CN109999101A - A kind of extracting method and its application of tobacco antitumor component - Google Patents
A kind of extracting method and its application of tobacco antitumor component Download PDFInfo
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Abstract
The present invention provides the extracting method and its application of a kind of tobacco antitumor component, wherein method includes: by pulverizing to tobacco material, and room temperature extraction is successively carried out for the tobacco material after pulverizing, high temperature extracts, methanol extracts, acetone extraction four times operations, and successively use highly polar solvent purification water, methanol, acetone is as Extraction solvent, tobacco acetone extract concentrated extract is taken to carry out chromatographic isolation by preparation liquid phase, component is obtained further to separate by the most strong component of cell activity experiment screening activity, the most strong component of activity obtained again by cell activity experiment screening is the object of the invention component.This method is reproducible, and obtained antitumor component can be used for preparing anti-tumor drug.
Description
Technical field
The present invention relates to tobacco products to process extractive technique field, and in particular to a kind of tobacco antitumor component mentions
Take method and its application
Background technique
Tobacco is also referred to as mesona, is Chinese medicine traditional simply, its inorganic constituents includes water, mineral element；Organic principle
It mainly include carbohydrate, alkaloid, heterocyclic, pigment, phenols, terpene, organic acid, lipid, alcohols, aldoketones etc..Wherein,
Organic principle has antibacterial, antiviral and antitumor, antioxidant activity, removes the multiple functions such as interior free yl in tobacco.
Currently, generally using means of supercritical extraction, Soxhlet extraction, ultrasonic wave to realize the extraction to tobacco active component
Extracting mode.Using dehydrated alcohol, n-hexane, acetone, ethyl acetate, petroleum ether, methylene chloride as Extraction solvent.It is wherein low
Polar solvent such as petroleum ether, n-hexane, middle polar solvent acetone, methylene chloride, these solvents are mostly volatile, inflammable, toxic
Solvent requires height to the equipment of industrial product, personnel etc., and the content of obtained tobacco active component is few, this is just greatly limited
The further research and application of tobacco active component.
Summary of the invention
In view of the above-mentioned problems, the present invention provides the extracting method and its application of a kind of tobacco antitumor component, to mention
The extracted amount of high tobacco active component.
In a first aspect, the present invention provides a kind of extracting method of tobacco antitumor component, this method includes following
Step:
(1) it pre-processes: drying and processing being carried out to tobacco material, and the tobacco material after drying and processing is subjected to Ultramicro-powder
Broken processing obtains tobacco Ultramicro-powder；
(2) normal-temperature water mentions: Extraction solvent being added in tobacco Ultramicro-powder, extracts operation under normal temperature conditions；To mentioning
Tobacco Ultramicro-powder after extract operation carries out centrifugal treating, and collects the sediment after centrifugal treating；Extraction solvent in step (2)
For purified water；
(3) high-temperature water mentions: Extraction solvent is added in the sediment collected in step (2), in 90-100 DEG C of extraction temperature
Degree is lower to carry out concentration extraction operation；Cooling processing, centrifugal treating are carried out to the concentrated extracting solution obtained after concentration extraction operation, and
Sediment after collecting centrifugal treating；Extraction solvent in step (3) is purified water；
(4) methanol extracts: Extraction solvent is added in the sediment obtained in step (3), under 0-4 DEG C of Extracting temperature
Extraction extraction operation is carried out, centrifugal treating is carried out to Soakage extraction object, and collect the supernatant after centrifugal treating, by the supernatant of collection
It carries out rotary evaporation and drying and processing is concentrated；The methanol that Extraction solvent in step (4) is 85-100%；
(5) acetone extraction: Extraction solvent is added in the sediment obtained in step (4), under 0-4 DEG C of Extracting temperature
Extraction extraction operation is carried out, centrifugal treating is carried out to Soakage extraction object, and collect the supernatant after centrifugal treating, by the supernatant of collection
It carries out rotary evaporation and drying and processing is concentrated, obtain tobacco active component；Extraction solvent in step (5) is the third of 85-100%
Ketone；
(6) tobacco active component separates: the supernatant obtained in step (5) carries out concentration drying and processing, prepares sample
Preparation liquid phase is separated, and isolated component carries out screening active ingredients, and active component further separates；
(7) sample of the active component obtained in step (6), preparation liquid phase are separated, isolated group
Divide and carry out screening active ingredients, chooses the strongest component of activity；
(8) mass spectral analysis: preparation liquid phase separates component with anti-tumor activity in the step (7), mass spectrum sample introduction,
Obtain its potential structural formula of institute.
Wherein, the drying and processing in step (1) is to dry tobacco material placement in an oven, so that drying and processing
The water content of tobacco material afterwards is less than 5%, and drying temperature is 50-60 DEG C, baking time 4-8h；
And/or the ultramicro crushing treatment in step (1) is that the tobacco material after drying and processing is used 50-80 mesh screen
Pulverizer carry out pulverization process, and to the tobacco material after pulverization process, the pulverizer of 200-400 mesh screen is used to carry out powder
Broken processing obtains the tobacco Ultramicro-powder.
Wherein, the middle quality that Extraction solvent is added of step (2) is 2-4 times of the quality of the tobacco Ultramicro-powder；
And/or the extraction operation in step (2) refers to the tobacco Ultramicro-powder and Extraction solvent is placed in extractor,
Extraction time is 0.5-3h；
And/or the centrifugal treating in step (2) refers to: centrifuging temperature is 8-12 DEG C, centrifugation time 0.5-1.5h, is turned
Speed is 3000-6000rpm/min.
Wherein, the middle quality that Extraction solvent is added of step (3) is 1.2-2.4 times of the quality of sediment；
And/or the extraction time of the concentration extraction operation in step (3) is 0.5-1.5h；
And/or the centrifugal treating in step (3) refer to will cooling treated that the concentrated extracting solution is placed in centrifugal bottle
In, centrifuging temperature is 8-12 DEG C, centrifugation time 0.5-1.5h, revolving speed 3000-6000rpm/min.
Wherein, the middle quality that Extraction solvent is added of step (4) is 1.5-2.5 times of the quality of sediment；
And/or the Soakage extraction operation in step (4) refers to, extraction time 12-24h primary every 0.5h stirring；
And/or the centrifugal treating in step (4) refers to the Soakage extraction object is placed in centrifugal bottle, centrifuging temperature are as follows:
3-5 DEG C, centrifugation time 0.5-1.5h, revolving speed 3000-6000rpm/min；
And/or the temperature of the concentration drying and processing in step (4) is 60-70 DEG C.
Wherein, the middle quality that Extraction solvent is added of step (5) is 1.5-2.5 times of the quality of sediment；
And/or the Soakage extraction operation in step (5) refers to, extraction time 12-24h primary every 0.5h stirring；
And/or the centrifugal treating in step (5) refers to the Soakage extraction object is placed in centrifugal bottle, centrifuging temperature are as follows:
3-5 DEG C, centrifugation time 0.5-1.5h, revolving speed 3000-6000rpm/min；
And/or the temperature of the concentration drying and processing in step (5) is 60-70 DEG C.
Wherein, the C18 that the chromatographic column filler that preparation liquid phase uses in step (6) is 10 μm of diameter, size be 150mm ×
250mm, type of elution are as follows: 5% methanol-water solution isocratic elution 10min, 15% methanol-water solution isocratic elution 5min, 20%
Methanol-water solution isocratic elution 5min, 30% methanol-water solution isocratic elution 5min, 40% methanol-water solution isocratic elution
5min, 50% methanol-water solution isocratic elution 5min, 55% methanol-water solution isocratic elution 5min, 65% methanol-water solution
Isocratic elution 5min, 100% methanol isocratic elution 12min, Detection wavelength 260nm, applied sample amount 200mL, flow velocity are
600mL/min。
Wherein, the silica gel that the chromatographic column filler that preparation liquid phase uses in step (7) is 10 μm of diameter, size be 150mm ×
250mm, type of elution are as follows: type of elution is 100% n-hexane isocratic elution 5min, and 85% n-hexane-ethanol solution is isocratic to be washed
De- 15min, 70% n-hexane-ethanol solution isocratic elution 23min, 60% n-hexane-ethanol solution isocratic elution 12min, inspection
Survey wavelength is 260nm, applied sample amount 80mL, flow velocity 450mL/min.
Wherein, step (6), anti tumor activity in vitro screening refers to and inhibit typeⅡ pneumocyte growth examination in (7)
It tests.
Second aspect, the present invention provides a kind of according to extracting method described above extraction, the cigarette that separation screening obtains again
Careless active component.
The present invention have it is following the utility model has the advantages that
1, the present invention is by successively carrying out room temperature extraction, high temperature extraction, methanol extraction, acetone extraction four times to tobacco material
Operation, and successively use highly polar solvent purification water, methanol, acetone as Extraction solvent, take the concentration leaching of tobacco acetone extract
Cream carries out chromatographic isolation by preparation liquid phase, improves the extraction content of tobacco active component；
2, Extraction solvent be water, ethyl alcohol, methanol, acetone, it is low in cost；
3, the present invention can be used as anti-tumor drug exploitation by the extract that specific process extracts to tobacco material, have wide
Wealthy application prospect.Meanwhile the preparation method of tobacco extract of the invention is simple, is suitble to industrialized production.
The above description is only an overview of the technical scheme of the present invention, in order to better understand the technical means of the present invention,
And can be implemented in accordance with the contents of the specification, below with presently preferred embodiments of the present invention.
Detailed description of the invention
Fig. 1 is the flow chart of tobacco active component extracting method；
Fig. 2 is tobacco acetone extract (TA) preparation liquid phase separation chromatogram；
Fig. 3 is that tobacco acetone extract preparation liquid phase (TA-150-12) component further separates liquid chromatogram；
Fig. 4 is tobacco active component (TA-150-12-pin) to human lung cancer's A549 cell growth inhibition schematic diagram；
Fig. 5 is tobacco active component (TA-150-12-pin) ion massspectrum figure；
The above description is only an overview of the technical scheme of the present invention, in order to better understand the technical means of the present invention,
And can be implemented in accordance with the contents of the specification, below with presently preferred embodiments of the present invention.
Specific embodiment
The present invention is further described in detail with reference to embodiments.It is noted that detailed description below is all to illustrate
Property, it is intended to further instruction is provided to the present invention.Unless otherwise indicated, all scientific and technical terms that the present invention uses
With with the normally understood identical meanings of the technical field of the invention personnel.
The present invention provides a kind of extraction separation method of tobacco active component, which please refers to shown in FIG. 1
Flow chart, the extracting method may comprise steps of:
(1) it pre-processes: drying and processing being carried out to tobacco material, and the tobacco material after drying and processing is subjected to Ultramicro-powder
Broken processing obtains tobacco Ultramicro-powder；
(2) normal-temperature water mentions: Extraction solvent being added in tobacco Ultramicro-powder, extracts operation under normal temperature conditions；To mentioning
Tobacco Ultramicro-powder after extract operation carries out centrifugal treating, and collects the sediment after centrifugal treating；Extraction solvent in step (2)
For purified water；
(3) high-temperature water mentions: Extraction solvent is added in the sediment collected in step (2), in 90-100 DEG C of extraction temperature
Degree is lower to carry out concentration extraction operation；Cooling processing, centrifugal treating are carried out to the concentrated extracting solution obtained after concentration extraction operation, and
Sediment after collecting centrifugal treating；Extraction solvent in step (3) is purified water；
(4) methanol extracts: Extraction solvent is added in the sediment obtained in step (3), under 0-4 DEG C of Extracting temperature
Extraction extraction operation is carried out, centrifugal treating is carried out to Soakage extraction object, and collect the supernatant after centrifugal treating, by the supernatant of collection
It carries out rotary evaporation and drying and processing is concentrated；The methanol that Extraction solvent in step (4) is 85-100%；
(5) acetone extraction: Extraction solvent is added in the sediment obtained in step (4), under 0-4 DEG C of Extracting temperature
Extraction extraction operation is carried out, centrifugal treating is carried out to Soakage extraction object, and collect the supernatant after centrifugal treating, by the supernatant of collection
It carries out rotary evaporation and drying and processing is concentrated, obtain tobacco active component；Extraction solvent in step (5) is the third of 85-100%
Ketone；
(6) tobacco active component separates: the supernatant obtained in step (5) carries out concentration drying and processing, prepares sample
Preparation liquid phase is separated, and isolated component carries out screening active ingredients, and active component further separates；
(7) sample of the active component obtained in step (6), preparation liquid phase are separated, isolated group
Divide and carry out screening active ingredients, chooses the strongest component of activity；
(8) mass spectral analysis: preparation liquid phase separates component with anti-tumor activity in the step (7), mass spectrum sample introduction,
Obtain its potential structural formula of institute.
Extracting and developing method of the invention is further described with specific embodiment separately below.
Embodiment 1
The present invention provides a kind of extracting and developing methods of tobacco active component, comprising the following steps:
(1) it pre-processes.
30Kg tobacco material is weighed, placement is dried in an oven, and 50 DEG C of drying temperature, baking time 4h is dried to cigarette
Careless water content is taken out less than 5%；Tobacco material after taking drying and processing after being crushed using the pulverizer of 50 mesh screens, and is put into
Ultramicro crushing treatment is carried out in the pulverizer of 200 mesh screens, obtains tobacco Ultramicro-powder.
(2) normal-temperature water mentions.
It weighs 30Kg tobacco Ultramicro-powder to be added in 200L extractor, 100Kg purified water is added, stirring at normal temperature is extracted
0.5h；Stirring is transferred in centrifugal bottle after extracting, 8 DEG C of centrifugations 0.5h, revolving speed 3000rpm/min；Collect centrifugal treating
Sediment afterwards.
(3) high-temperature water mentions.
The sediment 25Kg in step (2) is weighed, is added in 100L concentration extraction tank；60Kg purified water is added, opens
Concentration extraction tank heater switch；After being heated to 90 DEG C, in 90 DEG C of at a temperature of concentration extraction 0.5h；Into concentration extraction tank collet
It is passed through recirculated water, to carry out cooling processing to concentrated extracting solution, it is made to be cooled to room temperature；Concentrated extracting solution is transferred to centrifugal bottle
Middle carry out centrifugal treating, 8 DEG C of centrifugations 0.5h, revolving speed 3000rpm/min；And collect the sediment after centrifugal treating.
(4) methanol extracts.
Sediment obtained in step (3) is added in stainless steel barrel, every barrel of 10Kg；The first of 15Kg is added in every barrel
Alcohol, the mass fraction of methanol are 92%, and Soakage extraction operates under 0 DEG C of Extracting temperature；Wherein, extraction time 12h, every
0.5h stirs substance in bucket primary；Soakage extraction object is placed in centrifugal bottle, 3 DEG C of centrifugations 1.5h, revolving speed 3000rpm/
min；The supernatant of collection is transferred to 60 DEG C of concentrations in 50L revolving, after the completion of concentration by the supernatant after collecting centrifugal treating
It is transferred in baking pan, 60 DEG C are baked into medicinal extract, and collect the sediment after centrifugal treating.
(5) acetone extraction
Sediment obtained in step (4) is added in stainless steel barrel, every barrel of 10Kg；The third of 15Kg is added in every barrel
Ketone, the mass fraction of acetone are 92%, and Soakage extraction operates under 0 DEG C of Extracting temperature；Wherein, extraction time 12h, every
0.5h stirs substance in bucket primary；Soakage extraction object is placed in centrifugal bottle, 3 DEG C of centrifugations 1.5h, revolving speed 3000rpm/
min；The supernatant of collection is transferred to 60 DEG C of concentrations in 50L revolving, after the completion of concentration by the supernatant after collecting centrifugal treating
It is transferred in baking pan, 60 DEG C are baked into medicinal extract, and tobacco acetone extract (TA), i.e. tobacco active constituent are obtained.
(6) tobacco acetone extract prepares liquid phase separation
It takes and obtains acetone extraction supernatant concentrated extract (TA) in (5), with chromatography alcohol, ultrapure water is according to methanol: water=3:2
Than column dissolution to get；(C=0.25g/mL), 0.45 μm of organic mocromembrane filter membrane is crossed, chromatography point is then carried out by preparation liquid phase
From；Mobile phase methanol and water, using gradient elution；Type of elution is that 5% methanol elutes 10min, and 15% methanol elutes 5min,
20% methanol elutes the methanol of 5min, 30% methanol 5min, 40% 5min, 50% methanol 5min, 55% methanol 5min, 65% methanol
5min, 100% methanol 12min, Detection wavelength 260nm choose activity after obtaining the screening of each group lease making anti tumor activity in vitro
It is dried in vacuo after strongest component (TA-150-12) concentrated by rotary evaporation.
Experimental result: as shown in Fig. 2, tobacco acetone extract liquid phase separation chromatogram
(7) further liquid phase separation takes step to the isolated component (TA-150-12) of tobacco acetone extract preparation liquid phase
Suddenly it obtains active component (TA-150-12) in (6) to be dissolved with dehydrated alcohol, obtaining concentration is 0.25g/mL sample, with system
Standby liquid phase is separated, and type of elution is 100% n-hexane isocratic elution, 85% n-hexane-ethanol solution isocratic elution, and 70%
N-hexane-ethanol solution isocratic elution, 60% n-hexane-ethanol solution isocratic elution, Detection wavelength 260nm obtain each group
After the screening of lease making anti tumor activity in vitro, the strongest component of activity, i.e. purpose component (TA-150-12-pin) concentrated by rotary evaporation are chosen
After be dried in vacuo.
Experimental result: as shown in figure 3, tobacco acetone extract prepares liquid phase component (TA-150-12) further separating liquid
Phase chromatogram.
Embodiment 2: inhibit the test of human lung cancer A549 cell growth
Cell culture: in 5%CO2, 37 DEG C, under saturated humidity, trained with (10%FBS+1%PS) culture medium of RPMI 1640
Human lung cancer A549 cell is supported, chooses the cell of logarithmic phase growth as experimental cell.It is diluted to after cell count with culture medium
Certain density cell suspension.
Cell growth status monitoring: cell real-time monitor is put into 37 DEG C, 5%CO2, in saturated humidity incubator.Take 8
Orifice plate, every hole are added 1640 culture medium of 150mL RPMI, are put into cell real-time monitor and walk baseline, take out after covering baseline
Octal plate, dilute good A549 cell suspension 345mL is added in every hole, until every hole cell number about 2 × 104It is a, 30min is stood, is being fallen
Whether uniform set microscopically observation cell.Drug (the tobacco active component that embodiment 1 obtains that 5mL has diluted is added in every hole
TA-150-12-pin) to final concentration of 50 μ g/mL, it is real-time to be put into cell as blank control group for the culture medium containing 1%DMSO
It is detected in monitor, detection photographs to record when finishing.
Blank control wells: being not added drug, adds 1%DMSO；
Experimental port: dosing object
Experimental result is as shown in Figure 4: top line is blank control wells, lower section line is experimental port.
According to the proliferation growth curve of A549 cell, illustrate that tobacco active component (TA-150-12-pin) of the present invention is right
A549 cell growth inhibition.
Embodiment 3: tobacco active component mass spectral analysis
Mass spectral analysis: it takes and obtains component (TA-150-12-pin) progress LC-MS analysis in step (7).Mobile phase methanol and
Water, using gradient elution, elution process is that 5% methanol to 100% methanol elutes 10min, and 100% methanol elutes 5min.Mass spectrum
Condition: positive ion mode；First mass spectrometric；Reference ion mass-to-charge ratio: 121.050873 and 922.009798 find this by analysis
Active component may be terpenoid, and experimental result is as shown in Fig. 5 figure.
The above is merely a preferred embodiment of the present invention, it is noted that for those skilled in the art
For, under the premise of not departing from core of the invention technology, improvements and modifications can also be made, these improvements and modifications are also answered
Belong to scope of patent protection of the invention, and any change in the comparable meaning and scope of claims of the present invention, all
It is considered as including in Claims scope.
Claims (10)
1. a kind of extracting method of tobacco antitumor component, which comprises the following steps:
(1) it pre-processes: drying and processing being carried out to tobacco material, and the tobacco material after drying and processing is carried out at ultramicro grinding
Reason, obtains tobacco Ultramicro-powder；
(2) normal-temperature water mentions: Extraction solvent being added in tobacco Ultramicro-powder, extracts operation under normal temperature conditions；It is grasped to extracting
Tobacco Ultramicro-powder after work carries out centrifugal treating, and collects the sediment after centrifugal treating；Extraction solvent in step (2) is pure
Change water；
(3) high-temperature water mentions: Extraction solvent is added in the sediment collected in step (2), under 90-100 DEG C of Extracting temperature
Carry out concentration extraction operation；Cooling processing, centrifugal treating are carried out to the concentrated extracting solution obtained after concentration extraction operation, and collected
Sediment after centrifugal treating；Extraction solvent in step (3) is purified water；
(4) methanol extracts: Extraction solvent is added in the sediment obtained in step (3), carries out under 0-4 DEG C of Extracting temperature
Extraction operation is extracted, centrifugal treating is carried out to Soakage extraction object, and collect the supernatant after centrifugal treating, the supernatant of collection is carried out
Drying and processing is concentrated in rotary evaporation；The methanol that Extraction solvent in step (4) is 85-100%；
(5) acetone extraction: Extraction solvent is added in the sediment obtained in step (4), is carried out under 0-4 DEG C of Extracting temperature
Extraction operation is extracted, centrifugal treating is carried out to Soakage extraction object, and collect the supernatant after centrifugal treating, the supernatant of collection is carried out
Drying and processing is concentrated in rotary evaporation, obtains tobacco active component；The acetone that Extraction solvent in step (5) is 85-100%；
(6) tobacco active component separates: the supernatant obtained in step (5) carries out concentration drying and processing, prepares sample preparation
Liquid phase is separated, and isolated component carries out screening active ingredients, and active component further separates；
(7) sample of the active component obtained in step (6), preparation liquid phase separated, isolated component into
Row screening active ingredients choose the strongest component of activity；
(8) mass spectral analysis: preparation liquid phase separates component with anti-tumor activity in step (7), and mass spectrum sample introduction obtains
Its potential structural formula of institute.
2. extracting method according to claim 1, which is characterized in that
Drying and processing in step (1) is to dry tobacco material placement in an oven, so that the tobacco after drying and processing
The water content of raw material is less than 5%, and drying temperature is 50-60 DEG C, baking time 4-8h；
And/or the ultramicro crushing treatment in step (1) is that the tobacco material after drying and processing is used the powder of 50-80 mesh screen
Broken machine carries out pulverization process, and to the tobacco material after pulverization process, is carried out at crushing using the pulverizer of 200-400 mesh screen
Reason, obtains the tobacco Ultramicro-powder.
3. extracting method according to claim 1, which is characterized in that
The quality of addition Extraction solvent is 2-4 times of the quality of the tobacco Ultramicro-powder in step (2)；
And/or the extraction operation in step (2) refers to the tobacco Ultramicro-powder and Extraction solvent is placed in extractor, extracts
Time is 0.5-3h；
And/or the centrifugal treating in step (2) refers to: centrifuging temperature is 8-12 DEG C, centrifugation time 0.5-1.5h, and revolving speed is
3000-6000rpm/min。
4. extracting method according to claim 1, which is characterized in that
The quality of addition Extraction solvent is 1.2-2.4 times of the quality of sediment in step (3)；
And/or the extraction time of the concentration extraction operation in step (3) is 0.5-1.5h；
And/or the centrifugal treating in step (3) refer to will cooling treated that the concentrated extracting solution is placed in centrifugal bottle, from
Heart temperature is 8-12 DEG C, centrifugation time 0.5-1.5h, revolving speed 3000-6000rpm/min.
5. extracting method according to claim 1, which is characterized in that
The quality of addition Extraction solvent is 1.5-2.5 times of the quality of sediment in step (4)；
And/or the Soakage extraction operation in step (4) refers to, extraction time 12-24h primary every 0.5h stirring；
And/or the centrifugal treating in step (4) refers to the Soakage extraction object is placed in centrifugal bottle, centrifuging temperature are as follows: 3-5
DEG C, centrifugation time 0.5-1.5h, revolving speed 3000-6000rpm/min；
And/or the temperature of the concentration drying and processing in step (4) is 60-70 DEG C.
6. extracting method according to claim 1, which is characterized in that
The quality of addition Extraction solvent is 1.5-2.5 times of the quality of sediment in step (5)；
And/or the Soakage extraction operation in step (5) refers to, extraction time 12-24h primary every 0.5h stirring；
And/or the centrifugal treating in step (5) refers to the Soakage extraction object is placed in centrifugal bottle, centrifuging temperature are as follows: 3-5
DEG C, centrifugation time 0.5-1.5h, revolving speed 3000-6000rpm/min；
And/or the temperature of the concentration drying and processing in step (5) is 60-70 DEG C.
7. extracting method according to claim 1, which is characterized in that
The C18 that the chromatographic column filler that preparation liquid phase uses in step (6) is 10 μm of diameter, size are 150mm × 250mm, elution
Mode are as follows: 5% methanol-water solution isocratic elution 10min, 15% methanol-water solution isocratic elution 5min, 20% methanol-water are molten
Liquid isocratic elution 5min, 30% methanol-water solution isocratic elution 5min, 40% methanol-water solution isocratic elution 5min, 50% first
Alcohol-water solution isocratic elution 5min, 55% methanol-water solution isocratic elution 5min, 65% methanol-water solution isocratic elution
5min, 100% methanol isocratic elution 12min, Detection wavelength 260nm, applied sample amount 200mL, flow velocity 600mL/min.
8. extracting method according to claim 1, which is characterized in that
The silica gel that the chromatographic column filler that preparation liquid phase uses in step (7) is 10 μm of diameter, size are 150mm × 250mm, elution
Mode are as follows: type of elution be 100% n-hexane isocratic elution 5min, 85% n-hexane-ethanol solution isocratic elution 15min,
70% n-hexane-ethanol solution isocratic elution 23min, 60% n-hexane-ethanol solution isocratic elution 12min, Detection wavelength are
260nm, applied sample amount 80mL, flow velocity 450mL/min.
9. the method according to claim 1 for extracting antitumor component from tobacco, it is characterised in that: the step
(6), in (7) anti tumor activity in vitro screening refer to carry out inhibit typeⅡ pneumocyte growth test.
10. the tobacco active component that any extracting method obtains in -9 according to claim 1.
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| CN109223897A (en) * | 2018-11-12 | 2019-01-18 | 贵州贵安精准医学研究院股份有限公司 | A kind of tobacco active component, extracting method and its application |
| CN109453260A (en) * | 2018-12-07 | 2019-03-12 | 贵州贵安精准医学研究院股份有限公司 | The extracting method and its application of tobacco antitumor component |
| CN109464536A (en) * | 2018-12-07 | 2019-03-15 | 贵州贵安精准医学研究院股份有限公司 | The extracting method and its application of tobacco antitumor component |
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| CN109223897A (en) * | 2018-11-12 | 2019-01-18 | 贵州贵安精准医学研究院股份有限公司 | A kind of tobacco active component, extracting method and its application |
| CN109453260A (en) * | 2018-12-07 | 2019-03-12 | 贵州贵安精准医学研究院股份有限公司 | The extracting method and its application of tobacco antitumor component |
| CN109464536A (en) * | 2018-12-07 | 2019-03-15 | 贵州贵安精准医学研究院股份有限公司 | The extracting method and its application of tobacco antitumor component |
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