CN104151140B - A kind of method of comprehensive extraction plurality of active ingredients from tobacco leaf - Google Patents

A kind of method of comprehensive extraction plurality of active ingredients from tobacco leaf Download PDF

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CN104151140B
CN104151140B CN201410330343.4A CN201410330343A CN104151140B CN 104151140 B CN104151140 B CN 104151140B CN 201410330343 A CN201410330343 A CN 201410330343A CN 104151140 B CN104151140 B CN 104151140B
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extraction
tobacco leaf
aqueous solution
nicotine
protein
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CN104151140A (en
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刘治田
张旗
郑乐驰
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武汉工程大学
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    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07CACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C29/00Preparation of compounds having hydroxy or O-metal groups bound to a carbon atom not belonging to a six-membered aromatic ring
    • C07C29/74Separation; Purification; Use of additives, e.g. for stabilisation
    • C07C29/76Separation; Purification; Use of additives, e.g. for stabilisation by physical treatment
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07CACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C67/00Preparation of carboxylic acid esters
    • C07C67/48Separation; Purification; Stabilisation; Use of additives
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07DHETEROCYCLIC COMPOUNDS
    • C07D401/00Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/02Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
    • C07D401/04Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings directly linked by a ring-member-to-ring-member bond
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07HSUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H1/00Processes for the preparation of sugar derivatives
    • C07H1/06Separation; Purification
    • C07H1/08Separation; Purification from natural products
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K1/00General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14Extraction; Separation; Purification
    • C07K1/30Extraction; Separation; Purification by precipitation

Abstract

The invention discloses a kind of method of comprehensive extraction plurality of active ingredients from tobacco leaf, the method is by carrying out twice ultrasonic and circulated extraction to tobacco leaf, obtain containing the thick cream of Salanesol, nicotine, chlorogenic acid and the extracting solution containing protein, sugar respectively, and be separated or means of purification with precipitation etc. in conjunction with liquid-liquid two-phase extraction, column chromatography, recrystallization, wet distillation, five kinds of effective constituent Salanesols, nicotine, chlorogenic acid, protein and sugars in tobacco leaf are separated one by one.Present method achieves the continuous Extraction and isolation of plurality of active ingredients in tobacco leaf, substantially increase the utilization ratio of raw tobacco material, have extraction efficiency high, separated product is many, the advantage that waste discharge is few is a kind of efficient and environmentally friendly comprehensive extractive technique of tobacco leaf multicomponent.

Description

A kind of method of comprehensive extraction plurality of active ingredients from tobacco leaf

Technical field

The invention belongs to tobacco leaf deep process technology field, be specifically related to a kind of progressively extraction from tobacco leaf and the method for separating effective ingredient Salanesol, nicotine, chlorogenic acid, protein and sugar.

Background technology

There is effective constituent that is medicinal or edibleness, as Salanesol, nicotine, chlorogenic acid, protein and sugar class etc. containing multiple in tobacco leaf.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, and widely, its purposes mainly comprises agricultural chemicals and medical two broad aspect in application.Nicotine series pesticide platymiscium sterilant, have steam smoke, the feature of stomach toxicity, function of tagging and rapidly degraded noresidue, be desirable efficient green sterilant and biological agricultural chemicals.Meanwhile, high purity nicotine medicine industry is developed 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 white cell, hepatic cholagogic, antitumor, hypotensive, reducing blood-fat, the effect such as scavenging free radicals and stimulating central nervous system system.Tobacco leaf protein is containing 18 seed amino acids, and comprise 8 kinds of essential amino acids, the adult's aminoacid pattern recommended 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, may be used for food-processing and medication chemistry.

For exploitation tobacco leaf, the particularly purposes of discarded tobacco leaf in non-cigarette, domestic and international researchist has carried out large quantity research to the extraction of effective constituent in tobacco leaf, there is the multinomial extraction process that patent discloses the composition such as solanesol in tobacco leaves, nicotine at present, but it is few all to there is extract component in these techniques, the defect that raw material availability is not high.In recent years, the comprehensive extraction and application of people to tobacco leaf is comparatively paid close attention to.Patent CN200610069846.6 discloses a kind of method cleaning 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 being separated, the purified separation of solanesol extract obtains high-purity solanesol, and the nicotine aqueous solution is purified extracts to obtain high purity nicotine.Patent CN200710034942.1 discloses a kind of method 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 nicotine extracting solution and filter residue respectively, nicotine extracting solution is concentrated to obtain nicotine medicinal extract, add phase-splitting agent phase-splitting after water-soluble and obtain nicotine products, filter residue obtains Salanesol product after treatment; Patent CN201110113343.5 discloses a kind of method extracting Salanesol and nicotine from tobacco leaf, its technical process is with alkali lye and the mixed extractant solvent with normal hexane after being pulverized by tobacco leaf, extract complete filtration, separatory, get upper strata normal hexane to extract with aqueous hydrochloric acid again, upper strata normal hexane layer is concentrated into paste and is Salanesol crude extract, and lower floor's aqueous phase is crude salt acid fume alkaline solution.Patent CN201110248504.1 discloses one and to grow tobacco the method extracted continuously, is specifically related to utilize ultrasonic wave to alkalize pre-treatment extraction nicotine, hemicellulose, xylogen, cellulosic method successively from tobacco.

At present about the report extracting 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, cause the waste of resource.

Summary of the invention

The object of the present invention is to provide and a kind ofly comprehensively extract from tobacco leaf and be separated the method for plurality of active ingredients, 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 comprehensive extraction plurality of active ingredients from tobacco leaf, comprises the steps:

(1) ethanolic soln ultrasonic and circulated extraction Salanesol, nicotine and chlorogenic acid: after tobacco leaf being carried out oven dry pulverizing, obtain tobacco leaf fragment, and be placed in circulating supersonic extractors, add ethanolic soln, carry out ultrasonic and circulated extraction, filter to obtain ethanol extract and tobacco leaf filter residue, ethanol extract is concentrated, the thick cream of Salanesol, nicotine and chlorogenic acid must be contained;

(2) alkaline aqueous solution ultrasonic and circulated extraction protein and sugar: the tobacco leaf filter residue that step (1) obtains is placed in circulating supersonic extractors, adds alkaline aqueous solution, carry out ultrasonic and circulated extraction, filters containing the extracting solution of protein and sugar;

(3) Salanesol, nicotine are separated with chlorogenic acid: add sherwood oil and acidic aqueous solution to containing in the thick cream of Salanesol, nicotine and chlorogenic acid, carry out liquid-liquid two-phase extraction, get upper strata petroleum ether layer to carry out concentrating to obtain the thick cream of Salanesol, then carry out column chromatography for separation, recrystallization obtains Salanesol; After lower floor's aqueous phase is extracted with ethyl acetate, gets upper strata ethyl acetate layer and carry out concentrating 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 aqueous phase through extraction into ethyl acetate gained is concentrated, add alkali adjust ph, then wet distillation is carried out, cut is collected with the receiving flask that acidic aqueous solution is housed, obtain the nicotine salt aqueous solution, the nicotine salt aqueous solution is concentrated, adds alkali adjust ph, then extract with methylene dichloride, get dichloromethane layer and carry out concentrating 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 leaving standstill, centrifugation supernatant liquid and protein precipitation, after protein precipitation absolute ethanol washing, carry out low-temperature vacuum drying, obtain described protein; Supernatant liquid adds ethanol and carries out alcohol precipitation, filters, obtain sugared filter cake, after sugared filter cake absolute ethanol washing, carry out low-temperature vacuum drying, obtain described sugar after leaving standstill.

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 to be volume fraction of ethanol be 80 ~ 95% the acidic ethanol aqueous solution, pH value is 3 ~ 4, and pH value is regulated by phosphoric acid or citric acid.

According to such scheme, in the ethanolic soln ultrasonic and circulated extraction process of described step (1), ultrasonic power is 100 ~ 300W, and supersound extraction temperature is 50 ~ 60 DEG C, 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 and circulated extraction process of described step (2), ultrasonic power is 100 ~ 300W, and supersound extraction temperature is 50 ~ 60 DEG C, 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 DEG C.

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 sample 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 to be volume fraction be 30% aqueous ethanolic solution, elution flow rate is 4 times of column volumes per hour.

According to such scheme, the separating step of described nicotine comprises: carried out being concentrated into 1/3 of original volume by the lower floor's aqueous phase in step (3) after extraction into ethyl acetate, then add NaOH or KOH adjust ph to 11 ~ 13; Nicotine distillate is absorbed with citric acid solution when carrying out wet distillation nicotine, and control receiving liquid pH and be less than 3, distillation is stopped when slipping out liquid massfraction and being the silicotungstic acid detection sediment-free generation of 1%, then the nicotine salt aqueous solution is concentrated to 1/3 ~ 1/2 of original volume, add NaOH or KOH adjust ph to 11 ~ 13, with dichloromethane extraction 1 ~ 2 time, namely evaporation removing methylene dichloride obtains 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 DEG C, and the time is 2 ~ 5h; Gained protein precipitation absolute ethanol washing 2 ~ 3 times; The amount of alcohol added in gained supernatant liquid is 3 ~ 4 times of its volume; Alcohol precipitation time of repose is 1 ~ 2h, and temperature is 4 ~ 20 DEG C; Gained sugar filter cake absolute ethanol washing 2 ~ 3 times.

Compared with prior art, the invention has the beneficial effects as follows:

(1) by the simultaneous extraction of twice ultrasonic and circulated extraction realization to 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, substantially increases the utilization ratio of tobacco material.

(2) to have extraction efficiency high for present method, and separated product is many, 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 clearly understand, below in conjunction with drawings and Examples, be further elaborated to the present invention, specific embodiment described herein only in order to explain the present invention, is not intended to limit the present invention.

The material used in following examples, reagent etc. if no special instructions, all can from commercial channels in obtain.

In following examples, Salanesol, nicotine and chlorogenic acid all adopt high effective liquid chromatography for measuring the finished product purity, and the micro-triumphant formula nitriding of lipidated protein measures, and sugar phend-sulphuric acid measures its purity.

The high performance liquid chromatography test parameter of Salanesol comprises:

Chromatographic column: AgilentZORBAXEclipsePlusC18 reversed-phase column (250mm × 4.6mm, 5 μm);

Moving phase: Virahol: acetonitrile=50%:50% (volume ratio);

Flow velocity: 1mL/min;

Ultraviolet detection wavelength: 210nm;

Column temperature: 25 DEG C;

Sample size: 10 μ L.

The high performance liquid chromatography test parameter of nicotine comprises:

Chromatographic column: AgilentZORBAXEclipsePlusC18 reversed-phase column (250mm × 4.6mm, 5 μm);

Moving phase: methyl alcohol: phosphate buffer solution=60%:40% (volume ratio);

Phosphate buffer solution is prepared: 2.420g Sodium phosphate dibasic, and 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 DEG C;

Sample size: 10 μ L.

The high performance liquid chromatography test parameter of chlorogenic acid comprises:

Chromatographic column: AgilentZORBAXEclipsePlusC18 reversed-phase column (250mm × 4.6mm, 5 μm);

Moving phase: acetonitrile: 2% acetic acid=13%:87% (volume ratio);

Flow velocity: 1mL/min;

Ultraviolet detection wavelength: 328nm;

Column temperature: 25 DEG C;

Sample size: 10 μ L.

Embodiment one:

The comprehensive method extracting plurality of active ingredients from tobacco leaf, comprises the following steps:

(1) ethanolic soln ultrasonic and circulated extraction solanesol in tobacco leaves, nicotine and chlorogenic acid: after Yunnan tobacco ovendry power is broken to 20 orders, take 100g tobacco leaf fragment, be placed in ultrasonic and circulated extraction device, then the acidic ethanol aqueous solution that 500mL volume fraction of ethanol is 80% is added, the pH value of solution is 3, is regulated by phosphoric acid.Be 50 DEG C in temperature, ultrasonic power is ultrasonic and circulated extraction 30min under the condition of 100W, filters and obtains acidic ethanol extraction liquid and tobacco leaf filter residue.Ethanol extract is carried out concentrate containing the thick cream 18.2g of Salanesol, nicotine and chlorogenic acid, wherein Salanesol extraction yield 48.2%, nicotine extraction yield 59.4%, chlorogenic acid yield 62.7%.

(2) water soluble protein in alkaline aqueous solution ultrasonic and circulated extraction tobacco leaf filter residue and sugar: the tobacco leaf filter residue that step (1) obtains is placed in circulating supersonic extractors, add the NaOH aqueous solution that 1000mLpH value is 7, it is 50 DEG C in temperature, ultrasonic power is ultrasonic and circulated extraction 30min under the condition of 100W, 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 is separated with chlorogenic acid: to 18.2g containing Salanesol, sherwood oil 100mL is added respectively and pH value is 2 phosphate aqueous solution 150mL in the thick cream of nicotine and chlorogenic acid, 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 carry out concentrating to obtain the thick cream 1.7g of Salanesol, column chromatography for separation is carried out again by 100 ~ 200 object silica gel, eluent is sherwood oil: the mixed solvent of ethyl acetate=9:1 (volume ratio), collect Salanesol enrichment section in elutriant, concentrate to obtain 0.42g Salanesol purified, then 5mL ethanol is added, recrystallization at-15 DEG C, filter gained crystal at room temperature in vacuo drying (20 DEG C), obtain refining Salanesol 0.29g, purity testing is 91%.

The lower layer of water that twice petroleum ether extraction is crossed continues to be extracted with ethyl acetate twice mutually, the consumption of each ethyl acetate is 100mL, the ethyl acetate layer that merging obtains also carries out concentrating to obtain the thick cream 1.42g of chlorogenic acid, then add the chlorogenic acid aqueous solution that distilled water is made into 9mg/mL and carry 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 to be volume fraction be 30% aqueous ethanolic solution, elution flow rate is 4 times of column volumes per hour, total co-elute 6 times of column volumes, with 1/2 times of column volume for recruiting unit, collect the elutriant that chlorogenic acid content is higher, concentrating under reduced pressure also carries out vacuum dehydrating at lower temperature (temperature is lower than 20 DEG C) and namely obtains chlorogenic acid 0.58g, purity 47%.

NaOH adjust ph to 11 is added after the 150mL lower floor aqueous phase obtained by extraction into ethyl acetate is concentrated to 50mL, then wet distillation is carried out, cut is collected with the receiving bottle that aqueous citric acid solution is housed in advance, and control pH in receiving bottle and be less than 3, distillation is stopped when the silicotungstic acid that distillate massfraction is 1% detects when sediment-free produces, now in receiving bottle, liquor capacity is about 200mL, then by the citric acid nicotine solution for vacuum concentration in receiving bottle to 100mL, about adding NaOH adjust ph to 11, with dichloromethane extraction twice, the consumption of each methylene dichloride extraction is 80mL, combined dichloromethane layer also carries out drying by anhydrous sodium sulphate, namely last underpressure distillation removing methylene dichloride obtains nicotine 1.63g, purity testing is 98%.

(4) separation of protein and sugar: the aqueous solution 1000mL containing protein and sugar of aqueous solution ultrasonic and circulated extraction gained in step (2) is concentrated to 250mL, then slowly stirring adds 5mL cm-chitosan (molecular weight is 50000) concentration is the solution of 0.5wt%, leave standstill 2h at 4 DEG C after, centrifuging and taking supernatant liquid 230mL, gained protein precipitation is with after absolute ethanol washing twice, carry out low-temperature vacuum drying (temperature is lower than 20 DEG C) and namely obtain water soluble protein 4.1g, purity 70%.

Gained supernatant liquid continues to add 800mL ethanol and carries out alcohol precipitation, leaves standstill after the 1h time and filter at 4 DEG C, after obtaining sugared filter cake absolute ethanol washing twice, carries out low-temperature vacuum drying (temperature is lower than 20 DEG C) and namely obtains water-soluble sugar 8.2g, purity 80%.

Embodiment two:

The comprehensive method extracting plurality of active ingredients from tobacco leaf, comprises the following steps:

(1) ethanolic soln ultrasonic and circulated extraction solanesol in tobacco leaves, nicotine and chlorogenic acid: after Yunnan tobacco ovendry power is broken to 20 orders, take 100g tobacco leaf fragment, be placed in ultrasonic and circulated extraction device, then the acidic ethanol aqueous solution that 1000mL volume fraction of ethanol is 90% is added, the pH value of solution is 4, is regulated by phosphoric acid.Be 55 DEG C in temperature, ultrasonic power is ultrasonic and circulated extraction 45min under the condition of 200W, filters and obtains acidic ethanol extraction liquid and tobacco leaf filter residue.Ethanol extract is carried out concentrate containing the thick cream 20.3g of Salanesol, nicotine and chlorogenic acid, wherein Salanesol extraction yield 68.2%, nicotine extraction yield 72.3%, chlorogenic acid yield 74.7%.

(2) water soluble protein in alkaline aqueous solution ultrasonic and circulated extraction tobacco leaf filter residue and sugar: the tobacco leaf filter residue that step (1) obtains is placed in circulating supersonic extractors, then the pH value adding 1500mL is the NaOH aqueous solution of 8, it is 55 DEG C in temperature, ultrasonic power is ultrasonic and circulated extraction 45min under the condition of 200W, 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 is separated with chlorogenic acid: to 20.3g containing Salanesol, sherwood oil 100mL is added respectively and pH value is 2 phosphate aqueous solution 150mL in the thick cream of nicotine and chlorogenic acid, 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 carry out concentrating to obtain the thick cream 2.2g of Salanesol, column chromatography for separation is carried out again by 100 ~ 200 object silica gel, eluent is sherwood oil: the mixed solvent of ethyl acetate=9:1 (volume ratio), collect Salanesol enrichment section in elutriant, concentrate to obtain 0.54g Salanesol purified, then 5mL ethanol is added, recrystallization at-15 DEG C, filter gained crystal at room temperature in vacuo drying (20 DEG C), obtain refining Salanesol 0.35g, purity testing is 90%.

The lower layer of water that twice petroleum ether extraction is crossed continues to be extracted with ethyl acetate twice mutually, the consumption of each ethyl acetate is 100mL, merge the ethyl acetate layer obtained to carry out concentrating to obtain the thick cream 1.72g of chlorogenic acid, then add the chlorogenic acid aqueous solution that distilled water is made into 9mg/mL and carry 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 to be volume fraction be 30% aqueous ethanolic solution, elution flow rate is 4 times of column volumes per hour, total co-elute 6 times of column volumes, with 1/2 times of column volume for recruiting unit, collect the elutriant that chlorogenic acid content is higher, concentrating under reduced pressure also carries out vacuum dehydrating at lower temperature (temperature is lower than 20 DEG C) and namely obtains chlorogenic acid 0.7g, purity 46%.

NaOH adjust ph to 12 is added after the 255mL lower floor aqueous phase that extraction into ethyl acetate obtains is concentrated to 85mL, then wet distillation is carried out, cut is collected with the receiving bottle that aqueous citric acid solution is housed in advance, and control pH in receiving bottle and be less than 3, distillation is stopped when the silicotungstic acid that distillate massfraction is 1% detects when sediment-free produces, now in receiving bottle, liquor capacity is about 200mL, then by the citric acid nicotine solution for vacuum concentration in receiving bottle to 100mL, about adding NaOH adjust ph to 11, with dichloromethane extraction twice, the consumption of each methylene dichloride is 80mL, combined dichloromethane also carries out drying by anhydrous sodium sulphate, namely last underpressure distillation removing methylene dichloride obtains nicotine 2.1g, purity testing is 97%.

(4) separation of protein and sugar: the aqueous solution 1500mL containing protein and sugar of aqueous solution ultrasonic and circulated extraction gained in step (2) is concentrated to 300mL, then slowly stir and add cm-chitosan (molecular weight the is 50000) solution that 10mL content is 0.5wt%, leave standstill 5h at 4 DEG C after, centrifuging and taking supernatant liquid 280mL, gained protein precipitation is with after absolute ethanol washing twice, carry out low-temperature vacuum drying (temperature is lower than 20 DEG C) and namely obtain water soluble protein 5.8g, purity 71%.

Gained supernatant liquid continues to add 900mL ethanol and carries out alcohol precipitation, leaves standstill after the 1h time and filter at 20 DEG C, after obtaining sugared filter cake absolute ethanol washing three times, carries out low-temperature vacuum drying (temperature is lower than 20 DEG C) and namely obtains water-soluble sugar 8.9g, purity 81%.

Embodiment three:

The comprehensive method extracting plurality of active ingredients from tobacco leaf, comprises the following steps:

(1) ethanolic soln ultrasonic and circulated extraction solanesol in tobacco leaves, nicotine and chlorogenic acid: after Yunnan tobacco ovendry power is broken to 20 orders, take 100g tobacco leaf fragment, be placed in ultrasonic and circulated extraction device, then the acidic ethanol aqueous solution that 2000mL volume fraction of ethanol is 95% is added, the pH value of solution is 4, is regulated by phosphoric acid.Be 60 DEG C in temperature, ultrasonic power is ultrasonic and circulated extraction 60min under the condition of 300W, filters and obtains acidic ethanol extraction liquid and tobacco leaf filter residue.Ethanol extract is carried out concentrate containing the thick cream 21.6g of Salanesol, nicotine and chlorogenic acid, wherein Salanesol extraction yield 81.2%, nicotine extraction yield 94.3%, chlorogenic acid yield 93.7%.

(2) water soluble protein in alkaline aqueous solution ultrasonic and circulated extraction tobacco leaf filter residue and sugar: the tobacco leaf filter residue obtained in step (1) is placed in ultrasonic and circulated extraction device, then the pH value adding 2000mL is the NaOH aqueous solution of 8, it is 60 DEG C in temperature, ultrasonic power is ultrasonic and circulated extraction 60min under the condition of 300W, 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 is separated with chlorogenic acid: to 21.6g containing Salanesol, sherwood oil 100mL is added respectively and pH value is 3 phosphate aqueous solution 150mL in the thick cream of nicotine and chlorogenic acid, 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, column chromatography for separation is carried out again by 100 ~ 200 object silica gel, eluent is sherwood oil: the mixed solvent of ethyl acetate=9:1 (volume ratio), collect Salanesol enrichment section in elutriant, carry out concentrating to obtain 0.61g Salanesol purified, then 5mL ethanol is added, recrystallization at-15 DEG C, filter gained crystal at room temperature in vacuo drying (20 DEG C), obtain refining Salanesol 0.4g, purity testing is 91%.

The lower layer of water crossed 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 obtained to carry out concentrating to obtain the thick cream 1.78g of chlorogenic acid, then add the chlorogenic acid aqueous solution that distilled water is made into 9mg/mL and carry 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 to be volume fraction be 30% aqueous ethanolic solution, elution flow rate is 4 times of column volumes per hour, total co-elute 6 times of column volumes, with 1/2 times of column volume for recruiting unit, collect the elutriant that chlorogenic acid content is higher, concentrating under reduced pressure also carries out vacuum dehydrating at lower temperature (temperature is lower than 20 DEG C) and namely obtains chlorogenic acid 0.73g, purity 46%.

NaOH adjust ph to 12 is added after the 255mL lower floor aqueous phase that extraction into ethyl acetate obtains is concentrated to 85mL, then wet distillation is carried out, cut is collected with the receiving bottle that aqueous citric acid solution is housed in advance, and control pH in receiving bottle and be less than 3, when distillate massfraction is that 1% silicotungstic acid detects stopping distillation when sediment-free produces, now in receiving bottle, liquor capacity is about 300mL, then by the citric acid nicotine solution for vacuum concentration in receiving bottle to 100mL, about adding NaOH adjust ph to 12, with dichloromethane extraction twice, the consumption of each methylene dichloride extraction is 80mL, combined dichloromethane also carries out drying by anhydrous sodium sulphate, namely last underpressure distillation removing methylene dichloride obtains nicotine 2.8g, purity testing is 97%.

(4) separation of protein and sugar: the aqueous solution 2000mL containing protein and sugar of aqueous solution ultrasonic and circulated extraction gained in step (2) is concentrated to 300mL, then slowly stir and add cm-chitosan (molecular weight the is 50000) solution that 15mL content is 0.5wt%, leave standstill 2h at 4 DEG C after, centrifuging and taking supernatant liquid 270mL, gained protein precipitation is with after absolute ethanol washing twice, carry out cryodrying (temperature is lower than 20 DEG C) and namely obtain water soluble protein 6.5g, purity 74%.

Gained supernatant liquid continues to add 950mL ethanol and carries out alcohol precipitation, leaves standstill after the 2h time and filter at 4 DEG C, after obtaining sugared filter cake absolute ethanol washing twice, carries out low-temperature vacuum drying (temperature is lower than 20 DEG C) and namely obtains water-soluble sugar 9.3g, purity 81%.

When without prejudice to essence of the present invention and spirit, those of ordinary skill in the art may make various change or distortion according to the present invention, but these change accordingly or are out of shape the protection domain that all should belong to the claim appended by the present invention.

Claims (4)

1. the comprehensive method extracting plurality of active ingredients from tobacco leaf, is characterized in that, comprise the following steps:
(1) ethanolic soln ultrasonic and circulated extraction Salanesol, nicotine and chlorogenic acid: after tobacco leaf being carried out oven dry pulverizing, obtain tobacco leaf fragment, and be placed in circulating supersonic extractors, add ethanolic soln, carry out ultrasonic and circulated extraction, filter to obtain ethanol extract and tobacco leaf filter residue, ethanol extract is concentrated, the thick cream of Salanesol, nicotine and chlorogenic acid must be contained;
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 to be volume fraction of ethanol be 80 ~ 95% the acidic ethanol aqueous solution, pH value is 3 ~ 4, and pH value is regulated by phosphoric acid or citric acid;
(2) alkaline aqueous solution ultrasonic and circulated extraction protein and sugar: the tobacco leaf filter residue that step (1) obtains is placed in circulating supersonic extractors, adds alkaline aqueous solution, carry out ultrasonic and circulated extraction, filters containing the extracting solution of protein and sugar;
(3) Salanesol, nicotine are separated with chlorogenic acid: add sherwood oil and acidic aqueous solution to containing in the thick cream of Salanesol, nicotine and chlorogenic acid, carry out liquid-liquid two-phase extraction, get upper strata petroleum ether layer to carry out concentrating to obtain the thick cream of Salanesol, then carry out column chromatography for separation, recrystallization obtains Salanesol; After lower floor's aqueous phase is extracted with ethyl acetate, gets upper strata ethyl acetate layer and carry out concentrating 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 aqueous phase through extraction into ethyl acetate gained is concentrated, add alkali adjust ph, then wet distillation is carried out, cut is collected with the receiving flask that acidic aqueous solution is housed, obtain the nicotine salt aqueous solution, the nicotine salt aqueous solution is concentrated, adds alkali adjust ph, then extract with methylene dichloride, get dichloromethane layer and carry out concentrating to obtain nicotine;
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 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 DEG C;
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 sample 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, eluent to be volume fraction be 30% aqueous ethanolic solution, elution flow rate is 4 times of column volumes per hour;
(4) separation of protein and sugar: the extracting solution containing protein and sugar of gained in step (2) is concentrated, add protein precipitant, after leaving standstill, centrifugation supernatant liquid and protein precipitation, after protein precipitation absolute ethanol washing, carry out low-temperature vacuum drying, obtain described protein; Supernatant liquid adds ethanol and carries out alcohol precipitation, filters, obtain sugared filter cake, after sugared filter cake absolute ethanol washing, carry out low-temperature vacuum drying, obtain described sugar after leaving standstill;
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 DEG C, and the time is 2 ~ 5h; Gained protein precipitation absolute ethanol washing 2 ~ 3 times; Gained supernatant liquid adds 3 ~ 4 times that amount of alcohol is filtrate volume; Alcohol precipitation time of repose is 1 ~ 2h, and temperature is 4 ~ 20 DEG C; Gained sugar filter cake absolute ethanol washing 2 ~ 3 times.
2. the method for a kind of comprehensive extraction plurality of active ingredients from tobacco leaf according to claim 1, it is characterized in that, in the ethanolic soln ultrasonic and circulated extraction process of described step (1), ultrasonic power is 100 ~ 300W, supersound extraction temperature is 50 ~ 60 DEG C, 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.
3. the method for a kind of comprehensive extraction plurality of active ingredients from tobacco leaf according to claim 1, it is characterized in that, in the alkaline aqueous solution ultrasonic and circulated extraction process of described step (2), ultrasonic power is 100 ~ 300W, supersound extraction temperature is 50 ~ 60 DEG C, 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.
4. the method for a kind of comprehensive extraction plurality of active ingredients from tobacco leaf according to claim 1, it is characterized in that, the separating step of described nicotine comprises: carried out being concentrated into 1/3 of original volume by the lower floor's aqueous phase in step (3) after extraction into ethyl acetate, then add NaOH or KOH adjust ph to 11 ~ 13; Nicotine distillate is absorbed with citric acid solution when carrying out wet distillation nicotine, and control receiving liquid pH and be less than 3, distillation is stopped when slipping out liquid massfraction and being the silicotungstic acid detection sediment-free generation of 1%, then the nicotine salt aqueous solution is concentrated to 1/3 ~ 1/2 of original volume, add NaOH or KOH adjust ph to 11 ~ 13, with dichloromethane extraction 1 ~ 2 time, namely evaporation removing methylene dichloride obtains nicotine.
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