CN106748779B - A method of extracting saturation Rosmarinic acid from tobacco - Google Patents
A method of extracting saturation Rosmarinic acid from tobacco Download PDFInfo
- Publication number
- CN106748779B CN106748779B CN201611119196.1A CN201611119196A CN106748779B CN 106748779 B CN106748779 B CN 106748779B CN 201611119196 A CN201611119196 A CN 201611119196A CN 106748779 B CN106748779 B CN 106748779B
- Authority
- CN
- China
- Prior art keywords
- extract
- tobacco
- chromatography
- thin
- eluent
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- 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
-
- 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
-
- 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
Abstract
The method that the invention discloses a kind of to extract saturation Rosmarinic acid from tobacco, it is characterised in that: this method includes following step: tobacco extract；By the water-soluble rear extraction of tobacco extract；Extract liquor is eluted in macroreticular resin chromatographic column with ethyl alcohol；It after collecting eluent, is concentrated under reduced pressure, postcapillary samples thin-layer chromatography, and the eluent containing similar component is merged；It is concentrated to dryness to obtain tobacco polar extract；Using thin layer chromatography, crude extract medicinal extract is obtained after merging the sample reduced pressure for showing brownish black spot；Crude extract medicinal extract and silica gel after mixing evenly, are separated using normal phase silica gel chromatography column, are eluted；Merge according to opposed polarity according to different Rf values by thin layer chromatography and cause fragrant substance, obtain thin extract；Normal phase column chromatography, elution are carried out to thin extract, TLC detection is segmented into A-E5 part, by B sections therein by the way that after reversed-phase silica gel column chromatography, sample introduction is separated into half preparing chromatography system；Under each chromatographic condition, retention time 7.12 minutes, the isolated monomeric compound.
Description
Technical field
The method that the present invention relates to a kind of to extract saturation Rosmarinic acid from tobacco, belongs to technical field of tobacco chemistry.
Background technique
Rosmarinic acid is a kind of natural, has stronger antioxidant activity, and antioxidant activity is better than dimension life
Plain E, caffeic acid, chlorogenic acid, folic acid etc. facilitate cell damage caused by preventing free radical, therefore reduce cancer and artery
The risk of hardening.Rosmarinic acid has stronger anti-inflammatory activity, while Rosmarinic acid also has antibacterial, antiviral and antitumor
Activity, and have and inhibit acute and chronic infection, uvioresistant, inhibit the characteristics such as elastin degradation that Rosmarinic acid has been made to become makeup
The additive of product.Currently, Rosmarinic acid has embodied its important application value in the fields such as pharmacy, food, cosmetics.
Rosmarinic acid itself is also a kind of flavor matter, and the flavor matter that needs to add in existing tobacco increases cigarette
Mouthfeel, mainly the compound is prepared in separation to the present invention from tobacco for the first time, and the flavor matter extracted from tobacco can be most
Big degree improves cigarette mouthfeel, and it was found that inherently has the substance compared with strong anti-oxidation in tobacco.
Summary of the invention
The technical problem to be solved by the present invention is providing a kind of method for extracting saturation Rosmarinic acid from tobacco, make
For tobacco essence spice additive, with overcome the deficiencies in the prior art.
Technical solution of the present invention: a method of extracting saturation Rosmarinic acid from tobacco, this method includes following steps
It is rapid:
(1) after extracting tobacco sample with ethanol water, by the way that tobacco extract is concentrated under reduced pressure to obtain；
(2) by tobacco extract it is water-soluble after, after extracting using organic solvent, extract liquor is concentrated under reduced pressure to 1/10th volumes,
Obtain extract liquor；
(3) it by after the activation of D101 macroreticular resin, is fitted into chromatographic column, pure water is rinsed to no alcohol taste, will be in previous step
Obtained extract liquor is poured into from top to down in macroreticular resin chromatographic column, then, with the ethyl alcohol of various concentration gradient to macroreticular resin
Column is eluted；
(4) after collecting eluent, eluent is concentrated under reduced pressure, progress capillary takes after being concentrated into 1/10th volumes
Sample thin-layer chromatography, development system are chloroform: methanol, and color developing agent is 5% ethanol solution of sulfuric acid, and display methods is heating colour developing, will
Eluent containing similar component merges；Reduced pressure eluent to obtain tobacco polar extract to dry；
(5) thin layer chromatography is used, solvent is chloroform: acetone, merges and shows that the sample decompression of brownish black spot is dense
It obtains causing fragrant class crude extract medicinal extract after contracting；
(6) fragrant class crude extract medicinal extract and silica gel are caused after mixing evenly, is separated using normal phase silica gel chromatography column, mobile phase
For chloroform: methanol elution gradient collects each section eluent；
(7) by thin layer chromatography, solvent is chloroform: acetone, according to different Rf values, merges according to opposed polarity and causes
Fragrant substance obtains causing the thin extract of fragrant class；
(8) normal phase column chromatography is carried out to the thin extract of tobacco, using methanol: chloroform is eluted as eluent gradient, TLC detection segmentation
For A-E5 part, after passing through reversed-phase silica gel column chromatography for B sections therein, sample introduction is separated into half preparing chromatography system；
In chromatographic condition: mobile phase is methanol: 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.
Color developing agent is 5% ethanol solution of alpha-Naphthol, colour developing in thin layer chromatography described in the step (5) and (7)
Method is heating colour developing.
The beneficial effects of the present invention are: the extraction means of each step are especially right in step 8 in present invention process
TLC detection segmentation in B section pass through reversed-phase silica gel column chromatography after, sample introduction is separated into half preparing chromatography system；In each chromatography
Under the conditions of, obtaining within retention time 7.12 minutes saturation Rosmarinic acid is and its important step retention time especially therein,
The difference that the length of retention time, even 0.1-0.2 are divided, can all lead to the variation of separation product, this is that inventor is testing
In constantly grope what repetition test obtained.
The present invention is extracted from tobacco by separation and extraction technology obtains causing fragrant substance to be saturated Rosmarinic acid, and saturation fan changes
Fragrant acid can reduce the gaseous phase free radical of cigarette as cigarette additive, and possible mechanism is that the effective component in Chinese herbal medicine blocks
Cigarette burning when free radical generation or the free radical of generation is rapidly quenched.Simultaneously as being tobacco itself
Substance will not be generated to cigarette product bad due to adding non-tobacco product substance after being added in cigarette product
The adverse effects such as smell.
Detailed description of the invention
Fig. 1 is the nuclear magnetic resonance spectroscopy (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 that the heteronuclear Multiple-Quantum Coherences of the compounds of this invention are composed；
Fig. 4 is the 1H 1H COSY spectrum 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 (the saturation Rosmarinic acid) and VC of YC-22 in the present invention；
Fig. 7 is that the compounds of this invention half prepares chromatography 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 attached drawing
Step ground detailed description.
A kind of saturation Rosmarinic acid, the saturation Rosmarinic acid have 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 method of extracting saturation Rosmarinic acid from tobacco comprising the steps of:
(1) after extracting tobacco sample with 75% ethanol water, by the way that tobacco extract is concentrated under reduced pressure to obtain；
(2) by tobacco extract water, after ultrasonic wave hydrotropy, after ethyl acetate or petroleum ether extraction, extraction is concentrated under reduced pressure
It takes liquid to 1/10th volumes, obtains extract liquor；
(3) it after D101 macroreticular resin being required activation to specifications, is fitted into the chromatographic column of appropriate size, pure water punching
It is washed till no alcohol taste, extract liquor obtained in previous step is poured into from top to down in macroreticular resin chromatographic column, then, with different dense
The ethyl alcohol (10%-100%) of degree gradient elutes macroporous resin column；
(4) after collecting more bottles of eluents, each bottle of eluent is concentrated under reduced pressure, it is laggard to be concentrated into 1/10th volumes
Row capillary samples thin-layer chromatography, and development system is chloroform: methanol, and color developing agent is 5% ethanol solution of sulfuric acid, and display methods is to add
Heat colour developing.Eluent containing similar component is merged；Reduced pressure eluent to obtain tobacco polar extract to dry；
(5) thin layer chromatography is used, solvent is chloroform: acetone (5:1), and color developing agent is 5% ethanol solution of alpha-Naphthol, is shown
Color method is heating colour developing, obtains causing fragrant class crude extract medicinal extract after merging the sample reduced pressure for showing brownish black spot；
(7) fragrant class crude extract medicinal extract and silica gel are caused after mixing evenly, is separated using normal phase silica gel chromatography column, mobile phase
For chloroform: methanol elution gradient, elution liquid proportional are 100:0-0:100；Collect each section eluent；
(8) by thin layer chromatography, solvent is chloroform: acetone (10:1), and color developing agent is 5% ethanol solution of alpha-Naphthol,
Coloration method is heating colour developing, according to different Rf values, merges according to opposed polarity and causes fragrant substance, obtain that fragrant class is caused carefully to mention
Object；
(9) normal phase column chromatography is carried out to the thin extract of tobacco, using methanol: chloroform is eluted as eluent gradient, TLC detection segmentation
For A-E5 part, after passing through gel filtration chromatography for B sections therein, sample introduction is separated into half preparing chromatography system；In color
Under spectral condition (mobile phase is methanol: water, 68:32), retention time 7.12 minutes, the isolated monomeric compound.
Antioxidant activity research
(1) preparation of DPPH stock solution accurately weighs 1,1- diphenyl -2- trinitrophenyl-hydrazine (DPPH) 0.078g, and use is anhydrous
Ethyl alcohol dissolution is settled in 100ml brown volumetric flask, is shaken up, and obtaining concentration is 2mmol/L mother liquor, 4 DEG C of preservations.Used time takes 10ml female
Liquid dilutes constant volume into 100ml volumetric flask, and obtaining concentration is 0.2mmol/L.
(2) the preparation precision of VC solution weighs VC 0.25g, water-soluble to solve mother liquid concentration 2.5mg/ml, takes mother liquor respectively
0.01ml, 0.02ml, 0.03ml, 0.05ml, 0.08ml, 0.1ml, 0.15ml are settled in 25ml volumetric flask, obtain a certain 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,
It is placed at room temperature for.2ml sample solution is drawn respectively to measure according to method under " 1.1.4 " item.
(3) test solution according to a certain concentration gradient dilution, then divides with a certain amount of separated monomeric compound is produced
2ml test solution is not taken, is measured according to method under " 1.1.4 " item.
(4) free radical scavenging activity measuring method takes 2ml various concentration test solution and 2ml 0.2mmol/L DPPH molten
Liquid is added in colorimetric cylinder, and 30min is placed at room temperature for after mixing, and A value is measured at 517nm, is measured in parallel three times, is positive right with VC
According to.Using the absorption of the feature aubergine group of DPPH solution, with after UV-VIS spectrophotometry measurement reaction for examination
The Scavenging activity that solution indicates it to organic free radical in the decline degree that 517nm absorbs.
Sample understands that rate is calculated according to following formula to DPPH:
DPPH clearance rate=1- (Ai- Aj)/A0
AiFor the average value of sample+DPPH absorbance；
AjFor the average value of sample solution absorbance；
A0For the average value of DPPH absorbance.
As a result
The antioxidation activity in vitro of 1 YC-22 of table (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 |
Being saturated Rosmarinic acid has certain antioxidation activity in vitro, which is to be prepared into for the first time using separation in tobacco
The saturation Rosmarinic acid arrived carries out anti-oxidant experiment, and demonstrating the substance that tobacco itself contains just has oxidation resistant function.
Claims (2)
1. a kind of method for extracting saturation Rosmarinic acid from tobacco, it is characterised in that: this method includes following step:
(1) after extracting tobacco sample with ethanol water, by the way that tobacco extract is concentrated under reduced pressure to obtain；
(2) by tobacco extract it is water-soluble after, after ethyl acetate or petroleum ether extraction, be concentrated under reduced pressure extract liquor to ten/one
Product, obtains extract liquor；
(3) it by after the activation of D101 macroreticular resin, is fitted into chromatographic column, pure water is rinsed to no alcohol taste, will be obtained in previous step
Extract liquor poured into macroreticular resin chromatographic column from top to down, then, with the ethyl alcohol of various concentration gradient to macroporous resin column into
Row elution；
(4) after collecting eluent, eluent is concentrated under reduced pressure, progress capillary sampling is thin after being concentrated into 1/10th volumes
Analyse layer by layer, development system is chloroform: methanol, color developing agent are 5% ethanol solution of sulfuric acid, and display methods is heating colour developing, will be contained
The eluent of similar component merges；Reduced pressure eluent to obtain tobacco polar extract to dry；
(5) thin layer chromatography is used, solvent is chloroform: acetone, is merged after showing that the sample of brownish black spot is concentrated under reduced pressure
It obtains causing fragrant class crude extract medicinal extract；
(6) fragrant class crude extract medicinal extract and silica gel are caused after mixing evenly, is separated using normal phase silica gel chromatography column, mobile phase is chlorine
Imitative: methanol elution gradient collects each section eluent；
(7) by thin layer chromatography, solvent is chloroform: acetone, according to different Rf values, merges according to opposed polarity and causes fragrant class
Substance obtains causing the thin extract of fragrant class；
(8) normal phase column chromatography is carried out to the thin extract of tobacco, using methanol: chloroform is eluted as eluent gradient, and TLC detection is segmented into A-
E5 part, by B sections therein by the way that after reversed-phase silica gel column chromatography, sample introduction is separated into half preparing chromatography system；In color
Spectral condition: mobile phase is methanol: water, volume ratio 68:32, retention time 7.12 minutes, isolated monomeric compound: described
The step of (5) solvent in chloroform: acetone volume ratio be 5:1；Chloroform in described step (7) solvent: acetone volume ratio is
10:1。
2. the method according to claim 1 for extracting saturation Rosmarinic acid from tobacco, it is characterised in that: the step
(5) and in thin layer chromatography described in (7) color developing agent is 5% ethanol solution of alpha-Naphthol, and coloration method is heating colour developing.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201611119196.1A CN106748779B (en) | 2016-12-08 | 2016-12-08 | A method of extracting saturation Rosmarinic acid from tobacco |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201611119196.1A CN106748779B (en) | 2016-12-08 | 2016-12-08 | A method of extracting saturation Rosmarinic acid from tobacco |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN106748779A CN106748779A (en) | 2017-05-31 |
| CN106748779B true CN106748779B (en) | 2019-10-15 |
Family
ID=58881223
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201611119196.1A Active CN106748779B (en) | 2016-12-08 | 2016-12-08 | A method of extracting saturation Rosmarinic acid from tobacco |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106748779B (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| 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 |
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 |
-
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 |
Also Published As
| Publication number | Publication date |
|---|---|
| CN106748779A (en) | 2017-05-31 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Çelik et al. | Determination of antioxidants by a novel on-line HPLC-cupric reducing antioxidant capacity (CUPRAC) assay with post-column detection | |
| CN106749143A (en) | A kind of method that scopolactone compound is extracted from tobacco | |
| CN102749348B (en) | Method for identifying active components in medicinal plant | |
| Nerantzaki et al. | Novel determination of the total phenolic content in crude plant extracts by the use of 1H NMR of the–OH spectral region | |
| CN107589203A (en) | A kind of method for detecting three kinds of cannabinol compounds in Chinese fiber crops simultaneously using SPE HPLC | |
| Julianti et al. | Quantification of the antiplasmodial alkaloid carpaine in papaya (Carica papaya) leaves | |
| CN110407897B (en) | Extraction method and detection method of lycium barbarum acid in russian fruits | |
| CN110632203A (en) | Synchronous and rapid detection of vitamin A and vitamin D3And vitamin E | |
| CN110632208B (en) | Detection method for main components of traditional Chinese medicine composition for clearing lung, eliminating phlegm, relieving cough and asthma | |
| CN106748779B (en) | A method of extracting saturation Rosmarinic acid from tobacco | |
| CN109674917A (en) | A kind of extraction process and its antioxidation application of response phase method optimization sedge general flavone | |
| Tesio et al. | Analytical determinations of luteolin | |
| Çiçek et al. | Quantification of diterpene acids in Copaiba oleoresin by UHPLC-ELSD and heteronuclear two-dimensional qNMR | |
| CN109503696A (en) | A kind of triterpene compound with antibacterial functions and preparation method thereof and the application in electronic cigarette | |
| CN109030664A (en) | The method for measuring the content of 7 kinds of polyphenol active ingredients in different cultivars sweet osmanthus simultaneously by HPLC-MS-MS method | |
| CN106442788B (en) | The method of discrimination of fresh tobacco leaves sample quality in a kind of tobacco metabolism group based on pigment | |
| CN107703232A (en) | A kind of detection method of content astaxanthin | |
| CN107703080A (en) | A kind of assay method of dandelion extract general flavone content | |
| CN104777256B (en) | Analysis method for simultaneously determining six polyphenol contents in flue-cured tobacco | |
| CN109521123B (en) | Application of PMP-HPLC method in identification of garden ginseng and forest ginseng | |
| CN109632781A (en) | The measuring method of anticoccidial feedstuff additive product Content of Chlorogenic Acid and coffee acid content | |
| CN103616339A (en) | Detection method for preparation containing ganoderan | |
| Avula et al. | Rapid analysis of lignans from leaves of Podophyllum peltatum L. samples using UPLC‐UV‐MS | |
| Li et al. | Simultaneous determination of fifteen polyphenols in fruit juice using ultrahigh-performance liquid chromatography-tandem mass spectrometry combining dispersive liquid-liquid microextraction | |
| CN110272459A (en) | Two kinds of noval chemical compounds and its antioxidant activity position in root of Paeonia sinjiangensis |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |