CN104151140A - Method for comprehensively extracting multiple effective components from tobacco leaves - Google Patents
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- CN104151140A CN104151140A CN201410330343.4A CN201410330343A CN104151140A CN 104151140 A CN104151140 A CN 104151140A CN 201410330343 A CN201410330343 A CN 201410330343A CN 104151140 A CN104151140 A CN 104151140A
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Abstract
The invention discloses a method for comprehensively extracting multiple effective components from tobacco leaves. The method comprises the following steps: performing twice ultrasonic circulation extraction on the tobacco leaves to respectively obtain crude ointment containing solanesol, nicotine and chlorogenic acid and an extract containing protein and sugar, and with the combination of separation and purification methods such as liquid-liquid two-phase extraction, column chromatography, recrystallization, steam distillation and precipitation, separating five effective components, namely, solanesol, nicotine, chlorogenic acid, protein and sugar from the tobacco leaves one by one. The method can continuously extract and separate the multiple effective components form the tobacco leaves, can greatly improve the utilization rate of a tobacco leaf raw material, has the advantages that the extraction efficiency is high, a great number of separation products are obtained and a small amount of wastes are discharged, and is an efficient and environment-friendly comprehensive tobacco leaf multi-component extraction technique.
Description
Technical field
The invention belongs to tobacco leaf deep process technology field, be specifically related to a kind of also method of separating effective ingredient Salanesol, nicotine, chlorogenic acid, protein and sugar of progressively extracting from tobacco leaf.
Background technology
In tobacco leaf, contain the multiple effective constituent with medicinal or edibleness, as Salanesol, nicotine, chlorogenic acid, protein and sugar class etc.Salanesol is a kind of medicinal ingredients of high added value, has the pharmacological actions such as antibacterial, anti-inflammatory and hemostasis, is also the necessary raw material of synthesise vitamins K2 and Coenzyme Q10 99.0 simultaneously.Nicotine is a kind of alkaloid in tobacco leaf, applies very extensively, and its purposes mainly comprises agricultural chemicals and medical two broad aspect.Nicotine series pesticide platymiscium sterilant, have steam smoke, the feature of stomach toxicity, the function of tagging and the noresidue of degrading rapidly, be desirable efficient green sterilant and biological agricultural chemicals.Meanwhile, high purity nicotine is on medicine industry, to develop the extraordinary raw material of the illness medicines such as treatment is cardiovascular, skin, snake bite and insect sting.Chlorogenic acid is a kind of important biologically active substance, have antibacterial, antiviral, increase the effects such as white cell, hepatic cholagogic, antitumor, hypotensive, reducing blood-fat, removing free radical and stimulating central nervous system system.Tobacco leaf protein, containing 18 seed amino acids, comprises 8 kinds of essential amino acids, and adult's aminoacid pattern of recommending with Food and Argriculture OrganizationFAO conforms to substantially, and particularly lysine content is higher.In tobacco leaf, contained water-soluble sugar more than 90% is reducing sugar, and the effect of reducing sugar and purposes are very extensive, can be for food-processing and medication chemistry.
For exploitation tobacco leaf, the purposes of discarded tobacco leaf aspect non-cigarette particularly, researchist has carried out large quantity research to the extraction of effective constituent in tobacco leaf both at home and abroad, there is at present multinomial patent to disclose the extraction process of the compositions such as solanesol in tobacco leaves, nicotine, but these techniques all exist extraction composition few, the defect that raw material availability is not high.In recent years, people comparatively pay close attention to the comprehensive extraction and application of tobacco leaf.Patent CN200610069846.6 discloses a kind of method that cleans synchronous high-efficiency extraction of high purity solanesol alcohol and nicotine, it is characterized in that adopting diacolation circumfluence method to prepare crude extract, crude extract obtains solanesol extract and the nicotine aqueous solution after separation, the purified separation of solanesol extract obtains high-purity solanesol, the purified high purity nicotine that extracts to obtain of the nicotine aqueous solution.Patent CN200710034942.1 discloses a kind of method of simultaneously extracting high pure natural nicotine and Salanesol from abandoned tobacco, specifically adopt mixed solution heating and refluxing extraction, the filtration of second alcohol and water, obtain respectively nicotine extracting solution and filter residue, nicotine extracting solution is concentrated to obtain to nicotine medicinal extract, after water-soluble, add phase-splitting agent phase-splitting to obtain nicotine products, filter residue obtains Salanesol product after treatment; Patent CN201110113343.5 discloses a kind of method of extracting Salanesol and nicotine from tobacco leaf, its technical process is with alkali lye and the mixed extractant solvent of using normal hexane after tobacco leaf is pulverized, extract complete filtration, separatory, getting upper strata normal hexane extracts with aqueous hydrochloric acid again, upper strata normal hexane layer is concentrated into paste and is Salanesol crude extract, and lower floor's water is crude salt acid fume alkaline solution.Patent CN201110248504.1 discloses a method that grows tobacco and extract continuously, is specifically related to utilize ultrasonic wave alkalization pre-treatment extraction nicotine, hemicellulose, xylogen, cellulosic method successively from tobacco.
About extract the report of Multiple components in tobacco simultaneously, be for Salanesol and these two kinds of materials of nicotine mostly, and other effective constituents in tobacco, as protein, polyphenol and carbohydrate are not fully used, have caused the waste of resource at present.
Summary of the invention
The object of the present invention is to provide a kind of also method of separated plurality of active ingredients of comprehensively extracting from tobacco leaf, the method extraction efficiency is high, and waste discharge is few, is the comprehensive extractive technique of a kind of efficient and environmentally friendly tobacco leaf.
For achieving the above object, the technical solution used in the present invention is: a kind of method of comprehensively extracting plurality of active ingredients from tobacco leaf, comprises the steps:
(1) ethanolic soln ultrasonic circulating is extracted Salanesol, nicotine and chlorogenic acid: tobacco leaf is dried after pulverizing, obtain tobacco leaf fragment, and be placed in circulating supersound extraction device, add ethanolic soln, carry out ultrasonic circulating extraction, filter to obtain ethanol extract and tobacco leaf filter residue, ethanol extract is concentrated, must contain the thick cream of Salanesol, nicotine and chlorogenic acid;
(2) alkaline aqueous solution ultrasonic circulating is extracted protein and sugar: the tobacco leaf filter residue that step (1) is obtained is placed in circulating supersound extraction device, adds alkaline aqueous solution, carries out ultrasonic circulating extraction, filters containing the extracting solution of protein and sugar;
(3) Salanesol, nicotine and chlorogenic acid is separated: to adding sherwood oil and acidic aqueous solution containing in the thick cream of Salanesol, nicotine and chlorogenic acid, carry out liquid-liquid two-phase extraction, get upper strata petroleum ether layer and concentrate to obtain the thick cream of Salanesol, then carry out column chromatography for separation, recrystallization obtains Salanesol; After lower floor's water is extracted with ethyl acetate, gets upper strata ethyl acetate layer and concentrate to obtain the thick cream of chlorogenic acid, then be made into the chlorogenic acid aqueous solution and obtain chlorogenic acid by column chromatography for separation; Lower floor's water through ethyl acetate extraction gained is concentrated, add alkali to regulate pH value, then carry out wet distillation, with the receiving flask that acidic aqueous solution is housed, collect cut, obtain the nicotine salt aqueous solution, the nicotine salt aqueous solution is concentrated, add alkali to regulate pH value, then with methylene dichloride, extract, get dichloromethane layer and concentrate to obtain nicotine;
(4) separation of protein and sugar: the extracting solution containing protein and sugar of gained in step (2) is concentrated, add protein precipitant, after standing, centrifugation supernatant liquid and protein precipitation, protein precipitation is with after absolute ethanol washing, carry out low-temperature vacuum drying, obtain described protein; Supernatant liquid adds ethanol to carry out alcohol precipitation, filters after standing, obtains sugared filter cake, and sugared filter cake, with after absolute ethanol washing, is carried out to low-temperature vacuum drying, obtains described sugar.
According to such scheme, in described step (1), tobacco leaf drying is pulverized rear gained tobacco leaf fragment and is of a size of 20 orders; Described ethanolic soln is that volume fraction of ethanol is 80～95% the acidic ethanol aqueous solution, and pH value is that 3～4, pH value regulates by phosphoric acid or citric acid.
According to such scheme, in the ethanolic soln ultrasonic circulating leaching process of described step (1), ultrasonic power is 100～300W, and supersound extraction temperature is 50～60 ℃, and extraction time is 30～60min; The solid-liquid ratio of tobacco leaf fragment and ethanolic soln is 1:(5～20), solid-liquid ratio unit is g/mL.
According to such scheme, in the alkaline aqueous solution ultrasonic circulating leaching process of described step (2), ultrasonic power is 100～300W, and supersound extraction temperature is 50～60 ℃, and extraction time is 30～60min; Described alkaline aqueous solution is the alkaline aqueous solution of NaOH or KOH, and pH value is 7～8; The solid-liquid ratio of tobacco leaf filter residue and alkaline aqueous solution is 1:(10～20), solid-liquid ratio unit is g/mL.
According to such scheme, in the separating step of described Salanesol, in step (3), liquid-liquid two-phase extraction acidic aqueous solution used is phosphate aqueous solution, and pH value is 2～3, and the volume ratio of sherwood oil and acidic aqueous solution is 2:3, and extraction times is 2～3 times; The column chromatography for separation sorbing material used of described Salanesol is 100～200 object silica gel, and eluent is petrol ether/ethyl acetate mixed solvent, and the volume ratio of sherwood oil and ethyl acetate is 9:1; The recrystallization solvent of described Salanesol is ethanol, and recrystallization temperature is-15 ℃.
According to such scheme, in the separating step of described chlorogenic acid, column chromatography for separation adopts polyamide column as chromatographic separation post, polyamide column is of a size of 80～100 orders, the loading concentration of the chlorogenic acid aqueous solution is 9mg/mL, and applied sample amount is 5 times of column volumes, and loading flow velocity is 4 times of column volumes per hour, eluent is that volume fraction is 30% aqueous ethanolic solution, and elution flow rate is 4 times of column volumes per hour.
According to such scheme, the separating step of described nicotine comprises: the lower floor's water after ethyl acetate extraction in step (3) is concentrated into 1/3 of original volume, then adds NaOH or KOH to regulate pH value to 11～13; While carrying out wet distillation nicotine, with citric acid solution, absorb nicotine distillate, and control receiving liquid pH and be less than 3, when slipping out the silicotungstic acid detection sediment-free generation that liquid is 1% with massfraction, stop distillation, then the nicotine salt aqueous solution is concentrated to 1/3～1/2 of original volume, add NaOH or KOH and regulate pH value to 11～13, with dichloromethane extraction 1～2 time, evaporation is removed methylene dichloride and is obtained nicotine.
According to such scheme, in described step (4), the extracting solution containing protein and sugar of gained is concentrated into 1/8～1/3 of original volume; Described protein precipitant is cm-chitosan, and molecular weight is 50000～100000, and consumption is 0.01～0.05%wt; Precipitation temperature is 4 ℃, and the time is 2～5h; Absolute ethanol washing 2～3 times of gained protein precipitation; The amount of alcohol adding in gained supernatant liquid is 3～4 times of its volume; Alcohol precipitation time of repose is 1～2h, and temperature is 4～20 ℃; Absolute ethanol washing 2～3 times of gained sugar filter cake.
Compared with prior art, the invention has the beneficial effects as follows:
(1) by twice ultrasonic circulating, extract the simultaneous extraction realizing tobacco leaf plurality of active ingredients, and in conjunction with separation or means of purification such as liquid-liquid two-phase extraction, column chromatography, recrystallization, wet distillation, precipitations, five kinds of effective constituent Salanesols, nicotine, chlorogenic acid, protein and sugars in tobacco leaf are separated one by one, greatly improved the utilization ratio of tobacco material.
(2) to have extraction efficiency high for present method, and separated product is many, and the advantage that waste discharge is few belongs to a kind of efficient and environmentally friendly comprehensive sequential extraction method of tobacco leaf multicomponent.
Accompanying drawing explanation
Below in conjunction with accompanying drawing, the invention will be further described, in accompanying drawing:
Fig. 1 is process flow sheet of the present invention.
Embodiment
For making object of the present invention, technical scheme and advantage clearer, below in conjunction with drawings and Examples, the present invention is further elaborated, specific embodiment described herein only, in order to explain the present invention, is not intended to limit the present invention.
The material using in following examples, reagent etc. if no special instructions, obtain in all can be from commercial channels.
In following examples, Salanesol, nicotine and chlorogenic acid all adopt high effective liquid chromatography for measuring the finished product purity, and lipidated protein is measured with micro-triumphant formula nitriding, and sugar is measured its purity by phenolsulfuric acid method.
The high performance liquid chromatography test parameter of Salanesol comprises:
Chromatographic column: Agilent ZORBAX Eclipse Plus C18 reversed-phase column (250mm * 4.6mm, 5 μ m);
Moving phase: Virahol: acetonitrile=50%:50% (volume ratio);
Flow velocity: 1mL/min;
Ultraviolet detection wavelength: 210nm;
Column temperature: 25 ℃;
Sample size: 10 μ L.
The high performance liquid chromatography test parameter of nicotine comprises:
Chromatographic column: Agilent ZORBAX Eclipse Plus C18 reversed-phase column (250mm * 4.6mm, 5 μ m);
Moving phase: methyl alcohol: phosphate buffer solution=60%:40% (volume ratio);
Phosphate buffer solution preparation: 2.420g Sodium phosphate dibasic, 3.5mL phosphoric acid and 6.5mL triethylamine, add water and be settled to 1000mL;
Flow velocity: 1mL/mim;
Ultraviolet detection wavelength: 259nm;
Column temperature: 30 ℃;
Sample size: 10 μ L.
The high performance liquid chromatography test parameter of chlorogenic acid comprises:
Chromatographic column: Agilent ZORBAX Eclipse Plus C18 reversed-phase column (250mm * 4.6mm, 5 μ m);
Moving phase: acetonitrile: 2% acetic acid=13%:87% (volume ratio);
Flow velocity: 1mL/min;
Ultraviolet detection wavelength: 328nm;
Column temperature: 25 ℃;
Sample size: 10 μ L.
Embodiment mono-:
A comprehensive method of extracting plurality of active ingredients from tobacco leaf, comprises the following steps:
(1) ethanolic soln ultrasonic circulating is extracted solanesol in tobacco leaves, nicotine and chlorogenic acid: Yunnan tobacco ovendry power is broken to after 20 orders, take 100g tobacco leaf fragment, be placed in ultrasonic circulating extractor, then adding 500mL volume fraction of ethanol is 80% the acidic ethanol aqueous solution, the pH value of solution is 3, by phosphoric acid, regulates.In temperature, be 50 ℃, under the condition that ultrasonic power is 100W, ultrasonic circulating is extracted 30min, filters and obtains acidic ethanol extraction liquid and tobacco leaf filter residue.Ethanol extract is concentrated containing the thick cream 18.2g of Salanesol, nicotine and chlorogenic acid, and wherein Salanesol extraction yield 48.2%, nicotine extraction yield 59.4%, chlorogenic acid yield 62.7%.
(2) alkaline aqueous solution ultrasonic circulating is extracted water soluble protein and the sugar in tobacco leaf filter residue: the tobacco leaf filter residue that step (1) is obtained is placed in circulating supersound extraction device, add the NaOH aqueous solution that 1000mL pH value is 7, in temperature, it is 50 ℃, ultrasonic power is that under the condition of 100W, ultrasonic circulating is extracted 30min, filter to obtain the extracting solution of secondary filter residue and containing water-soluble protein and sugar, water soluble protein extraction yield is 52.4%, and water-soluble sugar extraction yield is 78%.
(3) Salanesol, nicotine and chlorogenic acid separated: to 18.2g containing Salanesol, in the thick cream of nicotine and chlorogenic acid, adding respectively sherwood oil 100mL and pH value is 2 phosphate aqueous solution 150mL, carry out liquid-liquid two-phase extraction, get upper strata petroleum ether layer, lower layer of water continues to use 100mL petroleum ether extraction the 2nd time mutually, merge twice petroleum ether layer and concentrate to obtain the thick cream 1.7g of Salanesol, by 100～200 object silica gel, carry out column chromatography for separation again, eluent is the mixed solvent of sherwood oil: ethyl acetate=9:1 (volume ratio), collect Salanesol enrichment section in elutriant, concentrate to obtain 0.42g Salanesol purified, then add 5mL ethanol, recrystallization at-15 ℃, filter gained crystal at room temperature vacuum-drying (20 ℃), must refine Salanesol 0.29g, purity testing is 91%.
The lower layer of water that twice petroleum ether extraction crossed continues to be extracted with ethyl acetate twice mutually, the consumption of each ethyl acetate is 100mL, merge the ethyl acetate layer obtaining and concentrate to obtain the thick cream 1.42g of chlorogenic acid, then the chlorogenic acid aqueous solution that adds distilled water to be made into 9mg/mL carries out polyamide column chromatography separation, polyamide column is of a size of 80～100 orders, applied sample amount is 5 times of column volumes, loading flow velocity is 4 times of column volumes per hour, eluent is that volume fraction is 30% aqueous ethanolic solution, elution flow rate is 4 times of column volumes per hour, total 6 times of column volumes of co-elute, the 1/2 times of column volume of take is recruiting unit, collect the higher elutriant of chlorogenic acid content, concentrating under reduced pressure also carries out vacuum dehydrating at lower temperature (temperature is lower than 20 ℃) and obtains chlorogenic acid 0.58g, purity 47%.
The 150mL lower floor water that ethyl acetate extraction is obtained adds NaOH to regulate pH value to 11 after being concentrated to 50mL, then carry out wet distillation, with the receiving bottle that aqueous citric acid solution is housed in advance, collect cut, and control pH in receiving bottle and be less than 3, when detecting, the distillate silicotungstic acid that is 1% with massfraction when sediment-free produces, stops distillation, the about 200mL of liquor capacity in receiving bottle now, then the citric acid nicotine solution for vacuum concentration in receiving bottle is arrived to 100mL, add NaOH to regulate pH value to 11 left and right, with twice of dichloromethane extraction, the consumption of each methylene dichloride extraction is 80mL, combined dichloromethane layer is also dried by anhydrous sodium sulphate, last underpressure distillation is removed methylene dichloride and is obtained nicotine 1.63g, purity testing is 98%.
(4) separation of protein and sugar: the aqueous solution 1000mL containing protein and sugar that aqueous solution ultrasonic circulating in step (2) is extracted to gained is concentrated to 250mL, then slowly stir and add the solution that 5mL cm-chitosan (molecular weight is 50000) concentration is 0.5wt%, at 4 ℃ after standing 2h, centrifuging and taking supernatant liquid 230mL, gained protein precipitation is with after twice of absolute ethanol washing, carry out low-temperature vacuum drying (temperature is lower than 20 ℃) and obtain water soluble protein 4.1g, purity 70%.
Gained supernatant liquid continues to add 800mL ethanol to carry out alcohol precipitation, and at 4 ℃, standing 1h filtered after the time, obtains sugared filter cake with after twice of absolute ethanol washing, carries out low-temperature vacuum drying (temperature is lower than 20 ℃) and obtains water-soluble sugar 8.2g, purity 80%.
Embodiment bis-:
A comprehensive method of extracting plurality of active ingredients from tobacco leaf, comprises the following steps:
(1) ethanolic soln ultrasonic circulating is extracted solanesol in tobacco leaves, nicotine and chlorogenic acid: Yunnan tobacco ovendry power is broken to after 20 orders, take 100g tobacco leaf fragment, be placed in ultrasonic circulating extractor, then adding 1000mL volume fraction of ethanol is 90% the acidic ethanol aqueous solution, the pH value of solution is 4, by phosphoric acid, regulates.In temperature, be 55 ℃, under the condition that ultrasonic power is 200W, ultrasonic circulating is extracted 45min, filters and obtains acidic ethanol extraction liquid and tobacco leaf filter residue.Ethanol extract is concentrated containing the thick cream 20.3g of Salanesol, nicotine and chlorogenic acid, and wherein Salanesol extraction yield 68.2%, nicotine extraction yield 72.3%, chlorogenic acid yield 74.7%.
(2) alkaline aqueous solution ultrasonic circulating is extracted water soluble protein and the sugar in tobacco leaf filter residue: the tobacco leaf filter residue that step (1) is obtained is placed in circulating supersound extraction device, then the NaOH aqueous solution that the pH value that adds 1500mL is 8, in temperature, it is 55 ℃, ultrasonic power is that under the condition of 200W, ultrasonic circulating is extracted 45min, filter to obtain the extracting solution of secondary filter residue and containing water-soluble protein and sugar, water soluble protein extraction yield is 72.4%, and water-soluble sugar extraction yield is 82%.
(3) Salanesol, nicotine and chlorogenic acid separated: to 20.3g containing Salanesol, in the thick cream of nicotine and chlorogenic acid, adding respectively sherwood oil 100mL and pH value is 2 phosphate aqueous solution 150mL, carry out liquid-liquid two-phase extraction, get upper strata petroleum ether layer, lower layer of water continues to use 100mL petroleum ether extraction the 2nd time mutually, merge twice petroleum ether layer and concentrate to obtain the thick cream 2.2g of Salanesol, by 100～200 object silica gel, carry out column chromatography for separation again, eluent is the mixed solvent of sherwood oil: ethyl acetate=9:1 (volume ratio), collect Salanesol enrichment section in elutriant, concentrate to obtain 0.54g Salanesol purified, then add 5mL ethanol, recrystallization at-15 ℃, filter gained crystal at room temperature vacuum-drying (20 ℃), must refine Salanesol 0.35g, purity testing is 90%.
The lower layer of water that twice petroleum ether extraction crossed continues to be extracted with ethyl acetate twice mutually, the consumption of each ethyl acetate is 100mL, merge the ethyl acetate layer obtaining and concentrate to obtain the thick cream 1.72g of chlorogenic acid, then the chlorogenic acid aqueous solution that adds distilled water to be made into 9mg/mL carries out polyamide column chromatography separation, polyamide column is of a size of 80～100 orders, applied sample amount is 5 times of column volumes, loading flow velocity is 4 times of column volumes per hour, eluent is that volume fraction is 30% aqueous ethanolic solution, elution flow rate is 4 times of column volumes per hour, total 6 times of column volumes of co-elute, the 1/2 times of column volume of take is recruiting unit, collect the higher elutriant of chlorogenic acid content, concentrating under reduced pressure also carries out vacuum dehydrating at lower temperature (temperature is lower than 20 ℃) and obtains chlorogenic acid 0.7g, purity 46%.
The 255mL lower floor water that ethyl acetate extraction obtains adds NaOH to regulate pH value to 12 after being concentrated to 85mL, then carry out wet distillation, with the receiving bottle that aqueous citric acid solution is housed in advance, collect cut, and control pH in receiving bottle and be less than 3, when detecting, the distillate silicotungstic acid that is 1% with massfraction when sediment-free produces, stops distillation, now in receiving bottle, liquor capacity is about 200mL, then the citric acid nicotine solution for vacuum concentration in receiving bottle is arrived to 100mL, add NaOH to regulate pH value to 11 left and right, with twice of dichloromethane extraction, the consumption of each methylene dichloride is 80mL, combined dichloromethane is also dried by anhydrous sodium sulphate, last underpressure distillation is removed methylene dichloride and is obtained nicotine 2.1g, purity testing is 97%.
(4) separation of protein and sugar: the aqueous solution 1500mL containing protein and sugar that aqueous solution ultrasonic circulating in step (2) is extracted to gained is concentrated to 300mL, then slowly stir that to add 10mL content be cm-chitosan (molecular weight the is 50000) solution of 0.5wt%, at 4 ℃ after standing 5h, centrifuging and taking supernatant liquid 280mL, gained protein precipitation is with after twice of absolute ethanol washing, carry out low-temperature vacuum drying (temperature is lower than 20 ℃) and obtain water soluble protein 5.8g, purity 71%.
Gained supernatant liquid continues to add 900mL ethanol to carry out alcohol precipitation, and at 20 ℃, standing 1h filtered after the time, obtains sugared filter cake with after absolute ethanol washing three times, carries out low-temperature vacuum drying (temperature is lower than 20 ℃) and obtains water-soluble sugar 8.9g, purity 81%.
Embodiment tri-:
A comprehensive method of extracting plurality of active ingredients from tobacco leaf, comprises the following steps:
(1) ethanolic soln ultrasonic circulating is extracted solanesol in tobacco leaves, nicotine and chlorogenic acid: Yunnan tobacco ovendry power is broken to after 20 orders, take 100g tobacco leaf fragment, be placed in ultrasonic circulating extractor, then adding 2000mL volume fraction of ethanol is 95% the acidic ethanol aqueous solution, the pH value of solution is 4, by phosphoric acid, regulates.In temperature, be 60 ℃, under the condition that ultrasonic power is 300W, ultrasonic circulating is extracted 60min, filters and obtains acidic ethanol extraction liquid and tobacco leaf filter residue.Ethanol extract is concentrated containing the thick cream 21.6g of Salanesol, nicotine and chlorogenic acid, and wherein Salanesol extraction yield 81.2%, nicotine extraction yield 94.3%, chlorogenic acid yield 93.7%.
(2) alkaline aqueous solution ultrasonic circulating is extracted water soluble protein and the sugar in tobacco leaf filter residue: the tobacco leaf filter residue obtaining in step (1) is placed in to ultrasonic circulating extractor, then the NaOH aqueous solution that the pH value that adds 2000mL is 8, in temperature, it is 60 ℃, ultrasonic power is that under the condition of 300W, ultrasonic circulating is extracted 60min, filter to obtain the extracting solution of secondary filter residue and containing water-soluble protein and sugar, water soluble protein extraction yield is 81.2%, and water-soluble sugar extraction yield is 84%.
(3) Salanesol, nicotine and chlorogenic acid separated: to 21.6g containing Salanesol, in the thick cream of nicotine and chlorogenic acid, adding respectively sherwood oil 100mL and pH value is 3 phosphate aqueous solution 150mL, carry out liquid-liquid two-phase extraction, get upper strata petroleum ether layer, lower layer of water continues to use 100mL petroleum ether extraction the 2nd time mutually, merge twice petroleum ether layer and concentrate to obtain the thick cream 2.4g of Salanesol, by 100～200 object silica gel, carry out column chromatography for separation again, eluent is the mixed solvent of sherwood oil: ethyl acetate=9:1 (volume ratio), collect Salanesol enrichment section in elutriant, concentrate to obtain 0.61g Salanesol purified, then add 5mL ethanol, recrystallization at-15 ℃, filter gained crystal at room temperature vacuum-drying (20 ℃), must refine Salanesol 0.4g, purity testing is 91%.
The lower layer of water of crossing through twice petroleum ether extraction continues to be extracted with ethyl acetate twice mutually, the consumption of each ethyl acetate is 100mL, merge the ethyl acetate layer obtaining and concentrate to obtain the thick cream 1.78g of chlorogenic acid, then the chlorogenic acid aqueous solution that adds distilled water to be made into 9mg/mL carries out polyamide column chromatography separation, polyamide column is of a size of 80～100 orders, applied sample amount is 5 times of column volumes, loading flow velocity is 4 times of column volumes per hour, eluent is that volume fraction is 30% aqueous ethanolic solution, elution flow rate is 4 times of column volumes per hour, total 6 times of column volumes of co-elute, the 1/2 times of column volume of take is recruiting unit, collect the higher elutriant of chlorogenic acid content, concentrating under reduced pressure also carries out vacuum dehydrating at lower temperature (temperature is lower than 20 ℃) and obtains chlorogenic acid 0.73g, purity 46%.
The 255mL lower floor water that ethyl acetate extraction obtains adds NaOH to regulate pH value to 12 after being concentrated to 85mL, then carry out wet distillation, with the receiving bottle that aqueous citric acid solution is housed in advance, collect cut, and control pH in receiving bottle and be less than 3, when being 1% silicotungstic acid detection sediment-free generation, distillate massfraction stops distillation, now in receiving bottle, liquor capacity is about 300mL, then the citric acid nicotine solution for vacuum concentration in receiving bottle is arrived to 100mL, add NaOH to regulate pH value to 12 left and right, with twice of dichloromethane extraction, the consumption of each methylene dichloride extraction is 80mL, combined dichloromethane is also dried by anhydrous sodium sulphate, last underpressure distillation is removed methylene dichloride and is obtained nicotine 2.8g, purity testing is 97%.
(4) separation of protein and sugar: the aqueous solution 2000mL containing protein and sugar that aqueous solution ultrasonic circulating in step (2) is extracted to gained is concentrated to 300mL, then slowly stir that to add 15mL content be cm-chitosan (molecular weight the is 50000) solution of 0.5wt%, at 4 ℃ after standing 2h, centrifuging and taking supernatant liquid 270mL, gained protein precipitation is with after twice of absolute ethanol washing, carry out cryodrying (temperature is lower than 20 ℃) and obtain water soluble protein 6.5g, purity 74%.
Gained supernatant liquid continues to add 950mL ethanol to carry out alcohol precipitation, and at 4 ℃, standing 2h filtered after the time, obtains sugared filter cake with after twice of absolute ethanol washing, carries out low-temperature vacuum drying (temperature is lower than 20 ℃) and obtains water-soluble sugar 9.3g, purity 81%.
Without prejudice to essence of the present invention and spirit in the situation that, those of ordinary skill in the art may make according to the present invention various changes or distortion, but these corresponding changes or distortion all should belong to the protection domain of the appended claim of the present invention.
Claims (8)
1. a comprehensive method of extracting plurality of active ingredients from tobacco leaf, is characterized in that, comprises the following steps:
(1) ethanolic soln ultrasonic circulating is extracted Salanesol, nicotine and chlorogenic acid: tobacco leaf is dried after pulverizing, obtain tobacco leaf fragment, and be placed in circulating supersound extraction device, add ethanolic soln, carry out ultrasonic circulating extraction, filter to obtain ethanol extract and tobacco leaf filter residue, ethanol extract is concentrated, must contain the thick cream of Salanesol, nicotine and chlorogenic acid;
(2) alkaline aqueous solution ultrasonic circulating is extracted protein and sugar: the tobacco leaf filter residue that step (1) is obtained is placed in circulating supersound extraction device, adds alkaline aqueous solution, carries out ultrasonic circulating extraction, filters containing the extracting solution of protein and sugar;
(3) Salanesol, nicotine and chlorogenic acid is separated: to adding sherwood oil and acidic aqueous solution containing in the thick cream of Salanesol, nicotine and chlorogenic acid, carry out liquid-liquid two-phase extraction, get upper strata petroleum ether layer and concentrate to obtain the thick cream of Salanesol, then carry out column chromatography for separation, recrystallization obtains Salanesol; After lower floor's water is extracted with ethyl acetate, gets upper strata ethyl acetate layer and concentrate to obtain the thick cream of chlorogenic acid, then be made into the chlorogenic acid aqueous solution and obtain chlorogenic acid by column chromatography for separation; Lower floor's water through ethyl acetate extraction gained is concentrated, add alkali to regulate pH value, then carry out wet distillation, with the receiving flask that acidic aqueous solution is housed, collect cut, obtain the nicotine salt aqueous solution, the nicotine salt aqueous solution is concentrated, add alkali to regulate pH value, then with methylene dichloride, extract, get dichloromethane layer and concentrate to obtain nicotine;
(4) separation of protein and sugar: the extracting solution containing protein and sugar of gained in step (2) is concentrated, add protein precipitant, after standing, centrifugation supernatant liquid and protein precipitation, protein precipitation is with after absolute ethanol washing, carry out low-temperature vacuum drying, obtain described protein; Supernatant liquid adds ethanol to carry out alcohol precipitation, filters after standing, obtains sugared filter cake, and sugared filter cake, with after absolute ethanol washing, is carried out to low-temperature vacuum drying, obtains described sugar.
2. a kind of method of comprehensively extracting plurality of active ingredients from tobacco leaf according to claim 1, is characterized in that, in described step (1), tobacco leaf drying is pulverized rear gained tobacco leaf fragment and is of a size of 20 orders; Described ethanolic soln is that volume fraction of ethanol is 80～95% the acidic ethanol aqueous solution, and pH value is that 3～4, pH value regulates by phosphoric acid or citric acid.
According to claim 1 a kind of from tobacco leaf the comprehensive method of extracting plurality of active ingredients, it is characterized in that, in the ethanolic soln ultrasonic circulating leaching process of described step (1), ultrasonic power is 100～300W, supersound extraction temperature is 50～60 ℃, and extraction time is 30～60min; The solid-liquid ratio of tobacco leaf fragment and ethanolic soln is 1:(5～20), solid-liquid ratio unit is g/mL.
According to claim 1 a kind of from tobacco leaf the comprehensive method of extracting plurality of active ingredients, it is characterized in that, in the alkaline aqueous solution ultrasonic circulating leaching process of described step (2), ultrasonic power is 100～300W, supersound extraction temperature is 50～60 ℃, and extraction time is 30～60min; Described alkaline aqueous solution is the alkaline aqueous solution of NaOH or KOH, and pH value is 7～8; The solid-liquid ratio of tobacco leaf filter residue and alkaline aqueous solution is 1:(10～20), solid-liquid ratio unit is g/mL.
According to claim 1 a kind of from tobacco leaf the comprehensive method of extracting plurality of active ingredients, it is characterized in that, in the separating step of described Salanesol, in step (3), liquid-liquid two-phase extraction acidic aqueous solution used is phosphate aqueous solution, pH value is 2～3, the volume ratio of sherwood oil and acidic aqueous solution is 2:3, and extraction times is 2～3 times; The column chromatography for separation sorbing material used of Salanesol is 100～200 object silica gel, and eluent is petrol ether/ethyl acetate mixed solvent, and the volume ratio of sherwood oil and ethyl acetate is 9:1; The recrystallization solvent of described Salanesol is ethanol, and recrystallization temperature is-15 ℃.
According to claim 1 a kind of from tobacco leaf the comprehensive method of extracting plurality of active ingredients, it is characterized in that, in the separating step of described chlorogenic acid, column chromatography for separation adopts polyamide column as chromatographic separation post, and polyamide column is of a size of 80～100 orders, and the loading concentration of the chlorogenic acid aqueous solution is 9mg/mL, applied sample amount is 5 times of column volumes, loading flow velocity is 4 times of column volumes per hour, and eluent is that volume fraction is 30% aqueous ethanolic solution, and elution flow rate is 4 times of column volumes per hour.
According to claim 1 a kind of from tobacco leaf the comprehensive method of extracting plurality of active ingredients, it is characterized in that, the separating step of described nicotine comprises: the lower floor's water after ethyl acetate extraction in step (3) is concentrated into 1/3 of original volume, then adds NaOH or KOH to regulate pH value to 11～13; While carrying out wet distillation nicotine, with citric acid solution, absorb nicotine distillate, and control receiving liquid pH and be less than 3, when slipping out the silicotungstic acid detection sediment-free generation that liquid is 1% with massfraction, stop distillation, then the nicotine salt aqueous solution is concentrated to 1/3～1/2 of original volume, add NaOH or KOH and regulate pH value to 11～13, with dichloromethane extraction 1～2 time, evaporation is removed methylene dichloride and is obtained nicotine.
8. a kind of method of comprehensively extracting plurality of active ingredients from tobacco leaf according to claim 1, is characterized in that, in described step (4), the extracting solution containing protein and sugar of gained is concentrated into 1/8～1/3 of original volume; Described protein precipitant is cm-chitosan, and molecular weight is 50000～100000, and consumption is 0.01～0.05%wt; Precipitation temperature is 4 ℃, and the time is 2～5h; Absolute ethanol washing 2～3 times of gained protein precipitation; It is 3～4 times of filtrate volume that gained supernatant liquid adds amount of alcohol; Alcohol precipitation time of repose is 1～2h, and temperature is 4～20 ℃; Absolute ethanol washing 2～3 times of gained sugar filter cake.
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