CN108689803A - A kind of method of waste tobacco leaf comprehensive utilization - Google Patents
A kind of method of waste tobacco leaf comprehensive utilization Download PDFInfo
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- CN108689803A CN108689803A CN201810683811.4A CN201810683811A CN108689803A CN 108689803 A CN108689803 A CN 108689803A CN 201810683811 A CN201810683811 A CN 201810683811A CN 108689803 A CN108689803 A CN 108689803A
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C29/00—Preparation of compounds having hydroxy or O-metal groups bound to a carbon atom not belonging to a six-membered aromatic ring
- C07C29/74—Separation; Purification; Use of additives, e.g. for stabilisation
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C29/00—Preparation of compounds having hydroxy or O-metal groups bound to a carbon atom not belonging to a six-membered aromatic ring
- C07C29/74—Separation; Purification; Use of additives, e.g. for stabilisation
- C07C29/76—Separation; Purification; Use of additives, e.g. for stabilisation by physical treatment
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C29/00—Preparation of compounds having hydroxy or O-metal groups bound to a carbon atom not belonging to a six-membered aromatic ring
- C07C29/74—Separation; Purification; Use of additives, e.g. for stabilisation
- C07C29/76—Separation; Purification; Use of additives, e.g. for stabilisation by physical treatment
- C07C29/86—Separation; Purification; Use of additives, e.g. for stabilisation by physical treatment by liquid-liquid treatment
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C67/00—Preparation of carboxylic acid esters
- C07C67/48—Separation; Purification; Stabilisation; Use of additives
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C67/00—Preparation of carboxylic acid esters
- C07C67/48—Separation; Purification; Stabilisation; Use of additives
- C07C67/58—Separation; Purification; Stabilisation; Use of additives by liquid-liquid treatment
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D401/00—Heterocyclic 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
- C07D401/02—Heterocyclic 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
- C07D401/04—Heterocyclic 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
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H1/00—Processes for the preparation of sugar derivatives
- C07H1/06—Separation; Purification
- C07H1/08—Separation; Purification from natural products
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H17/00—Compounds containing heterocyclic radicals directly attached to hetero atoms of saccharide radicals
- C07H17/04—Heterocyclic radicals containing only oxygen as ring hetero atoms
- C07H17/06—Benzopyran radicals
- C07H17/065—Benzo[b]pyrans
- C07H17/07—Benzo[b]pyran-4-ones
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- C—CHEMISTRY; METALLURGY
- C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
- C11B—PRODUCING, e.g. BY PRESSING RAW MATERIALS OR BY EXTRACTION FROM WASTE MATERIALS, REFINING OR PRESERVING FATS, FATTY SUBSTANCES, e.g. LANOLIN, FATTY OILS OR WAXES; ESSENTIAL OILS; PERFUMES
- C11B1/00—Production of fats or fatty oils from raw materials
- C11B1/10—Production of fats or fatty oils from raw materials by extracting
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- C—CHEMISTRY; METALLURGY
- C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
- C11B—PRODUCING, e.g. BY PRESSING RAW MATERIALS OR BY EXTRACTION FROM WASTE MATERIALS, REFINING OR PRESERVING FATS, FATTY SUBSTANCES, e.g. LANOLIN, FATTY OILS OR WAXES; ESSENTIAL OILS; PERFUMES
- C11B9/00—Essential oils; Perfumes
- C11B9/02—Recovery or refining of essential oils from raw materials
- C11B9/022—Refining
Abstract
The invention discloses a kind of methods of waste tobacco leaf comprehensive utilization, after being extracted to waste tobacco leaf using organic solvent, the precipitation of fragrance, chlorogenic acid and nicotine medicinal extract and rutin has been obtained using the method for solvent fractional extraction, means of purification is detached using solvent extraction, column chromatography and crystallization etc., obtain nicotine, fragrance, Salanesol, rutin and the chlorogenic acid of high-purity, it ferments to the tobacco leaf residue of effective component extracting, is prepared for microbial-bacterial fertilizer.Method provided by the invention; the higher value application of a variety of waste tobacco leaf active ingredients can be achieved; technique is simple and direct; it is at low cost; meet scale industrial production requirement, the yield and purity of active ingredient are high, and nicotine is free of in fragrance; bacterial manure is with short production cycle, viable count is high, is a kind of efficient, the comprehensive utilization of good economy performance, environment amenable tobacco leaf technology.
Description
Technical field
The present invention relates to the methods that active ingredient in tobacco leaf comprehensively utilizes, and can prepare nicotine, Salanesol, fragrance, reed simultaneously
Fourth, chlorogenic acid and microbial-bacterial fertilizer;Belong to bioengineering field.
Background technology
The tobacco planting area and yield in China are in world forefront, but due to by Cultivate administration technology and processing technology
Limitation, about 25% tobacco leaf, leftover bits and pieces are not fully utilized and by discarded during cultivation, transport and making cigarette.By this
A little waste tobacco leafs are burned or are arbitrarily abandoned, and are not only wasted, but also pollute environment.Through research, identified more than 300 in tobacco leaf
Kind compound, the wherein important compounds such as nicotine, Salanesol, fragrance, rutin and chlorogenic acid are the main components in tobacco leaf, are being changed
Work, medicine, agricultural etc. extensive application.
Contain abundant tobacco flavor material in tobacco, these flavor components are mostly low pole substance, wherein neophytadiene
It is the non-pigmented terpenoid in tobacco, the content highest in tobacco volatile fragrance matter is flavor matter important in tobacco leaf
One of, content is between 0.05-0.2% in tobacco leaf.In addition Megastigmatrienone, very little visit alcohol, palmitic acid and ethyl palmitate etc.
To causing perfume also to play an important role.Waste tobacco leaf recycles fragrance component, and main addition at present reaches reduction nicotine in cigarette
Content simultaneously, does not influence the effect of its aromatic style.
Salanesol is a kind of four sesquiterpenoids, and no optically active accounts for the 0.5-4.0% of tobacco leaf dry weight, with free state and change
It closes state to be present in tobacco leaf, compound state exists in the form of aliphatic ester.Salanesol has antibacterial anti-inflammatory and anticancer bioactive,
To in congestive heart disease therapeutic process stethemia, edema, angina pectoris have good therapeutic effect and to subacute liver it is bad
Extremely, the diseases such as acute, chronic hepatitis, duodenal ulcer, treatment scurvy, gangrenosum acne periodontitis, gastric ulcer blood coagulation have good doctor
Therapeutic effect.Salanesol can also be used to synthesize biologically active ubiquinone substance, such as ubiquinone10And vitamin K2。
Chlorogenic acid is a kind of phenylpropanoids that plant generates during aerobic respiration through shikimic acid pathway, is
The highest polyphenols of content in tobacco leaf, accounts for about the 0.5-2% of tobacco leaf dry weight, includes mainly that chlorogenic acid and its derivative are hidden green
Ortho acid, neochlorogenic acid and caffeic acid etc..Chlorogenic acid is a kind of important physiological activator, has antibacterial, antiviral, anti-swollen
Tumor, hypoglycemic cholagogic, increases the multiple pharmacological effects such as white blood cell at immunological regulation, has extensively in fields such as health products, drug and cosmetics
General application.
Nicotine belongs to binary organic weak base.The content of nicotine accounts for about the 95% of total alkaloid in tobacco leaf, the content in tobacco leaf
Generally 1-6% is the important source material for making environment friendly agricultural, can be used for producing efficient green insecticide, has low toxicity efficient, residual
The effect phase is long, to crop safety, without poisoning, insecticidal spectrum is wide, interior suction and infiltration are stronger the advantages that.
Rutin also known as rutin sophorin, rutin are a kind of flavone compounds that source is very wide, and content is about in tobacco leaf
0.5~1.5% (Hunan University's journal (natural science edition) 2005,31,616-619).Rutin has extensive bioactivity, main
To include anti-oxidizing activities, Anti-lipoid peroxide effect, the effect of citrin sample, anti-inflammatory effect, cardiovascular protection be made
With, always platelet activation effect etc., purposes is very extensive.
Because fragrance and nicotine, Salanesol, rutin and chlorogenic acid content and chemical property differ larger, extracting method
Also there is bigger difference, the extracting method of fragrance component includes mainly steam distillation, solvent extraction method and overcritical titanium dioxide
Carbon extraction method, the yield that steam distillation method extracts fragrance is higher, while because nicotine also has certain volatility, tobacco leaf middle part
Point nicotine can be also extracted, but because the condition is unfavorable for the release of plant cell wall breaking and nicotine, the yield of fragrance component also compared with
Low, it is higher that extraction obtains nicotine content in fragrance component, while while extracting needs to use vapor, and there is also extraction process energy consumptions
High disadvantage.Fragrance component can also be extracted using weak polar solvent, the solvent used includes petroleum ether, n-hexane, acetic acid second
Ester etc., the fragrance purity extracted is preferable, but yield is relatively low, and nicotine residual quantity is high.Carbon dioxide supercritical extraction method at
This height, equipment investment are big, also constrain its application in production practice.CN 101940361B disclose one kind in discarded cigarette
The method that composite plant hydrolase, cellulase and neutral proteinase extraction fragrance component are added in leaf, can improve index at
Divide the content of neophytadiene, but the enzyme added is of high cost, complex process, and also the content of neophytadiene only has in the medicinal extract obtained
The content of 1.366-1.799 μ g/g, i.e. neophytadiene only have 0.0001%-0.0002%, while this method can not integrate
Utilize the active ingredients such as nicotine, Salanesol, rutin and chlorogenic acid.
In the prior art, the extraction of nicotine is mainly water extraction, then passes through the way of distillation, ion-exchange, organic solvent
Or supercritical carbon dioxide extracting is purified.The extraction of Salanesol, generally selects weakly polar organic solvent, such as n-hexane, stone
Oily ether etc.;Then acidity alcohol solution extracts for chlorogenic acid extraction, and rutin mainly uses water or alcohol extracting.It follows that carrying simultaneously
These types of active ingredient is taken to be not easy to, have some reports, extracting method that nicotine and Salanesol extract altogether include mainly using
The yield of sulfuric acid water and acetone two-step method, nicotine and Salanesol be respectively 80.2% and 83.4% (University of Fuzhou's journal, 2008,
36,308-312) and n-hexane and 4% sodium hydroxide/n-hexane two-step method, the yield of nicotine and Salanesol is respectively 75% He
70% (Chinese Journal of Pharmaceuticals 2006,37,458-459) etc., but rutin, chlorogenic acid and fragrance can not be comprehensively utilized.
CN104151140B and CN104326912B is utilized respectively acid ethanol solution ultrasonic wave added and petroleum ether/phosphoric acid water two-phase mixture
System can chlorogenic acid extracting, Salanesol and nicotine simultaneously, but these methods cannot comprehensively utilize fragrance and rutin ingredient.And
Yield is also unsatisfactory, for example, in CN104151140B patents, the medicinal extract extract yield point of Salanesol, nicotine and chlorogenic acid
Not Zhi You 48.2%, 59.4% and 62.7%, in addition subsequently refined step, yield will be lower.What CN104326912B was obtained
The purity of chlorogenic acid is not higher than 47%, and yield is not higher than 46%, while must use ultrasonic wave added in the process, and equipment cost is high,
Amplification is difficult.In nicotine purifying, it is necessary to the method extracted using steam distillation combination dichloromethane, the energy of steam distillation
Consumption is high, the toxicity of dichloromethane is big, these factors limit it and applied in industrial practice.CN106496034A discloses one kind
Simultaneously green original is carried out using macroreticular resin from the method for tobacco leaf kind separating chlorogenic acid and rutin with water dissolution, 50 DEG C of stirring extractions
The purifying of acid and rutin has obtained the higher chlorogenic acid of purity and rutin, but yield only has 60% and 56.2% respectively, simultaneously should
Method can not comprehensively utilize nicotine, fragrance and Salanesol.CN1093540C disclose one kind from tobacco leaf simultaneously extraction nicotine
With the method for rutin, carried using water, the method for organic solvent extraction obtains nicotine, and carries out nicotine and rutin by macroreticular resin
Purifying, but because undetermined raw material in nicotine and rutin content, yield is unknown, and for nicotine and rutin, adopts
Purified with Amberlyst process, increase processing step, can also influence its yield.Li Xiaoqin etc. discloses one kind from tobacco leaf
The method for extracting Salanesol, chlorogenic acid, rutin and nicotine simultaneously obtains active ingredient using the method for ultrasonic wave assisted extraction
Extracting solution, then precipitate to obtain method separating solanesol using cooling, then other 3 kinds are obtained using fractional extraction and refined method
Active ingredient (combined extracting of tobacco main functional component and antioxidant activity research, Chinese Academy of Agricultural Sciences's academic dissertation
2015), the yield of the Salanesol of this method is higher, but because rutin solubility in low-temperature aqueous solution is poor, can be with eggplant Buddhist nun
Alcohol is precipitated together, affects rutin and obtains yield.Meanwhile filtering or centrifugation removal precipitate to obtain supernatant, when directly extracting chlorogenic acid,
Because rutin has dissolubility in ethyl acetate, and is extracted, the purity of chlorogenic acid and the yield of rutin are affected.Simultaneously as
When Salanesol detaches, being extracted without weak polar solvent, remain more impurity so that the purity of nicotine crude product is also relatively low,
Such as using more complicated these impurity obtained in method removing chlorogenic acid, nicotine, then it can reduce by two kinds of active ingredients and obtain yield.In addition
It also cannot achieve the comprehensive utilization of flavouring ingredient using this method.Although it follows that existing technology there is no method obtaining height
Nicotine, Salanesol, fragrance, rutin and the chlorogenic acid of purity simultaneously, and ensure that various effective ingredients all have higher yield.
On the other hand, a large amount of polysaccharide constituents, such as starch, cellulose, lignin and organic are contained in waste tobacco leaf
Acid and protein are such as used as waste material quilt even if can also account for 50% of waste tobacco leaf or more if the residue quality of effective component extracting
It abandons, ground source environment will be given to cause harmful effect.People also attempt to have carried out residue after waste tobacco leaf or waste tobacco leaf extraction
Higher value application such as prepares activated carbon (coal converts, 2004,27,64-66) using the residue of extraction nicotine, but activated carbon is attached
It is value added relatively low, it can not accomplish the higher value application of residue.Also have scientific worker by waste tobacco leaf or extracted effectively at
The residue divided carries out the preparation of microbial-bacterial fertilizer, but because nicotine has inhibiting effect to many microorganisms in tobacco leaf, if conduct
In the tobacco leaf or residue of microbial bacteria fertilizer ground substance, nicotine content is excessively high, then will produce microbial-bacterial fertilizer long preparation period, viable bacteria
Number is low.If CN103274784A discloses a kind of field processing waste tobacco leaf production organic fertilizer method, after waste tobacco leaf is crushed,
Be added wheat stalk, urea, horsehit etc., spontaneous fermentation prepares organic fertilizer, but because nicotine ingredient without removal, compost
Time needs 21-90 days, less efficient.
The existing technology of comprehensive analysis is it was found that major defect of the existing technology is:1) active ingredient utilizes
It is insufficient, it cannot achieve the coproduction of the nicotine, Salanesol, fragrance, rutin, chlorogenic acid and bacterial manure of waste tobacco leaf;2) active ingredient
Extraction process route is cumbersome, high energy consumption, amplification is difficult, environment is unfriendly, yield and purity are low;3) the microbial-bacterial fertilizer period is prepared
Length, viable count are low.So being badly in need of high yield in production practices, low cost prepares nicotine, eggplant Buddhist nun simultaneously from waste tobacco leaf raw material
Alcohol, fragrance, rutin, chlorogenic acid and microbial-bacterial fertilizer method, this method prepare fragrance yield and purity should all be higher, with
Improve fragrance component comprehensive utilization efficiency and added value, while the nicotine content in fragrance should also be as it is relatively low, in favor of its
Low nicotine, low tar content cigarette in apply, and nicotine, Salanesol, fragrance, rutin and chlorogenic acid content also should be compared with
Height, microbial-bacterial fertilizer is with short production cycle, viable count is high, in favor of its high-valued application.In addition prior art active ingredient utilizes
Insufficient, remaining compositional effect microorganism growth, can only could discharge after environmental protection treatment, also increase and be produced into waste liquid
This.
Invention content
It is an object of the present invention to provide it is a kind of using waste tobacco leaf simultaneously prepare high purity nicotine, Salanesol, fragrance, rutin,
The method of chlorogenic acid and microbial-bacterial fertilizer, to solve existing method active ingredient using insufficient, complex process, yield are low,
The problems such as content of nicotine is high in concrete, nicotine, Salanesol, fragrance, low rutin and chlorogenic acid purity.
For this purpose, the present invention provides a kind of method of waste tobacco leaf comprehensive utilization, include the following steps:
(1) it extracts:By after drying, crushing tobacco leaf and solution I heat flow back, hot reflux temperature is 35-80 DEG C, and the time is
10min~6 hour/time, it repeats 1-5 times, feed liquid mass ratio is 1:3~50, obtain extracting solution and tobacco leaf residue;The solution
I is the ethanol water of 70%-95% (v%);
(2) preparation of concrete and Salanesol:Remove extracting solution in ethyl alcohol, tunes pH be 1-6, with petroleum ether, just oneself
Alkane, hexamethylene, ethyl acetate, butyl acetate or kerosene are extracted, and extraction times are 1-6 times, obtain extract liquor I and raffinate
I;Extract liquor I is concentrated, dehydration, distills, obtains concrete crude product after drying;Concrete crude product is dissolved in 70%-90%
(v%) aqueous solution of ethyl alcohol, methanol, acetone or acetonitrile, is chromatographed with macroreticular resin, and uses the second of 80%-100% (v%)
The aqueous solution elution of alcohol, methanol, acetone or acetonitrile flows through component merging and is concentrated to dryness, obtains concrete fine work, elution fraction
Merging is concentrated to dryness, and is dissolved in ethyl alcohol, methanol, acetone or acetonitrile, and 10 to -20 DEG C of crystallizations obtain Salanesol;
(3) prepared by rutin:By raffinate I concentrations to be stood overnight after the 25%-80% of original volume, precipitation, centrifugation is precipitated
Or filtration method collects precipitation;And recrystallized with 70-95 DEG C of hot water or 50-75 DEG C of hot ethanol, feed liquid mass ratio is 1:10-100 is obtained
To rutin;
(4) prepared by chlorogenic acid:Raffinate in step (3) after recycling precipitation, is extracted, pH 1-6 with ethyl acetate, extraction time
Number is 1-6 times, obtains extract liquor II and raffinate II, and extract liquor II obtains chlorogenic acid medicinal extract after being concentrated to dryness, medicinal extract is dissolved in water,
Adjusting pH value is 1-6, is chromatographed with macroreticular resin, is eluted using the aqueous solution of the ethyl alcohol of 5%-70%, acetone, methanol or acetonitrile,
After eluent concentration, makes to be extracted with ethyl acetate, be concentrated to dryness, using water saturation ethyl acetate or alcohol crystal, obtain green original
Acid;
(5) prepared by nicotine:It is 8-14 by raffinate II tune pH, isometric petroleum ether, n-hexane, hexamethylene, second is added
Acetoacetic ester, butyl acetate or kerosene extraction, extraction times are 1-6 times, extract liquor III and raffinate III are obtained, by extract liquor
Nicotine sterling is obtained after III concentrations, dehydration, distillation, drying.
Beneficial effects of the present invention:The technology that active ingredient comprehensively utilizes in a kind of tobacco leaf is provided, extraction, solvent are passed through
The methods of extraction, column chromatography and crystallization, realize efficiently separating for active ingredient, and prepare the nicotine, chlorogenic acid, eggplant of high-purity
Buddhist nun's alcohol and not nicotine-containing concrete fine work utilize the residue fermenting and producing microbial bacterial agent after effective component extracting.This
The technology provided is invented, technique is simple and direct, at low cost, and the method for not using the high energy consumptions such as steam distillation in the process meets scale
Chemical industry production requirement.The yield and purity of active ingredient are high, and the waste liquid after effective component extracting can be used for microbial-bacterial fertilizer
Culture, the bacterial manure of preparation is with short production cycle, viable count is high, be a kind of good economy performance, efficient, environment amenable tobacco leaf effectively at
Divide the technology of comprehensive utilization.
Specific implementation mode
The present invention provides a kind of Comprehensive Utilization Schemes of tobacco leaf, in the technical side of tobacco leaf of the present invention comprehensive utilization
In case, each parameter can be optimized, to obtain the present invention more preferred embodiment.
One of specific implementation mode, hot reflux temperature is 45-70 DEG C in the step (1);Solution I is the second of 80-95%
Alcohol solution.
In another specific implementation mode, after the ethyl alcohol in the step (2) in removal extracting solution, pH1-4 is adjusted.
In another specific implementation mode, raffinate I is concentrated into the 25%-50% for original volume in the step (3).
In yet another embodiment, the pH value of the step (4) ethyl acetate extraction is 1-4;Carry out macroreticular resin
When chromatography, medicinal extract is dissolved in water tune pH 1-4.
In another specific implementation mode, pH value is 8-12 in the step (5).
Obviously, described in above-mentioned specific embodiment and can for obtaining the technical characteristic of more excellent technique effect
In any combination, to optimize obtained technique effect to a greater extent.The technical solution of optimization is provided as distance herein
One of:In the method for aforementioned present invention, hot reflux temperature is 45-70 DEG C in the step (1);Solution I is the second of 80-95%
Alcohol solution;After ethyl alcohol in the step (2) in removal extracting solution, pH1-4 is adjusted;
Raffinate I is concentrated into the 25%-50% for original volume in the step (3);Described step (4) ethyl acetate
The pH value of extraction is 1-4;When carrying out macroreticular resin chromatography, medicinal extract is dissolved in water tune pH 1-4;PH value is in the step (5)
8-12。
In technical scheme of the present invention, macroreticular resin used in step (2) and step (4) is nonpolarity or low pole
Macroreticular resin can separately select XDA-1,801,806, XR919C, HPD600, AB-8 or HZ802 in two steps.
More preferably in embodiment, the method for waste tobacco leaf comprehensive utilization of the present invention includes the following steps:
(1) it extracts:By the ethanol water of tobacco leaf and 90% (v%) after drying, crushing according to feed liquid mass ratio 1:10
Mixing, 50 DEG C of circumfluence distillation 30min obtain extracting solution and residue;
(2) preparation of concrete and Salanesol:The preparation of concrete and Salanesol:The ethyl alcohol in extracting solution is removed,
It is 3 to adjust pH, is extracted with petroleum ether, and extraction times are 3 times, obtain extract liquor I and raffinate I;Extract liquor I is concentrated, de-
Concrete crude product is obtained after water, distillation, drying;Concrete crude product is dissolved in 90% ethanol water, with macroreticular resin
Chromatography, and eluted using 100% ethanol solution, it flows through component merging and is concentrated to dryness, obtain concrete fine work, elution fraction
Merging is concentrated to dryness, and is dissolved in ethyl alcohol, methanol, acetone or acetonitrile, and 4 DEG C of crystallizations obtain Salanesol;
(3) prepared by rutin:It is stood overnight after being the 50% of original volume by raffinate I concentrations, precipitation, centrifugation or filtering is precipitated
Method collects precipitation;And recrystallized with 85 DEG C of hot water, feed liquid mass ratio is 1:20, obtain rutin;
(4) prepared by chlorogenic acid:Raffinate in step (3) after recycling precipitation, is extracted, pH 3, extraction times with ethyl acetate
It is 5 times, obtains extract liquor II and raffinate II, extract liquor II obtains chlorogenic acid medicinal extract after being concentrated to dryness, medicinal extract is dissolved in water, adjusts
PH value is 3, is chromatographed with macroreticular resin, is eluted using 35% ethanol water, after eluent concentration, is extracted using ethyl acetate
It takes, is concentrated to dryness, using alcohol crystal, obtain chlorogenic acid;
(5) prepared by nicotine:It is 10 by raffinate II tune pH, isometric petroleum ether extraction is added, extraction times are 3 times,
Extract liquor III and raffinate III are obtained, will obtain nicotine sterling after extract liquor III concentrations, dehydration, distillation, drying.
Based on technical solutions according to the invention and its preferred embodiment, the preparation-obtained fragrance of the present invention
In medicinal extract, using soxhlet extraction method as reference, the yield of neophytadiene is more than 75% in fragrance, and the content of neophytadiene is more than 7%,
Without nicotine;Nicotine after purification, Salanesol, rutin and chlorogenic acid quality purity be more than 90%, the quality purity of chlorogenic acid
More than 65%, the yield of nicotine is more than 70%, and the yield of Salanesol is more than 55%, and the yield of rutin is more than 68%, chlorogenic acid
Yield is more than 65%.
Further, further include the scheme comprehensively utilized to the residue after step (1) extraction in the present invention, specifically
Ground further includes the step that solid state rheology is carried out using the residue of step (1) as raw material microbe inoculation strain on the basis of the above method
Suddenly, the microorganism fungus kind is selected from bacillus subtilis (Bacillus subtilis), colloid bacillus cereus (Bacillus
Mucilaginosus), or mixtures thereof Paecilomyces lilacinus (Paecilomyces lilacinus).
Preferably, the microorganism fungus kind is bacillus subtilis (Bacillus subtilis), colloid gemma bar
Bacterium (Bacillus mucilaginosus), Paecilomyces lilacinus (Paecilomyces lilacinus) mixed bacteria;Its
In, calculated according to viable count, colloid bacillus cereus, bacillus subtilis, Paecilomyces lilacinus ratio be 1~10: 1~10:
1。
More specifically, the solid state fermentation includes the following steps:
(a) by bacillus subtilis, colloid bacillus cereus and Paecilomyces lilacinus respectively according to the inoculum concentration for meeting 1-15%
Be inoculated in seed culture medium, 30~37 DEG C, 150~200rpm shaking table cultures to OD620 be 4~6, obtain three kinds of strains kind
Sub- culture solution proportionally mixes to obtain mixed bacteria;
The seed culture medium of colloid bacillus cereus is 5~50 times of dilutions of molasses of the ammonium phosphate containing 0.2~1g/100ml;
Bacillus subtilis seed culture medium is 5~50 times of dilutions of molasses;
Paecilomyces lilacinus seed culture medium is 5~50 times of dilutions of molasses;
(b) by tobacco leaf residue and water in mass ratio 1:The mixture of 1.2-2.0 is as fermentation raw material, by the total matter of fermentation raw material
The mixed bacteria of amount inoculation 1~15% under the conditions of 10-37 DEG C, ferments 7-14 days, primary every 24~48h stirrings;
By the integration of above-mentioned scheme, multiple active components are obtained to one side high-efficiency high-quality, on the other hand, using carrying
Good microbial-bacterial fertilizer is prepared in tobacco leaf waste residue after taking, and wherein viable count is more than 8 × 108/g。
In the integration scenario of another further aspect, further includes the solvent in system and recycle to realize the energy-saving and emission-reduction of high standard
Target, raw material as microbial fermentation after the residue removal solvent of the step (1), the solvent recovery is for step (1)
Extraction;Recovered solvent is used for the extraction of step (2) and step (5) from extract liquor in the step (2);The step
(3) recovered solvent is used for the extraction of step (3) in.Further, the tobacco leaf residue is institute during raw material is fermented
Stating the water in fermentation raw material can partly be replaced using raffinate III, and it is 0.1 preferably to make the mass ratio of raffinate III and water:10
~7:3.
With reference to non-limiting embodiment, the following further describes the technical solution of the present invention, so that this field is general
The present invention, but do not limit the invention in any way is more fully understood in logical technical staff.
Unless otherwise instructed, in this specification, when addressing the mixed solution concentration of liquid and liquid, volumetric concentration is referred both to.
M% indicates mass percent.V% indicates percent by volume.
The tobacco leaf used in the present invention is that waste tobacco leaf is produced in Yunnan, will be crushed after drying tobacco, is crossed spare after 20 mesh sieve.It carries
It is analytical reagents to take solvent, and wherein petroleum ether boiling range is 30-60 DEG C, and methanol, ethyl alcohol, the acetonitrile in HPLC analyses are
Chromatography pure solvent.Nicotine, Salanesol, chlorogenic acid, rutin and neophytadiene standard items are purchased from Shanghai plumage piece biotechnology respectively to be had
Limit company and Dalian U.S. logical sequence Co., Ltd, purity are all higher than 95%, and macroreticular resin is purchased from Shanghai China of East China University of Science
Science and Technology Ltd. is shaken, Bacillus subtilis strain, colloid bacillus cereus and Paecilomyces lilacinus are that the screening of this seminar obtains.
In the present invention, the quantitative analysis method of nicotine, Salanesol and chlorogenic acid:It is all made of HPLC methods to be analyzed, chromatography
Column:Agilent 5HC-C18(2)150×4.6mm;The HPLC conditions of nicotine are:Flow velocity is 1mg/ml, mobile phase:Methanol-
Phosphate buffer (1:9), phosphate buffer solution is 0.02mol/L Na2HPO4, 0.01mol/L triethylamines adjust with phosphoric acid
PH is 6.5;Detection wavelength:260nm.Salanesol HPLC conditions are:Flow velocity is 1mg/ml, mobile phase:Methanol and ethyl alcohol (3:
2).Detection wavelength:210nm;The HPLC conditions of chlorogenic acid are:0.1% aqueous formic acid:Acetonitrile (7:3).Detection wavelength:
323nm.The HPLC conditions of rutin are:0.2% acetic acid water (A)-acetonitrile (B) gradient elution, elution requirement 0-10min, 15%-
30%B, Detection wavelength 254nm.Nicotine, Salanesol, chlorogenic acid and the standard items of rutin are diluted to various concentration, into rower
The measurement of directrix curve.Sample size analysis is carried out using obtained standard curve.
Neophytadiene quantitative analysis method:It is analyzed using GC methods, chromatographic condition is:HP-5MS columns (30m ×
0.25mm, 0.25 μm of grain size), carrier gas N2, 400KPa is pressed before column, and 50KPa is pressed before air column, 60KPa, sample introduction are pressed before hydrogen column
280 DEG C of temperature of mouth, 280 DEG C of detector temperature;1.0 μ l of sample size;Temperature program is:50 DEG C of initial temperature keeps 2min, with 6
DEG C/heating rate of min rises to 280 DEG C, keep 10min.Neophytadiene standard items are diluted to various concentration, it is bent to carry out standard
The measurement of line carries out sample size analysis.
When carrying out the purifying of Salanesol and chlorogenic acid using column chromatography, thin plate chromatography (TLC) method can be used to detect effective
The elution profile of ingredient, wherein the exhibition layer condition of Salanesol are petroleum ether:Ethyl acetate=9:1, the exhibition layer condition of chlorogenic acid be;
Butyl acetate:Methanol:Formic acid=8:1.5:1.5,5% phosphomolybdic acid ethanol solution is all made of as color developing agent.
The computational methods of yield are:
Each step yield (%)=[Active ingredient gross mass after the operation of active ingredient gross mass (mg)/this step before the operation of this step
(mg)]× 100%
Total recovery (%)=finished product kind effective ingredient gross mass/(the active constituent content * raw material matter that embodiment 1 measures
Amount) * 100%.
The assay of nicotine, Salanesol, chlorogenic acid and fragrance component in 1. tobacco leaf of embodiment
10g tobacco samples are weighed, soxhlet extraction is carried out, Extraction solvent is 90% ethyl alcohol of 70ml, and 70 DEG C of Extracting temperature carries
It is 4 hours to take the time.Parallel to be tested three times, the nicotine content mean value measured is 31.5mg/g tobacco leaves, the content of chlorogenic acid
For 6.66mg/g tobacco leaves, neophytadiene content is 1.46mg/g tobacco leaves, and rutin content is 5.0mg/g tobacco leaves, by soxhlet extraction liquid
70 DEG C of saponification 30min of NaOH to final concentration of 0.3M are added, the content for measuring Salanesol in saponification liquor is 9.1mg/g tobacco leaves, cigarette
Alkali, chlorogenic acid, Salanesol, rutin and neophytadiene content are respectively 3.15%, 0.66%, 0.91%, 0.50% and 0.15%.
The preparation 1 of 2. fragrance coarse extract of embodiment
It weighs 60g tobacco samples to be placed in 1L flask with three necks,round bottom, the ethanol water of 600mL 90%, 50 DEG C of heat is added
Reflux 30 minutes extracts 3 times, merges to obtain phegma and residue altogether.GC and HPLC is analysis shows in phegma, nicotine, eggplant
Buddhist nun's alcohol, chlorogenic acid, chlorogenic acid, neophytadiene and the extract yield of rutin are respectively 87.5%, 61.9%, 95.9%, 93.5%,
91.1% and 92%.Phegma evaporates ethyl alcohol at 35 DEG C of bath temperature with vacuum rotary evaporator, and pH is adjusted to dilute sulfuric acid
2, with isometric petroleum ether extraction 3 times, petroleum ether extraction liquid and raffinate I are obtained, after merging petroleum ether extraction liquid, 35 DEG C of water-baths
It is lower to be concentrated under reduced pressure, is dry to get concrete.GC and HPLC analysis shows, the quality purity of neophytadiene is in concrete
3.2%, the quality purity of total recovery 91.9%, Salanesol is 16.2%, and the quality purity of nicotine is 0.2%, chlorogenic acid
Quality purity is 0%, and the quality purity of rutin is 0%.
The preparation 2 of 3. fragrance coarse extract of embodiment
It weighs 60g tobacco samples to be placed in 1L flask with three necks,round bottom, the aqueous acetone solution of 600mL 80%, 50 DEG C of heat is added
Reflux 30 minutes extracts 3 times, merges to obtain phegma and residue altogether.GC and HPLC is analysis shows in phegma, nicotine, eggplant
Buddhist nun's alcohol, chlorogenic acid, chlorogenic acid, neophytadiene and the extract yield of rutin are respectively 90.0%, 51.0%, 96.0%, 88.0%
With 91%.Phegma evaporates acetone at 35 DEG C of bath temperature with vacuum rotary evaporator, and pH 3 is adjusted to dilute sulfuric acid, uses
Isometric n-hexane extraction 3 times, obtains n-hexane extract and raffinate I, after merging n-hexane extract, subtracts under 30 DEG C of water-baths
Pressure concentration, drying are to get spice extract.GC and HPLC analysis shows, the quality purity of neophytadiene is in concrete
2.8%, the quality purity of total recovery 88.9%, Salanesol is 16.0%, and the quality purity of nicotine is 0.3%, chlorogenic acid
Quality purity is 0%, and the quality purity of rutin is 0%.
The preparation of 4. rutin of embodiment
Embodiment 2 is extracted to the raffinate I after fragrance, 3 times is concentrated, is stored at room temperature 10h, there is thick yellow precipitate precipitation, mistake
The thick yellow precipitate obtained after filter and filtrate, it is 85% that rutin, which precipitates yield, according to 1:20 (w/w) are added hot water dissolving and carry out weight
Crystallization, is filtered to remove insoluble matter, and for 24 hours as room temperature environment, crystallization obtains light yellow crystal and obtains rutin after filtering, drying,
Quality purity 98.0%, total recovery 71%.
The preparation of 5. chlorogenic acid medicinal extract of embodiment
The filtrate that rutin in embodiment 4 is precipitated, makes to be extracted with ethyl acetate, and extraction time is 5 times, obtains ethyl acetate
Extract liquor and raffinate II, acetic acid ethyl acetate extract dehydration, are evaporated, can obtain chlorogenic acid medicinal extract, yield 95%, the quality of chlorogenic acid
Purity 13%, without active ingredients such as nicotine, Salanesol, neophytadiene, rutins.
The preparation 1 of 6. nicotine of embodiment
By the raffinate II after the chlorogenic acid extraction in embodiment 5, with sodium hydroxide tune pH 10, with isometric petroleum ether
Extraction 3 times obtains petroleum ether extraction liquid and raffinate III, merges petroleum ether extraction liquid, is concentrated under reduced pressure under 45 DEG C of water-baths, dry
Afterwards, obtain nicotine, HPLC analysis shows, the quality purity of nicotine is 99.0%, total recovery 79.9%, without neophytadiene,
Salanesol, chlorogenic acid and rutin.
The preparation 2 of 7. nicotine of embodiment
By the raffinate II sodium hydroxide tune pH 12 after the chlorogenic acid extraction in embodiment 5, extracted with isometric chloroform
3 times, chloroform extract liquor and raffinate III are obtained, merges chloroform extract liquor, is concentrated under reduced pressure under 35 DEG C of water-baths, after dry, obtains cigarette
Alkali, GC and HPLC analysis shows, the quality purity of nicotine is 99.2%, total recovery 79.5%, without Salanesol, chlorogenic acid,
Neophytadiene and rutin.
The preparation 1 of 8. Salanesol of embodiment
Concrete prepared by embodiment 2, according to volume ratio 1:50 ratio is dissolved in 90% ethyl alcohol, is added 0.3M's
PH is transferred to 2 by NaOH solution, mixing, 70 DEG C of saponification 30min, and filtering three times with petroleum ether extraction, obtains clear solution, by stone
Oily ether is evaporated, and obtains saponification medicinal extract, and after saponification, Salanesol saponification yield is 158%, and the Salanesol that extraction and saponification obtain is
97.9%.The medicinal extract is dissolved in the concentration of 20mg/ml in 90% ethyl alcohol, loading macroreticular resin AB-8, elution requirement 100%
Ethyl alcohol 10BV.Detect the Salanesol of each fraction with TLC HPLC methods, collect eluent, be concentrated to dryness after merging to get
To the purified of Salanesol.By the purified, 70 DEG C are dissolved in acetonitrile, and crystal is obtained by filtration, with freezing in 4 DEG C of crystallization 5h
Acetonitrile washes twice crystal, is dried crystal with vacuum desiccator.Dry crystal is still recrystallized as stated above, is used
Vacuum desiccator dries crystal.The quality purity of Salanesol is 97.9%, total recovery 78.32%.
The preparation 2 of 9. Salanesol of embodiment
Concrete prepared by embodiment 2,3, according to volume ratio 1:40 ratio is dissolved in 90% ethyl alcohol, is added 4%
Solid NaOH, mixing, pH is transferred to 2 by 70 DEG C of saponification 2h, and filtering three times with petroleum ether extraction, obtains clear solution, will just
Hexane is evaporated, and obtains saponification medicinal extract, after saponification, obtains saponification medicinal extract, saponification yield is 145%, and extraction and saponification obtain green
Ortho acid is 89.8%.The medicinal extract is dissolved in the concentration of 15mg/ml in 90% ethyl alcohol, loading macroreticular resin HPD600, elutes item
Part is 90% ethyl alcohol 2BV, 95% ethyl alcohol 12BV.The Salanesol of each fraction is detected with TLC HPLC methods, collects eluent, point
It is not concentrated to dryness to get to the purified of Salanesol.By the purified, it is dissolved in acetone, -10 DEG C of crystallizations in room-temperature dissolution
Crystal is obtained by filtration in 5h, and crystal is washed twice with the acetone of freezing, is dried crystal with vacuum desiccator.By dry crystal
It is recrystallized with acetonitrile, is dried crystal with vacuum desiccator.The quality purity of Salanesol is 95.2%, and total recovery is
61.1%.
The preparation of 10. fragrance fine work of embodiment
What macroreticular resin chromatographed in prepared by 8,9 Salanesol of embodiment flows through liquid collection merging, is evaporated, is dehydrated, can prepare
Obtain the fine work of fragrance, the quality purity of neophytadiene is 10.6%, total recovery 79.5%, without containing nicotine, chlorogenic acid and
The quality purity of rutin, Salanesol is less than 1%.
The preparation 1 of 11. chlorogenic acid of embodiment
The chlorogenic acid medicinal extract obtained in embodiment 5 is dissolved in 15% ethanol water, a concentration of 20mg/ml, pH 3 is carried out
AB-8 macroreticular resins chromatograph, and after end of the sample, with 6-8 times of column volume of 35% ethanol elution, are respectively evaporated with the detection of TLC HPLC methods
Point chlorogenic acid, collect the fraction containing chlorogenic acid, after recycling ethyl alcohol, by aqueous solution n-butyl acetate extraction 4 times, recycle acetic acid
Butyl ester obtains purifying resin object, which uses water dissolution again, adjusts pH3, with n-butyl acetate extraction 2 times, after drying and dehydrating, returns
It receives butyl acetate to be evaporated to dryness, obtains chlorogenic acid, quality purity 73%, total recovery 83.5%.50 DEG C of absolute ethyl alcohols are dissolved in,
It is cooled to room temperature, rearing crystal time is 4 hours, obtains chlorogenic acid pure product, quality purity 97.0%, total recovery 72.5%.
The preparation 2 of 12. chlorogenic acid of embodiment
The chlorogenic acid medicinal extract obtained in embodiment 5 is dissolved in water, a concentration of 10mg/ml, pH 2 carries out XDA-1 macropore trees
Lipid layer is analysed, and after end of the sample, with 6-8 times of column volume of 30% ethanol elution, the green original of each fraction is detected with TLC HPLC methods
Acid collects the fraction containing chlorogenic acid, and after recycling ethyl alcohol, aqueous solution is extracted with ethyl acetate 5 times, and recycling ethyl acetate obtains
Purifying resin object, the purified use water dissolution again, adjust pH2, are extracted with ethyl acetate 5 times, after drying and dehydrating, recycle acetic acid second
The purity that ester is evaporated to dryness chlorogenic acid is 76%.It is dissolving crystallized using water saturation ethyl acetate, obtain chlorogenic acid, quality purity
98.0%, total recovery 71.9%.
Embodiment 13. prepares the microorganism seed liquid for microbial-bacterial fertilizer
1mL bacillus subtilis bacterium solutions of activation culture 20h in LB culture mediums are taken to be inoculated in 100mL bacillus subtilises
Seed culture medium is enlarged culture, 200r/min, 37 DEG C of shaking table cultures 20h, OD620Reach 4~6, as bacillus subtilis
Seed liquor.
1mL colloid bacillus cereus bacterium solutions of activation culture 20h in LB culture mediums are taken to be inoculated in 100mL colloid bacillus cereus
Seed culture medium is enlarged culture, 200r/min, 30 DEG C of shaking table cultures 20h, OD620Reach 4~6, as colloid bacillus cereus
Seed liquor.
1mL Paecilomyces lilacinus bacterium solutions of activation culture 20h in PDA culture medium are taken to be inoculated in 100mL Paecilomyces lilacinus
Bacterium seed culture medium is enlarged culture, 200r/min, 30 DEG C of shaking table cultures 20h, OD620Reach 4~6, as Paecilomyces lilacinus
Bacterium seed liquor.
Residue prepares microbial-bacterial fertilizer -1 after embodiment 14. is extracted
Dry tobacco leaf residue 35g and water 49g are placed in the thin mouth conical flasks of 250mL, 115 DEG C of sterilizing 20min.Access embodiment
(ratio of three kinds of bacterium is 1 to the mixed bacterium seed liquor of preparation gross mass 10% in 13:1:1), the stationary culture in 30 DEG C of insulating boxs, often
It is primary every 48h stirrings.The sum of viable bacteria reached maximum value at the 8th day, added up to 1.5 × 1015Cfu/g, total spore content is the 10th
It reaches 2.5 × 1011Cfu/g, incubation time extend, and gemma number continues growing.
Residue prepares microbial-bacterial fertilizer -2 after embodiment 15. is extracted
By raffinate III, the 49g water in dry tobacco leaf residue 70g, 49g embodiment 6, it is placed in 500mL conical flasks, 115 DEG C
Sterilize 20min.It accesses and prepares bacillus subtilis seed liquor, the stationary culture in 30 DEG C of insulating boxs, every for 24 hours in embodiment 13
Stirring is primary, and the viable count of the 7th day bacillus subtilis is 9 × 108, incubation time extends, and gemma number continues growing.Because effectively
Composition extraction is more complete, and raffinate may be used for the preparation of microbial-bacterial fertilizer without processing, reduce the discharge of waste water.
1. weakly polar organic solvent extraction method of comparative example prepares fragrance
It weighing 10g tobacco samples to be placed in 1L flask with three necks,round bottom, 600mL petroleum ethers is added, 40 DEG C of heat flow back 30 minutes,
Extraction three times, merges to obtain phegma, GC and HPLC analysis shows the yield of neophytadiene and nicotine is respectively in phegma altogether
71.2% and 4.7%, less than the yield of two-component in embodiment 2 and 3 extracting solutions.It is obtained after phegma is merged distillation, drying
The yield of spice extract 0.35g, concrete are 3.5%, and the content of neophytadiene and nicotine is respectively in concrete
3.1% and 4.2%, the concrete that this method obtains, nicotine content height.
2. phegma cooling method of comparative example extraction separation nicotine, Salanesol, chlorogenic acid and rutin
(1) by after the phegma recycling ethyl alcohol in embodiment 1,4 DEG C is cooled to, precipitation is centrifuged to obtain, measures rutin in precipitation
Content be phegma in rutin total content 15%, which is dissolved with ethyl alcohol, according to volume mass than 20:1 is added NaOH,
55 DEG C of saponification 1h analyze active constituent content in saponification liquor, rutin are not detected, which is broken in saponification process
It is bad.
(2) in above-mentioned steps (1), the supernatant tune pH 2 after precipitation and separation is extracted with isometric ethyl acetate, is obtained green
Ortho acid crude product.The content of rutin is 25% of rutin total content in the supernatant after precipitation and separation in chlorogenic acid crude product, chlorogenic acid
The purity of crude product Content of Chlorogenic Acid is 8%, is far below the result (chlorogenic acid purity 13%) recorded in embodiment 5.Its reason is
Due to resolvability of the rutin in ethyl acetate, part rutin is caused to be extracted and then be mixed into chlorogenic acid crude product, serious shadow
Ring the total recovery of rutin in chlorogenic acid purity and later step.
(3) step (2) is extracted to the extraction raffinate after chlorogenic acid, it is to be precipitated 9 hours at Isosorbide-5-Nitrae DEG C to adjust pH, and acquisition is precipitated as rutin
Crude product, it is 68% that rutin, which precipitates yield, and the total recovery of rutin is 43.4%.As it was noted above, since rutin is in cooling preparation eggplant
Buddhist nun's alcohol and chlorogenic acid largely lose when extracting, and yield is caused to reduce, and yield 85% is settled well below the rutin in embodiment 4,
The result of total recovery 71%.
(4) by the extraction raffinate after rutin precipitation in step (3), it is 11 to adjust pH, and petroleum ether extraction 4 times obtains nicotine crude product, always
Yield is 80.5%, purity 66.6%.The Salanesol of comparative example detaches, and does not extract by acidic organic solvent such as embodiment 2,3
It takes, there is a large amount of low pole ingredients substance in solution, cause the purity of nicotine to be also far below and be substantially less than 99% in embodiment 6,7
Nicotine purity.
Claims (7)
1. a kind of method of waste tobacco leaf comprehensive utilization, includes the following steps:
(1) it extracts:By after drying, crushing tobacco leaf and solution I heat flow back, hot reflux temperature be 35-80 DEG C, time 10min
~6 hours/time repeat 1-5 times, and feed liquid mass ratio is 1:3~50, obtain extracting solution and tobacco leaf residue;The solution I is
The ethanol water of 70%-95%;
(2) preparation of concrete and Salanesol:The ethyl alcohol in extracting solution is removed, tune pH is 1-6, with petroleum ether, n-hexane, ring
Hexane, ethyl acetate, butyl acetate or kerosene are extracted, and extraction times are 1-6 times, obtain extract liquor I and raffinate I;Extraction
Concrete crude product is obtained after taking liquid I concentrated, dehydration, distillation, drying;Concrete crude product is dissolved in the second of 70%-90%
The aqueous solution of alcohol, methanol, acetone or acetonitrile, is chromatographed with macroreticular resin, and using the ethyl alcohol of 80%-100%, methanol, acetone or
The aqueous solution of acetonitrile elutes, and flows through component merging and is concentrated to dryness, and obtains concrete fine work, and elution fraction merging is concentrated to dryness,
It is dissolved in ethyl alcohol, methanol, acetone or acetonitrile, 10 to -20 DEG C of crystallizations obtain Salanesol;
(3) prepared by rutin:By raffinate I concentrations to be stood overnight after the 25%-80% of original volume, precipitation, centrifugation or mistake is precipitated
Filter method collects precipitation;And recrystallized with 70-95 DEG C of hot water or 50-75 DEG C of hot ethanol, feed liquid mass ratio is 1:10-100 obtains reed
Fourth;
(4) prepared by chlorogenic acid:Raffinate in step (3) after recycling precipitation, is extracted, pH 1-6, extraction times are with ethyl acetate
1-6 times, extract liquor II and raffinate II are obtained, extract liquor II obtains chlorogenic acid medicinal extract after being concentrated to dryness, medicinal extract is dissolved in water, adjusts
PH value is 1-6, is chromatographed with macroreticular resin, is eluted using the aqueous solution of the ethyl alcohol of 5%-70%, acetone, methanol or acetonitrile, elution
After liquid concentration, makes to be extracted with ethyl acetate, be concentrated to dryness, using water saturation ethyl acetate or alcohol crystal, obtain chlorogenic acid;
(5) prepared by nicotine:It is 8-14 by raffinate II tune pH, isometric petroleum ether, n-hexane, hexamethylene, acetic acid second is added
Ester, butyl acetate or kerosene extraction, extraction times are 1-6 times, obtain extract liquor III and raffinate III, and extract liquor III is dense
Nicotine sterling is obtained after contracting, dehydration, distillation, drying.
2. according to the method described in claim 1, it is characterized in that,
Hot reflux temperature is 45-70 DEG C in the step (1);Solution I is the ethanol water of 80-95%;
After ethyl alcohol in the step (2) in removal extracting solution, pH1-4 is adjusted;
Raffinate I is concentrated into the 25%-50% for original volume in the step (3);
The pH value of step (4) ethyl acetate extraction is 1-4;When carrying out macroreticular resin chromatography, medicinal extract is dissolved in water tune pH
1-4;
PH value is 8-12 in the step (5).
3. according to the method described in claim 1, including the following steps:
(1) it extracts:By after drying, crushing tobacco leaf and 90% ethanol water according to feed liquid mass ratio 1:10 mixing, 50 DEG C
Circumfluence distillation 30min, obtains extracting solution and residue;
(2) preparation of concrete and Salanesol:The preparation of concrete and Salanesol:The ethyl alcohol in extracting solution is removed, pH is adjusted
It is 3, is extracted with petroleum ether, extraction times is 3 times, obtain extract liquor I and raffinate I;Extract liquor I is concentrated, dehydration, steams
Evaporate, dry after obtain concrete crude product;Concrete crude product is dissolved in 90% ethanol water, is chromatographed with macroreticular resin,
And eluted using 100% ethanol solution, it flows through component merging and is concentrated to dryness, obtain concrete fine work, elution fraction merges
It is concentrated to dryness, is dissolved in ethyl alcohol, methanol, acetone or acetonitrile, 4 DEG C of crystallizations obtain Salanesol;
(3) prepared by rutin:It is stood overnight after being the 50% of original volume by raffinate I concentrations, precipitation is precipitated, centrifugation or filtration method are received
Collection precipitation;And recrystallized with 85 DEG C of hot water, feed liquid mass ratio is 1:20, obtain rutin;
(4) prepared by chlorogenic acid:Raffinate in step (3) after recycling precipitation, is extracted, pH 3, extraction times 5 with ethyl acetate
It is secondary, extract liquor II and raffinate II are obtained, extract liquor II obtains chlorogenic acid medicinal extract after being concentrated to dryness, medicinal extract is dissolved in water, adjusts pH
Value is 3, is chromatographed with macroreticular resin, is eluted using 35% ethanol water, after eluent concentration, makes to be extracted with ethyl acetate,
It is concentrated to dryness, using alcohol crystal, obtains chlorogenic acid;
(5) prepared by nicotine:It is 10 by raffinate II tune pH, isometric petroleum ether extraction is added, extraction times is 3 times, are obtained
Extract liquor III and raffinate III will obtain nicotine sterling after extract liquor III concentrations, dehydration, distillation, drying.
4. according to the method described in claim 1, it is characterized in that, further including being inoculated with micro- life by raw material of the residue of step (1)
Object strain carries out the step of solid state rheology, and the microorganism fungus kind is selected from bacillus subtilis, colloid bacillus cereus, pale purple quasi-
Or mixtures thereof Penicillium notatum.
5. according to the method described in claim 4, which is characterized in that the microorganism fungus kind is bacillus subtilis, glue
Matter bacillus, Paecilomyces lilacinus mixed bacteria;Wherein, it is calculated according to viable count, colloid bacillus cereus, bacillus subtilis
Bacterium, Paecilomyces lilacinus ratio be 1~10: 1~10: 1.
6. according to the method described in claim 5, it is characterized in that, the solid state fermentation includes the following steps:
(a) bacillus subtilis, colloid bacillus cereus and Paecilomyces lilacinus are inoculated with according to the inoculum concentration for meeting 1-15% respectively
In seed culture medium, 30~37 DEG C, 150~200rpm shaking table cultures to OD620 be 4~6, obtain the seed training of three kinds of strains
Nutrient solution proportionally mixes to obtain mixed bacteria;
The seed culture medium of colloid bacillus cereus is 5~50 times of dilutions of molasses of the ammonium phosphate containing 0.2~1g/100ml;
Bacillus subtilis seed culture medium is 5~50 times of dilutions of molasses;
Paecilomyces lilacinus seed culture medium is 5~50 times of dilutions of molasses;
(b) by tobacco leaf residue and water in mass ratio 1:The mixture of 1.2-2.0 is connect as fermentation raw material by fermentation raw material gross mass
Kind 1~15% mixed bacteria under the conditions of 10-37 DEG C, ferments 7-14 days, primary every 24~48h stirrings.
7. according to the method described in claim 4, it is characterized in that, as micro- after the residue removal solvent of the step (1)
The raw material of biofermentation, the solvent recovery are used for the extraction of step (1);In the step (2) from extract liquor recovered solvent
Extraction for step (2) and step (5);Recovered solvent is used for the extraction of step (3) in the step (3).
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