CN109464537A - The extracting method and its application of tobacco antitumor component - Google Patents
The extracting method and its application of tobacco antitumor component Download PDFInfo
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- 241000208125 Nicotiana Species 0.000 title claims abstract description 71
- 235000002637 Nicotiana tabacum Nutrition 0.000 title claims abstract description 71
- 238000000034 method Methods 0.000 title claims abstract description 33
- 230000000259 anti-tumor effect Effects 0.000 title claims abstract description 31
- 238000000605 extraction Methods 0.000 claims abstract description 61
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims abstract description 45
- 239000002904 solvent Substances 0.000 claims abstract description 26
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 22
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims abstract description 19
- 238000012545 processing Methods 0.000 claims abstract description 19
- 239000000463 material Substances 0.000 claims abstract description 17
- 238000003811 acetone extraction Methods 0.000 claims abstract description 13
- 206010028980 Neoplasm Diseases 0.000 claims abstract description 4
- 238000010829 isocratic elution Methods 0.000 claims description 61
- GBMDVOWEEQVZKZ-UHFFFAOYSA-N methanol;hydrate Chemical compound O.OC GBMDVOWEEQVZKZ-UHFFFAOYSA-N 0.000 claims description 41
- 239000006228 supernatant Substances 0.000 claims description 23
- 239000000284 extract Substances 0.000 claims description 21
- 238000001035 drying Methods 0.000 claims description 20
- 239000013049 sediment Substances 0.000 claims description 17
- 239000000843 powder Substances 0.000 claims description 16
- 238000010828 elution Methods 0.000 claims description 14
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 claims description 14
- 238000002360 preparation method Methods 0.000 claims description 12
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 11
- GCFHZZWXZLABBL-UHFFFAOYSA-N ethanol;hexane Chemical compound CCO.CCCCCC GCFHZZWXZLABBL-UHFFFAOYSA-N 0.000 claims description 11
- 230000008569 process Effects 0.000 claims description 11
- 238000001514 detection method Methods 0.000 claims description 10
- 239000007791 liquid phase Substances 0.000 claims description 10
- 238000012216 screening Methods 0.000 claims description 10
- 238000002390 rotary evaporation Methods 0.000 claims description 9
- 230000012010 growth Effects 0.000 claims description 7
- 238000000338 in vitro Methods 0.000 claims description 7
- 238000005119 centrifugation Methods 0.000 claims description 6
- 230000000694 effects Effects 0.000 claims description 6
- 238000004949 mass spectrometry Methods 0.000 claims description 6
- 238000001816 cooling Methods 0.000 claims description 5
- 201000007270 liver cancer Diseases 0.000 claims description 5
- 208000014018 liver neoplasm Diseases 0.000 claims description 5
- 238000010298 pulverizing process Methods 0.000 claims description 5
- 239000008213 purified water Substances 0.000 claims description 5
- 238000003756 stirring Methods 0.000 claims description 5
- 238000012360 testing method Methods 0.000 claims description 5
- 229960000935 dehydrated alcohol Drugs 0.000 claims description 4
- 229960004756 ethanol Drugs 0.000 claims description 4
- 239000000945 filler Substances 0.000 claims description 4
- 239000000401 methanolic extract Substances 0.000 claims description 4
- 238000007445 Chromatographic isolation Methods 0.000 claims description 3
- 239000002246 antineoplastic agent Substances 0.000 claims description 3
- 229940041181 antineoplastic drug Drugs 0.000 claims description 3
- 230000008859 change Effects 0.000 claims description 3
- 239000000203 mixture Substances 0.000 claims description 3
- 238000012916 structural analysis Methods 0.000 claims description 3
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 2
- 238000002701 cell growth assay Methods 0.000 claims description 2
- 210000004043 pneumocyte Anatomy 0.000 claims description 2
- 239000000741 silica gel Substances 0.000 claims description 2
- 229910002027 silica gel Inorganic materials 0.000 claims description 2
- 239000000243 solution Substances 0.000 claims 25
- 240000002853 Nelumbo nucifera Species 0.000 claims 1
- 235000006508 Nelumbo nucifera Nutrition 0.000 claims 1
- 235000006510 Nelumbo pentapetala Nutrition 0.000 claims 1
- 239000007864 aqueous solution Substances 0.000 claims 1
- OAYLNYINCPYISS-UHFFFAOYSA-N ethyl acetate;hexane Chemical compound CCCCCC.CCOC(C)=O OAYLNYINCPYISS-UHFFFAOYSA-N 0.000 claims 1
- 238000000227 grinding Methods 0.000 claims 1
- 239000003814 drug Substances 0.000 abstract description 6
- 229940079593 drug Drugs 0.000 abstract description 5
- 239000002798 polar solvent Substances 0.000 abstract description 4
- 239000007788 liquid Substances 0.000 abstract description 3
- 238000003808 methanol extraction Methods 0.000 abstract description 2
- 238000007781 pre-processing Methods 0.000 abstract description 2
- 238000000746 purification Methods 0.000 abstract description 2
- 238000000926 separation method Methods 0.000 abstract description 2
- 235000019505 tobacco product Nutrition 0.000 abstract description 2
- 238000002474 experimental method Methods 0.000 abstract 1
- 210000004027 cell Anatomy 0.000 description 19
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 12
- 239000001963 growth medium Substances 0.000 description 7
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 6
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 4
- 206010058467 Lung neoplasm malignant Diseases 0.000 description 4
- 230000010261 cell growth Effects 0.000 description 4
- 239000006285 cell suspension Substances 0.000 description 4
- 235000019441 ethanol Nutrition 0.000 description 4
- 201000005202 lung cancer Diseases 0.000 description 4
- 208000020816 lung neoplasm Diseases 0.000 description 4
- 229920006395 saturated elastomer Polymers 0.000 description 4
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 3
- 238000004113 cell culture Methods 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 238000005194 fractionation Methods 0.000 description 2
- 230000003834 intracellular effect Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012544 monitoring process Methods 0.000 description 2
- 239000003208 petroleum Substances 0.000 description 2
- 239000012071 phase Substances 0.000 description 2
- 229910001220 stainless steel Inorganic materials 0.000 description 2
- 239000010935 stainless steel Substances 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 150000003505 terpenes Chemical class 0.000 description 2
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 1
- 241001646834 Mesona Species 0.000 description 1
- 239000012980 RPMI-1640 medium Substances 0.000 description 1
- 239000006146 Roswell Park Memorial Institute medium Substances 0.000 description 1
- 238000000944 Soxhlet extraction Methods 0.000 description 1
- 150000001298 alcohols Chemical class 0.000 description 1
- 229930013930 alkaloid Natural products 0.000 description 1
- 150000003797 alkaloid derivatives Chemical class 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
- 230000000840 anti-viral effect Effects 0.000 description 1
- 230000003078 antioxidant effect Effects 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 235000019504 cigarettes Nutrition 0.000 description 1
- 239000000470 constituent Substances 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 125000000623 heterocyclic group Chemical group 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000002366 mineral element Substances 0.000 description 1
- 150000007524 organic acids Chemical class 0.000 description 1
- 150000002989 phenols Chemical class 0.000 description 1
- 239000000049 pigment Substances 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000000194 supercritical-fluid extraction Methods 0.000 description 1
- 235000007586 terpenes Nutrition 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 238000001291 vacuum drying Methods 0.000 description 1
Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/81—Solanaceae (Potato family), e.g. tobacco, nightshade, tomato, belladonna, capsicum or jimsonweed
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/10—Preparation or pretreatment of starting material
- A61K2236/15—Preparation or pretreatment of starting material involving mechanical treatment, e.g. chopping up, cutting or grinding
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/10—Preparation or pretreatment of starting material
- A61K2236/17—Preparation or pretreatment of starting material involving drying, e.g. sun-drying or wilting
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/30—Extraction of the material
- A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/30—Extraction of the material
- A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
- A61K2236/331—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using water, e.g. cold water, infusion, tea, steam distillation or decoction
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/30—Extraction of the material
- A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
- A61K2236/333—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using mixed solvents, e.g. 70% EtOH
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/50—Methods involving additional extraction steps
- A61K2236/51—Concentration or drying of the extract, e.g. Lyophilisation, freeze-drying or spray-drying
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/50—Methods involving additional extraction steps
- A61K2236/53—Liquid-solid separation, e.g. centrifugation, sedimentation or crystallization
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/50—Methods involving additional extraction steps
- A61K2236/55—Liquid-liquid separation; Phase separation
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Abstract
The present invention provides the extracting methods and its application of a kind of tobacco antitumor component, belong to tobacco product processing extractive technique field.The present invention is by pre-processing tobacco material, and the operations such as room temperature extraction, high temperature extraction, methanol extraction, acetone extraction, liquid chromatogram separation are successively carried out for pretreated tobacco material, and use highly polar solvent purification water, methanol, acetone as Extraction solvent, by using different solvents various extracting conditions, it may be implemented sufficiently to extract the tobacco active component in tobacco material, improve the extraction content of tobacco active component.Experiment finds that the purpose component has anti-tumor function, can be developed into prevent and treat the drug of tumour.
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, obtain methanol and extract supernatant concentrated extract (TMS)；Extraction solvent in step (4) is
The methanol of 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
Rotary evaporation concentration drying/frozen drying processing is carried out, acetone extraction supernatant concentrated extract/acetone extraction supernatant is obtained and freezes
Dry powder (TAS)；The acetone that Extraction solvent in step (5) is 85-100%；
(6) chromatographic isolation and composition activity screening:
(a) acetone extraction supernatant concentrated extract obtained in step (5)/acetone extraction supernatant freeze-dried powder (TAS) is taken to use
60% methanol-water solution is dissolved, and obtaining concentration is 0.25g/mL sample, is separated with preparation liquid phase, type of elution are as follows:
5% methanol-water solution isocratic elution, 15% methanol-water solution isocratic elution, 20% methanol-water solution isocratic elution, 30% first
Alcohol-water solution isocratic elution, 40% methanol-water solution isocratic elution, 50% methanol-water solution isocratic elution, 55% methanol-water
Solution isocratic elution, 65% methanol-water solution isocratic elution, 100% methanol isocratic elution, Detection wavelength 260nm are obtained each
After group lease making anti tumor activity in vitro screening, chooses active component 12 and carry out concentrated by rotary evaporation drying；
(b) active component 12 is taken to be dissolved with dehydrated alcohol, obtain concentration be 0.25g/ml sample, with preparation liquid phase into
Row separation, type of elution are 100% n-hexane isocratic elution, 85% n-hexane-ethanol solution isocratic elution, 70% n-hexane-
It is external to obtain each group lease making by ethanol solution isocratic elution, 60% n-hexane-ethanol solution isocratic elution, Detection wavelength 260nm
After antitumor activity screening, chooses active component TA-150-12-5 and carry out concentrated by rotary evaporation drying；
(7) it structural analysis: takes and obtains active component TA-150-12-5 in step (6) and be analyzed by mass spectrometry.Chromatographic condition:
5%-100% methanol-water solution 10min；100% methanol 5min；Mass Spectrometry Conditions: positive ion mode first mass spectrometric；Reference ion
Mass-to-charge ratio: 121.050873 and 922.009798.
Wherein, in step (1) in ultramicro crushing treatment be that the tobacco material after drying and processing is used into 50-80 mesh
The pulverizer of sieve carries out pulverization process, and to the tobacco material after pulverization process, carries out pulverization process using micronizer,
Obtain the tobacco Ultramicro-powder.
Wherein, the centrifugal treating in step (3) refer to will cooling treated that the concentrated extracting solution is placed in centrifugal bottle,
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.
Wherein, the Soakage extraction operation in step (4) refers to, extraction time 12-24h primary every 0.5h stirring.
Wherein, the temperature of the concentration drying and processing in step (4) is 60-70 DEG C.
Wherein, the C18, size 150mm that the chromatographic column filler that preparation liquid phase uses in step (6) (a) is 10 μm of diameter
× 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 is isocratic to be washed
De- 5min, 50% methanol-water solution isocratic elution 5min, 55% methanol-water solution isocratic elution 5min, 65% methanol-water are molten
Liquid 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 (6) (b) is 10 μm of diameter, size 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
Elute 15min, 70% n-hexane-ethanol solution isocratic elution 23min, 60% n-hexane-ethanol solution isocratic elution 1min, inspection
Survey wavelength is 260nm, applied sample amount 80mL, flow velocity 450mL/min.
Wherein, step (6), (a), anti tumor activity in vitro screening refers to and inhibit typeⅡ pneumocyte raw in (b)
Long test and inhibition human liver cancer Hep-G2 Cell Growth Assays.
Second aspect, the present invention provides a kind of according to the tobacco active component in the application prepared in anticancer drug.
The present invention have it is following the utility model has the advantages that
1, the present invention successively carries out room temperature by pre-processing to tobacco material, and for pretreated tobacco material
Extraction, high temperature are extracted, methanol extraction operates three times, and use highly polar solvent purification water, methanol as Extraction solvent, by adopting
With different solvents various extracting conditions, it is ensured that realize the tobacco active component in tobacco material and sufficiently extract, mention
The extraction content of high tobacco active component；
2, Extraction solvent be water, ethyl alcohol isometric(al), 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
Shown in FIG. 1 is the flow chart for the tobacco active component that the embodiment of the present invention 1 obtains；
Shown in Fig. 2 is the tobacco acetone extract chromatographic fractionation figure that the embodiment of the present invention 1 (6) (a) obtains；
Shown in Fig. 3 is 12 chromatographic fractionation figure of tobacco active component that the embodiment of the present invention 1 (6) (b) obtains；
Shown in Fig. 4 is the tobacco active component TA-150-12-5 Mass Spectrometer Method ion figure that the embodiment of the present invention 1 obtains；
Shown in fig. 5 is that the tobacco active component TA-150-12-5 that the embodiment of the present invention 1 obtains is thin to human lung cancer A549
Intracellular growth inhibits schematic diagram；
Shown in fig. 6 is that the tobacco active component TA-150-12-5 that the embodiment of the present invention 1 obtains is thin to human liver cancer Hep-G2
Intracellular growth inhibits schematic diagram；
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 extracting method of tobacco active component, which please refers to process shown in FIG. 1
Figure, the extracting method may comprise steps of:
Embodiment 1: the extraction of tobacco antitumor component
1, the extraction step of tobacco antitumor component is as follows:
(1) it pre-processes.
30Kg tobacco material is weighed, placement is dried in an oven, and 60 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 micronizer, 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 4000rpm/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 4000rpm/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 4000rpm/
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 methanolic extract (TMS) is obtained.
(5) acetone extraction.
It will be added in stainless steel barrel in sediment obtained in step (4), every barrel of 10kg；Every barrel of addition 15kg acetone,
The mass fraction of acetone is 95%, and extraction extraction operation is carried out under 4 DEG C of Extracting temperature, wherein extraction time 12h, often
Substance in bucket is stirred every 0.5h primary；Soakage extraction object is placed in centrifugal bottle, 3 DEG C of centrifugations 1.5h, revolving speed 4000rpm/
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 (TAS) is obtained.
(6) chromatographic isolation and composition activity screening.
(a) acetone extraction supernatant concentrated extract (TAS) obtained in step (5) is taken to be carried out with 60% methanol-water solution molten
Solution, obtaining concentration is 0.25g/mL sample, is separated with preparation liquid phase, type of elution are as follows: 5% methanol-water solution is isocratic to be washed
De- 10min, 15% methanol-water solution isocratic elution 5min, 20% methanol-water solution isocratic elution 5min, 30% methanol-water are molten
Liquid isocratic elution 5min, 40% methanol-water solution isocratic elution 5min, 50% methanol-water solution isocratic elution 5min, 55% first
Alcohol-water solution isocratic elution 5min, 65% methanol-water solution isocratic elution 5min, 100% methanol isocratic elution 12min, detection
Wavelength is 260nm, applied sample amount 200mL, flow velocity 600mL/min, is as a result swollen as shown in Fig. 2, obtaining each group lease making and resisting in vitro
After tumor activity screening, choose vacuum drying after the strongest component concentrated by rotary evaporation of activity obtain active component 12 do separate in next step it is pure
Change.
(b) it takes and obtains active component 12 in step (6) (a) and dissolved with dehydrated alcohol, obtaining concentration is 0.25g/mL
Sample is separated, type of elution with preparation liquid phase are as follows: type of elution is 100% n-hexane isocratic elution 5min, 85% just oneself
Alkane-ethanol solution isocratic elution 15min, 70% n-hexane-ethanol solution isocratic elution 23min, 60% n-hexane-ethanol solution
Isocratic elution 12min, Detection wavelength 260nm, applied sample amount 80mL, flow velocity 450mL/min, as a result as shown in figure 3, obtaining
After the screening of each group lease making anti tumor activity in vitro, the object of the invention antitumor component TA-150-12-5 is obtained.
(7) it structural analysis: takes and obtains active component TA-150-12-5 in step (6) (b) and be analyzed by mass spectrometry.Chromatography
Condition: 5%-100% methanol-water solution 10min；100% methanol 5min；Mass Spectrometry Conditions: positive ion mode first mass spectrometric；Reference
Ion mass-to-charge ratio: 121.050873 and 922.009798 may be terpenoid according to the active component is analyzed.
Embodiment 2: tobacco active component inhibits the test of human tumour A549 cell growth
Blank control wells: being not added drug, adds final concentration of 1% DMSO；
Experimental port: add 1 step of embodiment (6) (b) resulting antitumor component
Cell culture:
In 5%CO2, 37 DEG C, under saturated humidity, with RPMI 1640 (10%FBS+1%PS) culture medium culture human lung cancer
A549 cell chooses the cell of logarithmic phase growth as experimental cell.About 5.8 ten thousand every millis are diluted to culture medium after cell count
The cell suspension risen.
Cell growth status monitoring:
Cell real-time monitor is put into 5%CO2, in 37 DEG C of saturated humidity incubators.8 orifice plates are taken, 150 μ L are added in every hole
1640 culture medium of RPMI, is put into cell real-time monitor and walks baseline, takes out octal plate after covering baseline, it is dilute good that every hole is added
345 μ L of A549 cell suspension, until every hole cell number about 2 × 104It is a, 30min is stood, cell is observed under inverted microscope is
It is no uniform.Drug (the tobacco active component that embodiment 1 obtains) the extremely final concentration of 50 μ g/mL that 5 μ L have diluted is added in every hole, contains
The culture medium of 1%DMSO is put into cell real-time monitor and detects as blank control group, and detection photographs to record when finishing.It is real
It is as shown in Figure 5 to test result, the results showed that: the tobacco antitumor component TA-150-12-5 that the present invention extracts is in 50 μ g/
The basic growth for inhibiting human lung cancer cell A549 can be reached when mL.
Embodiment 3: inhibit the test of human liver cancer Hep-G2 cell growth
Blank control wells: being not added drug, adds final concentration of 1% DMSO；
Experimental port: add 1 step of embodiment (6) (b) resulting antitumor component
Cell culture:
In 5%CO2, 37 DEG C, under saturated humidity, with DMEM (10%FBS, 1%PS) culture medium culture human lung cancer Hep-G2
Cell chooses the cell of logarithmic phase growth as experimental cell.About 5.8 ten thousand every milliliter is diluted to culture medium after cell count
Cell suspension.
Cell growth status monitoring: cell real-time monitor is put into 5%CO2, in 37 DEG C of saturated humidity incubators.Take eight
Orifice plate, every hole are added 150 μ LDMEM culture mediums, are put into cell real-time monitor and walk baseline, take out octal plate after covering baseline,
The 345 μ L of Hep-G2 cell suspension diluted is added in every hole, until every hole cell number about 4 × 104It is a, 30min is stood, it is aobvious being inverted
Whether micro- microscopic observation cell is uniform.Drug (the tobacco active component that embodiment 1 obtains) that 5 μ L have diluted is added to end in every hole
Concentration is 50 μ g/mL, and the culture medium containing 1%DMSO is put into cell real-time monitor and detects, detected as blank control group
It is photographed to record when complete.Experimental result is as shown in fig. 6, top line is blank control wells, lower section line is experimental port.The result shows that: this
The tobacco antitumor component TA-150-12-5 that invention extraction obtains can reach basic in 50 μ g/mL and inhibit human liver cancer thin
The growth of born of the same parents Hep-G2.
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, obtains methanol and extracts supernatant concentrated extract (TMS)；Extraction solvent in step (4) is 85-
100% methanol；
(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/frozen drying processing is concentrated in rotary evaporation, obtains acetone extraction supernatant concentrated extract/acetone extraction supernatant freeze-dried powder
(TAS)；The acetone that Extraction solvent in step (5) is 85-100%；
(6) chromatographic isolation and composition activity screening:
(a) acetone extraction supernatant concentrated extract obtained in step (5)/60% first of acetone extraction supernatant freeze-dried powder (TAS) is taken
Alcohol-water solution is dissolved, and obtaining concentration is 0.25g/mL sample, is separated with preparation liquid phase, type of elution are as follows: 5% first
Alcohol-water solution isocratic elution, 15% methanol-water solution isocratic elution, 20% methanol-water solution isocratic elution, 30% methanol-water
Solution isocratic elution, 40% methanol-water solution isocratic elution, 50% methanol-water solution isocratic elution, 55% methanol-water solution
Isocratic elution, 65% methanol-water solution isocratic elution, 100% methanol isocratic elution, Detection wavelength 260nm obtain each component
After anti tumor activity in vitro screens, chooses active component 12 and carry out concentrated by rotary evaporation drying；
(b) active component 12 is taken to be dissolved with dehydrated alcohol, obtaining concentration is 0.25g/ml sample, is divided with preparation liquid phase
From type of elution is 100% n-hexane isocratic elution, 85% n-hexane-ethanol solution isocratic elution, 70% n-hexane-ethyl alcohol
It is anti-swollen in vitro to obtain each group lease making by solution isocratic elution, 60% n-hexane-ethanol solution isocratic elution, Detection wavelength 260nm
After tumor activity screening, chooses active component 5 (being referred to as TA-150-12-5 below) and carry out concentrated by rotary evaporation drying；
(7) it structural analysis: takes and obtains active component TA-150-12-5 in step (6) and be analyzed by mass spectrometry.Chromatographic condition: 5%-
100% methanol-water solution 10min；100% methanol 5min；Mass Spectrometry Conditions: positive ion mode first mass spectrometric；Reference ion matter lotus
Than: 121.050873 and 922.009798.
2. the method according to claim 1 for extracting antitumor component from tobacco, it is characterised in that: the step
(1) ultramicro crushing treatment in is that the tobacco material after drying and processing is carried out powder using the pulverizer of 50-80 mesh screen
Broken processing, and to the tobacco material after pulverization process, pulverization process is carried out using micronizer, obtains the tobacco ultra micro
Powder.
3. the method according to claim 1 for extracting antitumor component from tobacco, it is characterised in that: the step
(3) centrifugal treating in refer to will cooling treated that the concentrated extracting solution is placed in centrifugal bottle, centrifuging temperature is 8-12 DEG C,
Centrifugation time is 0.5-1.5h, revolving speed 3000-6000rpm/min.
4. the method according to claim 1 for extracting antitumor component from tobacco, it is characterised in that: the step
(4) quality of addition Extraction solvent is 1.5-2.5 times of the quality of sediment in.
5. the method according to claim 1 for extracting antitumor component from tobacco, it is characterised in that: the step
(4) the Soakage extraction operation in refers to, extraction time 12-24h primary every 0.5h stirring.
6. the method according to claim 1 for extracting antitumor component from tobacco, it is characterised in that: the step
(4) temperature of the concentration drying and processing in is 60-70 DEG C.
7. the method according to claim 1 for extracting antitumor component from tobacco, it is characterised in that: the step
(6) C18 that the chromatographic column filler that preparation liquid phase uses in (a) is 10 μm of diameter, size are 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 etc.
Degree elution 5min, 30% methanol-water solution isocratic elution 5min, 40% methanol-water solution isocratic elution 5min, 50% methanol-
Aqueous 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. the method according to claim 1 for extracting antitumor component from tobacco, it is characterised in that: the step
(6) silica gel that the chromatographic column filler that preparation liquid phase uses in (b) is 10 μm of diameter, size are 150mm × 250mm, type of elution
Are as follows: type of elution is 100% n-hexane isocratic elution 5min, and 85% n-hexane-ethanol solution isocratic elution 15min, 70% just
Hexane-EtOAc solution isocratic elution 23min, 60% n-hexane-ethanol solution isocratic elution 1min, Detection wavelength 260nm, on
Sample amount is 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), (a), anti tumor activity in vitro screening refers to and inhibit typeⅡ pneumocyte growth test and inhibit human liver cancer in (b)
Hep-G2 Cell Growth Assays.
10. the antitumor component described in claim 1 extracted from tobacco answering in the preparation of antitumor drugs
With.
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