CN106748779A - A kind of method that saturation Rosmarinic acid is extracted from tobacco - Google Patents
A kind of method that saturation Rosmarinic acid is extracted from tobacco Download PDFInfo
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- CN106748779A CN106748779A CN201611119196.1A CN201611119196A CN106748779A CN 106748779 A CN106748779 A CN 106748779A CN 201611119196 A CN201611119196 A CN 201611119196A CN 106748779 A CN106748779 A CN 106748779A
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- GMILNBIETOPRPK-MRXNPFEDSA-N OC([C@@H](Cc(cc1O)ccc1O)OC(CCc(cc1)cc(O)c1O)=O)=O Chemical compound OC([C@@H](Cc(cc1O)ccc1O)OC(CCc(cc1)cc(O)c1O)=O)=O GMILNBIETOPRPK-MRXNPFEDSA-N 0.000 description 1
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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
- C07C67/56—Separation; Purification; Stabilisation; Use of additives by solid-liquid treatment; by chemisorption
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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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- Organic Chemistry (AREA)
- Manufacture Of Tobacco Products (AREA)
- Fats And Perfumes (AREA)
- Medicines Containing Plant Substances (AREA)
Abstract
The invention discloses a kind of method that saturation Rosmarinic acid is extracted from tobacco, it is characterised in that：The method includes following step：Tobacco extract；By the water-soluble rear extraction of tobacco extract；Extract is eluted in macroreticular resin chromatographic column with ethanol；After collecting eluent, it is concentrated under reduced pressure, postcapillary sampling thin-layer chromatography merges the eluent containing similar component；It is concentrated under reduced pressure into dry tobacco polar extract；Using TLC, merging shows that the sample of brownish black spot obtains crude extract medicinal extract after concentrated under reduced pressure；After crude extract medicinal extract stirs with silica gel, separated using normal phase silica gel chromatography post, eluted；By TLC, according to different Rf values, merge according to opposed polarity and cause fragrant class material, obtain thin extract；Normal phase column chromatography is carried out to thin extract, is eluted, TLC detections are segmented into A E5 parts, by B sections therein by after reversed-phase silica gel column chromatography, being separated in sample introduction to half preparing chromatography system；Under each chromatographic condition, retention time 7.12 minutes, the isolated monomeric compound.
Description
Technical field
The present invention relates to a kind of method that saturation Rosmarinic acid is extracted from tobacco, belong to technical field of tobacco chemistry.
Background technology
Rosmarinic acid is a kind of natural, and with stronger antioxidation activity, its antioxidation activity is better than dimension life
Plain E, caffeic acid, chlorogenic acid, folic acid etc., contribute to the cell damage for preventing free radical from causing, therefore reduce cancer and artery
The risk of hardening.Rosmarinic acid has stronger anti-inflammatory activity, while Rosmarinic acid also has antibacterial, antiviral, antineoplastic
Activity, and there is suppression acute and chronic infection, uvioresistant, the characteristics such as elastin degradation that suppress turn into Rosmarinic acid and make up
The additive of product.At present, Rosmarinic acid has embodied its important application value in the fields such as pharmacy, food, cosmetics.
Rosmarinic acid is also in itself a kind of flavor matter, in existing tobacco the need for add flavor matter and increase cigarette
Mouthfeel, the present invention is mainly separated from tobacco and prepares the compound first, and the flavor matter extracted from tobacco can be most
Big degree improves cigarette mouthfeel, and it was found that inherently have the material compared with strong anti-oxidation in tobacco.
The content of the invention
The technical problem to be solved in the present invention is：A kind of method that saturation Rosmarinic acid is extracted from tobacco is provided, its work
It is tobacco essence perfume additive, to overcome the deficiencies in the prior art.
Technical scheme：A kind of method that saturation Rosmarinic acid is extracted from tobacco, the method includes following steps
Suddenly：
(1) after extracting tobacco sample with ethanol water, by the tobacco extract that is concentrated under reduced pressure to obtain；
(2) by tobacco extract it is water-soluble after, using organic solvent extract after, be concentrated under reduced pressure extract to 1/10th volumes,
Obtain extract；
(3) after D101 macroreticular resins are activated, it is fitted into chromatographic column, pure water is rinsed to without alcohol taste, by previous step
The extract for obtaining is poured into macroreticular resin chromatographic column from top to down, then, with the ethanol of various concentrations gradient to macroreticular resin
Post is eluted；
(4) after collecting eluent, eluent is concentrated under reduced pressure, being concentrated into after 1/10th volumes carries out capillary and take
Sample thin-layer chromatography, development system is chloroform：Methyl alcohol, developer is 5% ethanol solution of sulfuric acid, and display methods develops the color for heating, will
Eluent containing similar component is merged；The eluent that is concentrated under reduced pressure to obtain tobacco polar extract to dry；
(5) TLC is used, solvent is chloroform：Acetone, merging shows that the sample decompression of brownish black spot is dense
Obtain causing fragrant class crude extract medicinal extract after contracting；
(6) after causing fragrant class crude extract medicinal extract to be stirred with silica gel, separated using normal phase silica gel chromatography post, mobile phase
It is chloroform：Methanol elution gradient, collects each several part eluent；
(7) by TLC, solvent is chloroform：Acetone, according to different Rf values, merges according to opposed polarity and causes
Fragrant class material, obtains causing the thin extract of fragrant class；
(8) normal phase column chromatography is carried out to the thin extract of tobacco, with methyl alcohol：Chloroform is eluted for eluent gradient, TLC detection segmentations
It is A-E5 part, by B sections therein by after reversed-phase silica gel column chromatography, being separated in sample introduction to half preparing chromatography system；
In chromatographic condition：Mobile phase is methyl alcohol:Water, volume ratio 68:32, retention time 7.12 minutes, the isolated monomeric compound.
Chloroform in described step (5) solvent：Acetone volume ratio is 5:1.
Chloroform in described step (7) solvent：Acetone volume ratio is 10:1.
Developer is the ethanol solution of alpha-Naphthol 5%, colour developing in TLC described in described step (5) and (7)
Method develops the color for heating.
The beneficial effects of the invention are as follows：In present invention process, the extraction means of each step are especially right in step 8
TLC detection segmentation in B sections by after reversed-phase silica gel column chromatography, being separated in sample introduction to half preparing chromatography system；In each chromatogram
Under the conditions of, retention time obtains saturation Rosmarinic acid for 7.12 minutes is and its important step, especially retention time therein,
The difference that the length of retention time, even 0.1-0.2 are divided, can all cause the change of separation product, and this is inventor in experiment
In constantly grope what repetition test was obtained.
The present invention is extracted from tobacco by separation and extraction technology and obtains causing fragrant class material saturation Rosmarinic acid, and saturation fan change
Fragrant acid can reduce the gaseous phase free radical of cigarette as cigarette additive, and its possible mechanism is that the active ingredient in Chinese herbal medicine is blocked
The cigarette generation of free radical or the free radical to generating in burning are rapidly quenched.Simultaneously as being tobacco itself
Material, in added to cigarette product after, will not due to addition non-tobacco product class material and to cigarette product produce it is bad
The harmful effect such as smell.
Brief description of the drawings
Fig. 1 is the proton nmr spectra (1H-NMR) of the compounds of this invention；
Fig. 2 is the carbon-13 nmr spectra (13C-NMR) of the compounds of this invention；
Fig. 3 is composed for the heteronuclear Multiple-Quantum Coherences of the compounds of this invention；
Fig. 4 is the 1H 1H COSY spectrums of the double quantum filterings of the compounds of this invention；
Fig. 5 is the compounds of this invention heteronuclear polysaccharide Correlated Spectroscopy；
Fig. 6 is the antioxidation activity in vitro figure of YC-22 (saturation Rosmarinic acid) and VC in the present invention；
Fig. 7 is that the preparative chromatography of the compounds of this invention half analyzes testing result figure.
Specific embodiment
To make the object, technical solutions and advantages of the present invention clearer, the present invention is made into one below in conjunction with accompanying drawing
Step ground is described in detail.
A kind of saturation Rosmarinic acid, the saturation Rosmarinic acid has following structural formula：
White powder (CD3OD), C18H18O8Exact Mass:362.10Molecular Weight:362.33m/z:
362.10 (100.0%), 363.10 (19.8%), 364.11 (1.9%), 364.10 (1.6%) Elemental Analysis:
C,59.67；H,5.01；O,35.33.
A kind of method that saturation Rosmarinic acid is extracted from tobacco, comprises the steps of：
(1) after extracting tobacco sample with 75% ethanol water, by the tobacco extract that is concentrated under reduced pressure to obtain；
(2) by tobacco extract water, after ultrasonic wave hydrotropy, using ethyl acetate or petroleum ether extraction after, be concentrated under reduced pressure extraction
Liquid to 1/10th volumes is taken, extract is obtained；
(3) after D101 macroreticular resins being required into activation to specifications, it is fitted into the chromatographic column of appropriate size, pure water punching
It is washed till without alcohol taste, the extract that will be obtained in previous step is poured into macroreticular resin chromatographic column from top to down, it is then, dense with difference
The ethanol (10%-100%) for spending gradient is eluted to macroporous resin column；
(4) after collecting many bottles of eluents, each bottle of eluent is concentrated under reduced pressure, is concentrated into 1/10th volumes laggard
Row capillary samples thin-layer chromatography, and development system is chloroform：Methyl alcohol, developer is 5% ethanol solution of sulfuric acid, and display methods is to add
Heat colour developing.Eluent containing similar component is merged；The eluent that is concentrated under reduced pressure to obtain tobacco polar extract to dry；
(5) TLC is used, solvent is chloroform：Acetone (5:1), developer is the ethanol solution of alpha-Naphthol 5%, is shown
Color method develops the color for heating, and merging shows that the sample of brownish black spot obtains causing fragrant class crude extract medicinal extract after concentrated under reduced pressure；
(7) after causing fragrant class crude extract medicinal extract to be stirred with silica gel, separated using normal phase silica gel chromatography post, mobile phase
It is chloroform：Methanol elution gradient, wash-out liquid proportional is 100:0-0:100；Collect each several part eluent；
(8) by TLC, solvent is chloroform：Acetone (10:1), developer is the ethanol solution of alpha-Naphthol 5%,
Coloration method develops the color for heating, according to different Rf values, merges according to opposed polarity and causes fragrant class material, obtains causing fragrant class carefully to carry
Thing；
(9) normal phase column chromatography is carried out to the thin extract of tobacco, with methyl alcohol：Chloroform is eluted for eluent gradient, TLC detection segmentations
It is A-E5 part, by B sections therein by after gel filtration chromatography, being separated in sample introduction to half preparing chromatography system；In color
(mobile phase is methyl alcohol under spectral condition:Water, 68:32), retention time 7.12 minutes, the isolated monomeric compound.
Antioxidation activity is studied
(1) preparation of DPPH storing solutions accurately weighs 1,1- diphenyl -2- trinitrophenyl-hydrazine (DPPH) 0.078g, with anhydrous
Ethanol dissolving is settled in 100ml brown volumetric flasks, is shaken up, and obtains concentration for 2mmol/L mother liquors, 4 DEG C of preservations.It is female that used time takes 10ml
Liquid dilutes constant volume in 100ml volumetric flasks, obtaining concentration for 0.2mmol/L.
(2) the preparation precision of VC solution weighs VC 0.25g, and water dissolves obtain mother liquid concentration 2.5mg/ml, mother liquor is taken respectively
0.01ml, 0.02ml, 0.03ml, 0.05ml, 0.08ml, 0.1ml, 0.15ml are settled in 25ml volumetric flasks, obtain finite concentration
Gradient 0.001mg/L, 0.002mg/L, 0.003mg/L, 0.005mg/L, 0.008mg/L, 0.01mg/L, 0.015mg/L shake up,
Room temperature is placed.2ml sample solutions are drawn respectively to be determined according to method under " 1.1.4 " item.
(3) need testing solution according to finite concentration gradient dilution, then divides with a certain amount of separated monomeric compound is produced
2ml test solutions are not taken, are determined according to method under " 1.1.4 " item.
(4) to take 2ml various concentrations need testing solution molten with 2ml 0.2mmol/L DPPH for free radical scavenging activity assay method
Liquid is added in colorimetric cylinder, and room temperature places 30min after mixing, and A values are determined at 517nm, and parallel determination three times is right as the positive with VC
According to.Using the absorption of the feature aubergine group of DPPH solution, determine reacted for examination with UV-VIS spectrophotometry
Solution represents its Scavenging activity to organic free radical in the decline degree that 517nm absorbs.
Sample understands that rate is calculated according to below equation to DPPH：
DPPH clearance rates=1- (Ai- Aj)/A0
AiIt is the average value of sample+DPPH absorbances；
AjIt is the average value of sample solution absorbance；
A0It is the average value of DPPH absorbances.
As a result
The antioxidation activity in vitro of the YC-22 of table 1 (saturation Rosmarinic acid) and VC
| Sample | Linear regression | R2 | IC50/(mg·mL-1) |
| YC-22 | Y=363.46x-9.941 | 0.9979 | 0.1649 |
| VC | Y=6573.2x-3.1645 | 0.9996 | 0.0081 |
Saturation Rosmarinic acid has certain antioxidation activity in vitro, and the experiment is to be prepared into using separation in tobacco first
To saturation Rosmarinic acid carry out anti-oxidant experiment, demonstrating the material that tobacco contains in itself just has oxidation resistant function.
Claims (4)
1. it is a kind of from tobacco extract saturation Rosmarinic acid method, it is characterised in that：The method includes following step：
(1) after extracting tobacco sample with ethanol water, by the tobacco extract that is concentrated under reduced pressure to obtain；
(2) by tobacco extract it is water-soluble after, using organic solvent extract after, be concentrated under reduced pressure extract to 1/10th volumes, obtain
Extract；
(3) after D101 macroreticular resins are activated, it is fitted into chromatographic column, pure water is rinsed to without alcohol taste, will be obtained in previous step
Extract pour into from top to down in macroreticular resin chromatographic column, then, macroporous resin column is entered with the ethanol of various concentrations gradient
Row wash-out；
(4) after collecting eluent, eluent is concentrated under reduced pressure, is concentrated into after 1/10th volumes that to carry out capillary sampling thin
Analyse layer by layer, development system is chloroform：Methyl alcohol, developer is 5% ethanol solution of sulfuric acid, and display methods develops the color for heating, will contain
The eluent of similar component is merged；The eluent that is concentrated under reduced pressure to obtain tobacco polar extract to dry；
(5) TLC is used, solvent is chloroform：Acetone, after merging shows that the sample of brownish black spot is concentrated under reduced pressure
Obtain causing fragrant class crude extract medicinal extract；
(6) after causing fragrant class crude extract medicinal extract to be stirred with silica gel, separated using normal phase silica gel chromatography post, mobile phase is chlorine
It is imitative：Methanol elution gradient, collects each several part eluent；
(7) by TLC, solvent is chloroform：Acetone, according to different Rf values, merges according to opposed polarity and causes fragrant class
Material, obtains causing the thin extract of fragrant class；
(8) normal phase column chromatography is carried out to the thin extract of tobacco, with methyl alcohol：Chloroform is eluted for eluent gradient, and TLC detections are segmented into A-
E5 part, by B sections therein by after reversed-phase silica gel column chromatography, being separated in sample introduction to half preparing chromatography system；In color
Spectral condition：Mobile phase is methyl alcohol:Water, volume ratio 68:32, retention time 7.12 minutes, the isolated monomeric compound.
2. it is according to claim 1 from tobacco extract saturation Rosmarinic acid method, it is characterised in that：Described step
(5) chloroform in solvent：Acetone volume ratio is 5:1.
3. it is according to claim 1 from tobacco extract saturation Rosmarinic acid method, it is characterised in that：Described step
(7) chloroform in solvent：Acetone volume ratio is 10:1.
4. it is according to claim 1 from tobacco extract saturation Rosmarinic acid method, it is characterised in that：Described step
(5) developer is the ethanol solution of alpha-Naphthol 5% in the TLC and described in (7), and coloration method develops the color for heating.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201611119196.1A CN106748779B (en) | 2016-12-08 | 2016-12-08 | A method of extracting saturation Rosmarinic acid from tobacco |
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| CN201611119196.1A CN106748779B (en) | 2016-12-08 | 2016-12-08 | A method of extracting saturation Rosmarinic acid from tobacco |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108017515A (en) * | 2017-12-18 | 2018-05-11 | 中国烟草总公司郑州烟草研究院 | A kind of method for isolating and purifying ladanum Diterpenoids from bulbus in tobacco |
| CN113277947A (en) * | 2021-06-04 | 2021-08-20 | 安徽中烟工业有限责任公司 | Monomer spice 3- (2-hydroxyphenyl) propionic acid-2 hydroxypropyl ester for cigarette and synthetic method and application thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102675105A (en) * | 2012-04-24 | 2012-09-19 | 云南烟草科学研究院 | Phenolic compound in tobacco as well as preparation method and application thereof |
| CN102850219A (en) * | 2012-09-28 | 2013-01-02 | 中北大学 | Method for extracting rosmarinic acid from folia perillae acutae |
| CN102952018A (en) * | 2011-08-17 | 2013-03-06 | 湖北中烟工业有限责任公司 | Purification method of antioxidant rosmarinic acid for tobacco |
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2016
- 2016-12-08 CN CN201611119196.1A patent/CN106748779B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102952018A (en) * | 2011-08-17 | 2013-03-06 | 湖北中烟工业有限责任公司 | Purification method of antioxidant rosmarinic acid for tobacco |
| CN102675105A (en) * | 2012-04-24 | 2012-09-19 | 云南烟草科学研究院 | Phenolic compound in tobacco as well as preparation method and application thereof |
| CN102850219A (en) * | 2012-09-28 | 2013-01-02 | 中北大学 | Method for extracting rosmarinic acid from folia perillae acutae |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108017515A (en) * | 2017-12-18 | 2018-05-11 | 中国烟草总公司郑州烟草研究院 | A kind of method for isolating and purifying ladanum Diterpenoids from bulbus in tobacco |
| CN108017515B (en) * | 2017-12-18 | 2021-06-01 | 中国烟草总公司郑州烟草研究院 | Method for separating and purifying labdanum diterpenoid components in tobacco |
| CN113277947A (en) * | 2021-06-04 | 2021-08-20 | 安徽中烟工业有限责任公司 | Monomer spice 3- (2-hydroxyphenyl) propionic acid-2 hydroxypropyl ester for cigarette and synthetic method and application thereof |
| CN113277947B (en) * | 2021-06-04 | 2023-10-03 | 安徽中烟工业有限责任公司 | 3- (2-hydroxyphenyl) propionic acid-2-hydroxypropyl ester as cigarette monomer spice and synthesis method and application thereof |
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