl acetate to be followed successively by the gradient elution agent of 100: 0,75: 25,50: 50,25: 75,0: 100.
(4) positive cyano group column chromatography is separated: adopt positive cyano group preparative column to carry out gradient elution to the elutriant S3 that obtains, obtaining essential substance is α-4,8,13-west cypress triolefin-1,3-two pure and mild β-4,8, the western cypress triolefin-1 of 13-, the elutriant S4 of 3-glycol, described moving phase is selected increases the agent of the gradient elution of polarity gradually.
Described positive cyano group column chromatography separation specifically comprises the steps: to adopt positive cyano group preparative column, select the mixed system of normal hexane and ethyl acetate 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 the wash-out effluent liquid of 75: 25.
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 the eluent of 90: 10,75: 25,50: 50.
(5) anti-phase C18 column chromatography is separated: adopt anti-phase C18 preparative column to carry out gradient elution to the elutriant S4 that obtains, two strong absorption peaks successively appear at the 210nm place during wash-out, collect respectively two elutriant S5 and elutriant S6 that strong absorption peak is corresponding, elutriant S5 and elutriant S6 are obtained α-4,8 after the concentrating under reduced pressure drying respectively, the western cypress triolefin-1 of 13-, 3-two pure and mild β-4,8,13-west cypress triolefin-1, the 3-glycol.
Described anti-phase C18 column chromatography separation specifically comprises the steps:: use the C18 preparative column, select the mixed system of first alcohol and water as the gradient elution agent, the elutriant S4 that obtains is carried out gradient elution; Adopt simultaneously UV-detector monitoring stream fluid in the absorbancy at 210nm place, and record uv-absorbing gradient elution curve; Volume ratio is 90: 10-95: successively occur two strong absorption peaks when 5 methyl alcohol and water elution agent wash-out at the 210nm place, collect respectively two elutriant S5 and elutriant S6 that absorption peak is corresponding, 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 the methyl alcohol of 95: 5 and water elution agent wash-out, collect respectively successively corresponding elutriant S5 and the elutriant S6 of two strong absorption peaks of appearance of 210nm place.
Described anti-phase C18 column chromatography can adopt the volume ratio of first alcohol and water to be followed successively by the eluent of 75: 25,85: 15,95: 5,100: 0 in separating.
In step (1), described new fresh tobacco leaf is selected from flue-cured tobacco, Turkish tobaccos and the burley tobaccos of any kind.
In 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 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 advantages of the present invention is: can be effectively to α-4,8 in fresh tobacco leaf surface secretory product, and the western cypress triolefin-1 of 13-, 3-two pure and mild β-4,8, the western cypress triolefin-1 of 13-, the 3-glycol is realized extracting fully; Final product α-4,8, the western cypress triolefin-1 of 13-, 3-two pure and mild β-4,8, the western cypress triolefin-1 of 13-, 3-glycol purity can be used as standard substance and is applied in related science research all greater than 99%; Whole separation and purification product recovery rate is high, and technological operation is easy, and level of automation is high, easily realizes preparation in enormous quantities.
Description of drawings
Fig. 1: prepare α-4,8 in new fresh tobacco leaf, the western cypress triolefin-1 of 13-, 3-two pure and mild β-4,8, the western cypress triolefin-1 of 13-, 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, the western cypress triolefin-1 of 13-, the gas chromatography mass spectrometry figure of 3-glycol sterling.
Fig. 4: β-4,8, the western cypress triolefin-1 of 13-, the gas chromatography mass spectrometry figure of 3-glycol sterling.
Embodiment
Further set forth the present invention below in conjunction with specific embodiment, should be understood that these embodiment only are used for explanation the present invention and are not used in restriction protection scope of the present invention.
Embodiment 1
α in the separation and purification tobacco-4,8, the western cypress triolefin-1 of 13-, 3-two pure and mild β-4,8, the western cypress triolefin-1 of 13-, the method for 3-glycol, its technical process comprises the following steps as shown in Figure 1:
(1) leaching: in the Yuxi tobacco leaf place of production, Yunnan Province, take the K326 cured tobacco leaf as object, gather 1000 new fresh tobacco leafs.Blade is dipped in the 10L methylene dichloride 3 times continuously, and each 2s embathes the rear solution filter paper filtering that anhydrous sodium sulphate is housed.Filtrate reduce pressure under 40 ℃ (10-13kPa) be concentrated into driedly, obtain approximately 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, the western cypress triolefin-1 of 13-, 3-two pure and mild β-4,8, the western cypress triolefin-1 of 13-, the total content of 3-glycol is 67.6%.
(2) liquid-liquid extraction: take 20g tobacco leaf surface secretory product medicinal extract and be placed in 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 respectively lower floor's water and upper organic phase.Methanol-water phase and normal hexane are used respectively mutually with volume normal hexane and methanol-water (80: 20) mixing solutions repeatedly extract 3 times, merged all methanol-water phase extracting solution filter paper filterings, obtain approximately 300ml of filtrate.Detect through GC/MS, in the methanol/water extracting solution, target compound α-4,8, the western cypress triolefin-1 of 13-, 3-two pure and mild β-4,8, the western cypress triolefin-1 of 13-, 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 the n-hexane/ethyl acetate mixed system as eluent, with the volume ratio order that increases gradually polarity (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, be concentrated into the 200ml left and right with Rotary Evaporators (240mbar).Detect through GC/MS, the methanol/water 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 the n-hexane/ethyl acetate mixed system as eluent, with the volume ratio order that increases gradually polarity (90: 10,75: 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, be concentrated into the 200ml left and right with Rotary Evaporators (vacuum tightness 240mbar).Detect through GC/MS, after separating through the cyano group column chromatography, target compound α-4,8, the western cypress triolefin-1 of 13-, 3-two pure and mild β-4,8, the western cypress triolefin-1 of 13-, 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 the methanol/water 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 records uv-absorbing gradient elution curve in the absorbancy at 210nm place.Two strong absorption peaks successively appear at the 210nm place during 95: 5 wash-outs of volume ratio, collect respectively two effluent liquid that the peak is corresponding, under 40 ℃ of reduced pressure, be concentrated into driedly with Rotary Evaporators (vacuum tightness 72mbar), obtain respectively 2.3g α-4,8,13-west cypress triolefin-1, the pure and mild 760mg β-4,8 of 3-two, 13-west cypress triolefin-1, the sterling of 3-glycol.Detect through GC/MS, obtain α-4,8 as shown in Figure 3,13-west cypress triolefin-1, the gas of 3-glycol sterling/matter coupling figure and β as shown in Figure 4-4,8,13-west cypress triolefin-1, the gas of 3-glycol sterling/matter coupling figure, as we know from the figure, 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 99.54% and 99.21%; α-4,8, the western cypress triolefin-1 of 13-, 3-two pure and mild β-4,8, the western cypress triolefin-1 of 13-, 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, then are warmed up to 220 ℃ with 0.5 ℃/min, then are warmed up to 280 ℃ with 10 ℃/min, 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, the western cypress triolefin-1 of 13-, 3-two pure and mild β-4,8, the western cypress triolefin-1 of 13-, the method for 3-glycol, its technical process comprises the following steps as shown in Figure 1:
(1) leaching: in the Yuxi tobacco leaf place of production, Yunnan Province, take the K326 cured tobacco leaf as object, gather 1000 new fresh tobacco leafs.Blade is dipped in the 10L trichloromethane 3 times continuously, and each 2s embathes the rear solution filter paper filtering that anhydrous sodium sulphate is housed.Filtrate reduce pressure under 40 ℃ (10-13kPa) be concentrated into driedly, obtain approximately 80g of Vandyke brown medicinal extract.Detect through GC/MS, target compound α-4,8, the western cypress triolefin-1 of 13-, 3-two pure and mild β-4,8, the western cypress triolefin-1 of 13-, the total content of 3-glycol is 67.6%.
(2) liquid-liquid extraction: take 20g tobacco leaf surface secretory product medicinal extract and be placed in 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 respectively lower floor's water and upper organic phase.Methanol-water phase and normal hexane are used respectively mutually with volume normal hexane and methanol-water (50: 50) mixing solutions repeatedly extract 3 times, merged all methanol-water phase extracting solution filter paper filterings, obtain approximately 300ml of filtrate.Detect through GC/MS, in the methanol/water extracting solution, target compound α-4,8, the western cypress triolefin-1 of 13-, 3-two pure and mild β-4,8, the western cypress triolefin-1 of 13-, 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 the n-hexane/ethyl acetate mixed system as eluent, with the volume ratio order that increases gradually polarity (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, be concentrated into the 200ml left and right with Rotary Evaporators (240mbar).Detect through GC/MS, the methanol/water 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 the n-hexane/ethyl acetate mixed system as eluent, with the volume ratio order that increases gradually polarity (90: 10,80: 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, be concentrated into the 200ml left and right with Rotary Evaporators (vacuum tightness 240mbar).Detect through GC/MS, after separating through the cyano group column chromatography, target compound α-4,8, the western cypress triolefin-1 of 13-, 3-two pure and mild β-4,8, the western cypress triolefin-1 of 13-, 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 the methanol/water 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 records uv-absorbing gradient elution curve in the absorbancy at 210nm place.Two strong absorption peaks successively appear at the 210nm place during 95: 10 wash-outs of volume ratio, collect respectively two effluent liquid that the peak is corresponding, under 40 ℃ of reduced pressure, be concentrated into driedly with Rotary Evaporators (vacuum tightness 72mbar), obtain respectively 2.2g α-4,8,13-west cypress triolefin-1, the pure and mild 700mg β-4,8 of 3-two, 13-west cypress triolefin-1, the sterling of 3-glycol.Detect through GC/MS, after separating through the C18 column chromatography, gained target compound α-4,8, the western cypress triolefin-1 of 13-, 3-two pure and mild β-4,8, the western cypress triolefin-1 of 13-, the content of 3-glycol is respectively 99.50% and 99.20%; α-4,8, the western cypress triolefin-1 of 13-, 3-two pure and mild β-4,8, the western cypress triolefin-1 of 13-, 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, then are warmed up to 220 ℃ with 0.5 ℃/min, then are warmed up to 280 ℃ with 10 ℃/min, 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), in the hybrid extraction solvent, the volume ratio of normal hexane, first alcohol and water is 100: 90: 10, and in step (3) and step (4), the vacuum tightness of Rotary Evaporators is that the vacuum tightness of Rotary Evaporators in 300mbar and step (5) is beyond 60mbar, 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, the western cypress triolefin-1 of 13-, 3-two pure and mild β-4,8, the western cypress triolefin-1 of 13-, 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 (14)
1. α-4,8 in a 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 to carry out gradient elution to the extracting solution S2 that obtains, obtaining essential substance is α-4,8,13-west cypress triolefin-1,3-two pure and mild β-4,8, the western cypress triolefin-1 of 13-, the elutriant S3 of 3-glycol, described moving phase is selected increases the agent of the gradient elution of polarity gradually;
(4) positive cyano group column chromatography is separated: adopt positive cyano group preparative column to carry out gradient elution to the elutriant S3 that obtains, obtaining essential substance is α-4,8,13-west cypress triolefin-1,3-two pure and mild β-4,8, the western cypress triolefin-1 of 13-, the elutriant S4 of 3-glycol, described moving phase is selected increases the agent of the gradient elution of polarity gradually;
(5) anti-phase C18 column chromatography is separated: adopt anti-phase C18 preparative column to carry out gradient elution to the elutriant S4 that obtains, two strong absorption peaks successively appear at the 210nm place during wash-out, collect respectively two elutriant S5 and elutriant S6 that strong absorption peak is corresponding, elutriant S5 and elutriant S6 are obtained α-4,8 after the concentrating under reduced pressure drying respectively, the western cypress triolefin-1 of 13-, 3-two pure and mild β-4,8,13-west cypress triolefin-1, the 3-glycol.
2. separation purification method as claimed in claim 1, is characterized in that, in 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 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 respectively upper phase and lower floor's liquid phase; Then adopt the hybrid extraction solvent that upper phase and lower floor's liquid phase are extracted respectively repeatedly, merge the extracting solution of all lower floor's liquid phases, obtain containing the extracting solution S2 of western cypress alkyl compound; 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).
4. separation purification method as claimed in claim 1, it is characterized in that, in step (3), described normal phase silica gel column chromatography separation specifically comprises the steps: to adopt normal phase silicagel column, select normal hexane and ethyl acetate as the gradient elution agent, the extracting solution S2 that obtains is carried out gradient elution, collected volume is than being the wash-out effluent liquid of 60:40-40:60, it is α-4,8 that concentrating under reduced pressure obtains essential substance, the western cypress triolefin-1 of 13-, 3-two pure and mild β-4,8,13-west cypress triolefin-1, the elutriant S3 of 3-glycol.
5. separation purification method as claimed in claim 4, is characterized in that, in described step (3), the operational condition of concentrating under reduced pressure is: bath temperature 40-50 ℃, and vacuum tightness 240-300mbar.
6. separation purification method as claimed in claim 4, is characterized in that, during described normal phase silica gel column chromatography separates, the extracting solution S2 that obtains 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 6, is characterized in that, during described normal phase silica gel column chromatography separated, collected volume was than being the wash-out effluent liquid of 50:50.
8. separation purification method as claimed in claim 1, it is characterized in that, in step (4), described positive cyano group column chromatography separation specifically comprises the steps: to adopt positive cyano group preparative column, select the mixed system of normal hexane and ethyl acetate as the gradient elution agent, the elutriant S3 that obtains is carried out gradient elution, collected volume is than being the wash-out effluent liquid of 85:15-65:35, it is α-4,8 that concentrating under reduced pressure obtains essential substance, the western cypress triolefin-1 of 13-, 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 8, is characterized in that, in described step (4), the operational condition of concentrating under reduced pressure is: bath temperature 40-50 ℃, and vacuum tightness 240-300mbar.
10. separation purification method as claimed in claim 8, is characterized in that, during described positive cyano group column chromatography is separated, the elutriant S3 that obtains carried out gradient elution, and collected volume is than being (80-75): wash-out effluent liquid (20-25).
11. separation purification method as claimed in claim 10 is characterized in that, during described positive cyano group column chromatography was separated, collected volume was than being the wash-out effluent liquid of 75:25.
12. separation purification method as claimed in claim 1, it is characterized in that, in step (5), described anti-phase C18 column chromatography separation specifically comprises the steps: to use the C18 preparative column, select the mixed system of first alcohol and water as the gradient elution agent, the elutriant S4 that obtains is carried out gradient elution; Adopt simultaneously UV-detector monitoring stream fluid in the absorbancy at 210nm place, and record uv-absorbing gradient elution curve; When volume ratio is the methyl alcohol of 90:10-95:5 and water elution agent wash-out at the 210nm place, two strong absorption peaks successively appear, collect respectively two elutriant S5 and elutriant S6 that absorption peak is corresponding, 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.
13. separation purification method as claimed in claim 12, it is characterized in that, during described anti-phase C18 column chromatography is separated, when adopting volume ratio to be the methyl alcohol of 95:5 and water elution agent wash-out, collect respectively successively corresponding elutriant S5 and the elutriant S6 of two strong absorption peaks of appearance of 210nm place.
14. separation purification method as claimed in claim 1 is characterized in that, in 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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| CN102586020B (en) * | 2012-02-23 | 2013-08-21 | 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 |
| CN102759588B (en) * | 2012-07-16 | 2014-05-07 | 上海烟草集团有限责任公司 | Method for separating and preparing key acid flavor components in cigarette mainstream smoke and application of key acid volatile components |
| CN104655772B (en) * | 2014-12-30 | 2017-02-22 | 广东中烟工业有限责任公司 | Method for detecting alpha-2, 7, 11-cembrane triene-4, 6-diol (alpha-CBD) in tobacco |
| CN104569261A (en) * | 2014-12-30 | 2015-04-29 | 广东中烟工业有限责任公司 | Method for detecting beta-2, 7, 11-cembratriene-4, 6-diol in tobacco |
| CN105001052B (en) * | 2015-07-03 | 2016-11-16 | 中国农业科学院烟草研究所 | The method extracting western cypress alkane diterpene in Nicotiana tabacum L. inflorescence |
| CN105167175B (en) * | 2015-11-05 | 2016-08-17 | 河南农业大学 | A kind of enrichment method being applicable to prepare the pungent component of tobacco juice for electronic smoke |
| CN106008444B (en) * | 2016-06-14 | 2018-07-10 | 中国农业科学院烟草研究所 | A kind of method for extracting Salanesol, Cystatins C, vitamin E, phytosterol simultaneously from tobacco |
| CN108017515B (en) * | 2017-12-18 | 2021-06-01 | 中国烟草总公司郑州烟草研究院 | Method for separating and purifying labdanum diterpenoid components in tobacco |
| CN109275956B (en) * | 2018-09-20 | 2021-01-29 | 云南中烟工业有限责任公司 | Filter tip additive mainly containing refined perilla frutescens cool taste component and preparation method thereof |
| CN109846077A (en) * | 2018-12-12 | 2019-06-07 | 云南中烟工业有限责任公司 | A kind of method and application that characteristic component is screened and prepared from tobacco |
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| US4701570A (en) * | 1984-11-05 | 1987-10-20 | Japan Tobacco Inc. | Antitumor agent |
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