CN109999102A - 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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- CN109999102A CN109999102A CN201910167621.1A CN201910167621A CN109999102A CN 109999102 A CN109999102 A CN 109999102A CN 201910167621 A CN201910167621 A CN 201910167621A CN 109999102 A CN109999102 A CN 109999102A
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- 241000208125 Nicotiana Species 0.000 title claims abstract description 81
- 235000002637 Nicotiana tabacum Nutrition 0.000 title claims abstract description 81
- 238000000034 method Methods 0.000 title claims abstract description 36
- 230000000259 anti-tumor effect Effects 0.000 title claims abstract description 16
- 238000000605 extraction Methods 0.000 claims abstract description 99
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims abstract description 72
- 239000002904 solvent Substances 0.000 claims abstract description 40
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims abstract description 32
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 28
- 239000007791 liquid phase Substances 0.000 claims abstract description 25
- 239000000284 extract Substances 0.000 claims abstract description 22
- 239000000463 material Substances 0.000 claims abstract description 19
- 238000002360 preparation method Methods 0.000 claims abstract description 19
- 238000012216 screening Methods 0.000 claims abstract description 13
- 230000000694 effects Effects 0.000 claims abstract description 9
- 238000003811 acetone extraction Methods 0.000 claims abstract description 8
- 238000010298 pulverizing process Methods 0.000 claims abstract description 6
- 239000000401 methanolic extract Substances 0.000 claims abstract description 5
- 238000012545 processing Methods 0.000 claims description 32
- 238000001035 drying Methods 0.000 claims description 29
- 238000010829 isocratic elution Methods 0.000 claims description 28
- 239000013049 sediment Substances 0.000 claims description 27
- 239000000843 powder Substances 0.000 claims description 21
- 239000006228 supernatant Substances 0.000 claims description 20
- GBMDVOWEEQVZKZ-UHFFFAOYSA-N methanol;hydrate Chemical compound O.OC GBMDVOWEEQVZKZ-UHFFFAOYSA-N 0.000 claims description 15
- 238000005119 centrifugation Methods 0.000 claims description 12
- 238000010828 elution Methods 0.000 claims description 12
- 230000008569 process Effects 0.000 claims description 11
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 claims description 10
- 238000001514 detection method Methods 0.000 claims description 9
- GCFHZZWXZLABBL-UHFFFAOYSA-N ethanol;hexane Chemical compound CCO.CCCCCC GCFHZZWXZLABBL-UHFFFAOYSA-N 0.000 claims description 9
- 238000002390 rotary evaporation Methods 0.000 claims description 8
- 238000003756 stirring Methods 0.000 claims description 8
- 239000008213 purified water Substances 0.000 claims description 7
- 239000004480 active ingredient Substances 0.000 claims description 6
- 238000001816 cooling Methods 0.000 claims description 6
- 230000012010 growth Effects 0.000 claims description 6
- 238000001819 mass spectrum Methods 0.000 claims description 5
- 238000010183 spectrum analysis Methods 0.000 claims description 5
- 239000000945 filler Substances 0.000 claims description 4
- 238000000338 in vitro Methods 0.000 claims description 4
- 201000007270 liver cancer Diseases 0.000 claims description 4
- 208000014018 liver neoplasm Diseases 0.000 claims description 4
- 238000012360 testing method Methods 0.000 claims description 4
- 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
- 230000008859 change Effects 0.000 claims description 2
- 239000007788 liquid Substances 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
- 238000000227 grinding Methods 0.000 claims 1
- 239000002994 raw material Substances 0.000 claims 1
- 239000002798 polar solvent Substances 0.000 abstract description 4
- 238000007445 Chromatographic isolation Methods 0.000 abstract description 2
- 239000002246 antineoplastic agent Substances 0.000 abstract description 2
- 229940041181 antineoplastic drug Drugs 0.000 abstract description 2
- 238000000746 purification Methods 0.000 abstract description 2
- 238000002474 experimental method Methods 0.000 abstract 2
- 210000004027 cell Anatomy 0.000 description 20
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 12
- 230000010261 cell growth Effects 0.000 description 8
- 239000001963 growth medium Substances 0.000 description 8
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 6
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 6
- 230000005764 inhibitory process Effects 0.000 description 6
- 239000003814 drug Substances 0.000 description 5
- 238000005191 phase separation Methods 0.000 description 5
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 4
- 206010058467 Lung neoplasm malignant Diseases 0.000 description 4
- 239000006285 cell suspension Substances 0.000 description 4
- 229940079593 drug Drugs 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
- 150000002576 ketones Chemical class 0.000 description 3
- 239000012071 phase Substances 0.000 description 3
- 238000000926 separation method Methods 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 2
- 238000004113 cell culture Methods 0.000 description 2
- 238000004587 chromatography analysis Methods 0.000 description 2
- 235000019504 cigarettes Nutrition 0.000 description 2
- 239000000470 constituent Substances 0.000 description 2
- 229960000935 dehydrated alcohol Drugs 0.000 description 2
- 238000010586 diagram Methods 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
- 230000035755 proliferation Effects 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
- 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
- 239000006071 cream Substances 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 229960004756 ethanol Drugs 0.000 description 1
- 125000000623 heterocyclic group Chemical group 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 238000002386 leaching Methods 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 238000004895 liquid chromatography mass spectrometry Methods 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 238000003808 methanol extraction 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
- 150000003505 terpenes Chemical class 0.000 description 1
- 235000007586 terpenes Nutrition 0.000 description 1
- 235000019505 tobacco product Nutrition 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- -1 triterpene compound Chemical class 0.000 description 1
- 229910021642 ultra pure water Inorganic materials 0.000 description 1
- 239000012498 ultrapure water Substances 0.000 description 1
Classifications
-
- 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
-
- 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
-
- 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
-
- 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
- A61K2236/51—Concentration or drying of the extract, e.g. Lyophilisation, freeze-drying or spray-drying
-
- 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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- Health & Medical Sciences (AREA)
- Natural Medicines & Medicinal Plants (AREA)
- Life Sciences & Earth Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Veterinary Medicine (AREA)
- Medicinal Chemistry (AREA)
- Public Health (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Organic Chemistry (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Engineering & Computer Science (AREA)
- Alternative & Traditional Medicine (AREA)
- Biotechnology (AREA)
- Botany (AREA)
- Medical Informatics (AREA)
- Microbiology (AREA)
- Mycology (AREA)
- Epidemiology (AREA)
- Medicines Containing Plant Substances (AREA)
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
Process extractive technique field the present invention relates to tobacco product, a kind of extracting method of tobacco antitumor component and
It is applied.
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, a kind of extracting method of tobacco antitumor component, to improve mentioning for tobacco active component
Taken amount.
In a first aspect, this method includes following step the present invention provides a kind of extraction separation method of tobacco active component
It is rapid:
(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.
Preferably, the drying and processing in step (1) is to dry tobacco material placement in an oven, so that at drying
The water content of tobacco material after reason 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.
Preferably, 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.
Preferably, 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.
Preferably, 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.
Preferably, 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.
Preferably, 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。
Preferably, the silica gel that the chromatographic column filler that preparation liquid phase uses in step (7) 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
15min, 70% n-hexane-ethanol solution isocratic elution 23min, 60% n-hexane-ethanol solution isocratic elution 12min are eluted,
Detection wavelength is 260nm, applied sample amount 80mL, flow velocity 450mL/min.
Preferably, the step (6), anti tumor activity in vitro screening refers to and carries out inhibition typeⅡ pneumocyte in (7)
Growth test and inhibition human liver cancer Hep-G2 Cell Growth Assays.
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 prepares liquid phase component (TA-150-12) and further separates liquid chromatogram；
Fig. 4 is tobacco active component (TA-150-12-8) to human lung cancer's A549 cell growth inhibition schematic diagram；
Fig. 5 is tobacco active component (TA-150-12-8) to human liver cancer Hep-G2 cell growth inhibition schematic diagram；
Fig. 6 is tobacco active component (TA-150-12-8) 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), the organic mocromembrane filter membrane of 0.45um 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) tobacco acetone extraction ingredient prepares liquid phase separation component (TA-150-12) further liquid phase separation
It takes and obtains active component (TA-150-12) in step (6) and dissolved with dehydrated alcohol, obtaining concentration is 0.25g/
ML sample is separated with preparation liquid phase, and type of elution is 100% n-hexane isocratic elution, 85% n-hexane-ethanol solution etc.
Degree elution, 70% n-hexane-ethanol solution isocratic elution, 60% n-hexane-ethanol solution isocratic elution, Detection wavelength are
260nm after obtaining the screening of each group lease making anti tumor activity in vitro, chooses the strongest component of activity, i.e. purpose component (TA-150-
12-8) it is dried in vacuo after concentrated by rotary evaporation.
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-150-12-8) to final concentration of 50 μ g/mL, the culture medium containing 1%DMSO is put into cell and supervises in real time as blank control group
It surveys in instrument and detects, 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 tobacco active component (TA-150-12-8) of the present invention to A549
Cell growth inhibition.
Embodiment 3: inhibit the test of human liver cancer Hep-G2 cell growth
Cell culture: in 5%CO2, 37 DEG C, under saturated humidity, with DMEM (10%FBS, 1%PS) culture medium culture people's lung
Cancer Hep-G2 cell chooses the good cell of growth conditions as experimental cell.It is diluted to centainly after cell count with culture medium
The cell suspension of concentration.
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 150mLDMEM culture medium, are put into cell real-time monitor and walk baseline, take out octal plate after covering baseline,
The Hep-G2 cell suspension 345mL 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 TA- that embodiment 1 obtains that 5mL has diluted is added in every hole
150-12-8) to final concentration of 50 μ g/mL, the culture medium containing 1%DMSO is put into cell real-time monitor as blank control group
Middle detection, detection photograph to record when finishing.
Blank control wells: being not added drug, and 1%DMSO is added；
Experimental port: dosing object.
Experimental result is as shown in Figure 5: top line is blank control wells, lower section line is experimental port.
According to the proliferation growth curve of Hep-G2 cell, illustrate that tobacco active component (TA-150-12-8) of the present invention is right
Hep-G2 cell growth inhibition.
Embodiment 4: tobacco active component mass spectral analysis
Mass spectral analysis: it takes and obtains component (TA-150-12-8) 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 triterpene compound, and experimental result is as shown in Figure 6.
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 and inhibit human liver cancer Hep-
G2 Cell Growth Assays.
10. the tobacco active component that any extracting method obtains in -9 according to claim 1.
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| 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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| 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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