CN108379346A - The microwave auxiliary extracting method of total flavonoids of broussonetia papyrifera and its application - Google Patents

The microwave auxiliary extracting method of total flavonoids of broussonetia papyrifera and its application Download PDF

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CN108379346A
CN108379346A CN201810376604.4A CN201810376604A CN108379346A CN 108379346 A CN108379346 A CN 108379346A CN 201810376604 A CN201810376604 A CN 201810376604A CN 108379346 A CN108379346 A CN 108379346A
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broussonetia papyrifera
total flavonoids
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朱开梅
顾生玖
蒋运生
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Guilin Medical University
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    • 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
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Abstract

The invention discloses the microwave auxiliary extracting method of total flavonoids of broussonetia papyrifera and its applications, it is solvent that extracting method, which selects ethyl alcohol, using Orthogonal Experiment and Design microwave radiation exaraction total flavonoids of broussonetia papyrifera, having investigated influences the various factors of Microwave Extraction, and processing is optimized to it, the optimum process condition filtered out：Microwave power 350w, 45 DEG C of microwave temperature, microwave time 15min, solid-liquid ratio 1：10（g·mL‑1）Carry out 3 microwave radiation exaractions；Certain theoretical foundation is provided for reasonable utilize of feed with paper-mulberry leaf resource.Total flavonoids of broussonetia papyrifera extract application in preparation of anti-tumor drugs is also disclosed.The present invention utilizes microwave radiation exaraction total flavonoids of broussonetia papyrifera, compared with traditional handicraft, has extracted amount height, accurate, quick, operating cost is low, and stability is good, and the features such as environmental sound.

Description

The microwave auxiliary extracting method of total flavonoids of broussonetia papyrifera and its application
Technical field
The present invention relates to the extracting method of plant general flavone and its applications, the specifically Microwave-assisted Extraction of total flavonoids of broussonetia papyrifera Take method and its application on preparing antitumor drug.
Background technology
Paper mulberry is Moraceae paper mulberry platymiscium, is distributed mainly on the provinces and cities such as Guangxi, Guangdong, Hainan, Fujian, resource is very rich It is rich.Feed with paper-mulberry leaf is also known as paper mulberry leaf, can clearing heat and promoting diuresis, the effect of cooling blood and hemostasis, desinsection detoxifies.Domestic and foreign scholars are to structure in the latest 20 years Further research has been done in the chemical composition of Pterostyrax and pharmacological action, therefrom separates a variety of chemical compositions, mainly there is tonka-bean Element, triterpenes, alkaloid, fat oil and a large amount of flavonoids and diphenyl propane class compound.Currently, paper mulberry category extract quilt It was found that isoreactivity is formed with antimycotic, antiviral, anti-peroxidation, antiplatelet, and wherein play useful effect is flavonoids And alkaloids.
Flavone compound is a kind of polyphenols being widely present in fruits and vegetables, medicinal plant, has a variety of biologies Activity, it is such as anti-inflammatory antiviral, it is anti-oxidant etc..From the 1970s, it has been found that a variety of flavone compounds have anti- The bioactivity of tumour, such as：Quercetin inhibits the proliferation of nasopharyngeal carcinoma, the proliferation of mechanism of resveratrol inhibiting tumor in digestive tract cell yellow A kind of reed mentioned in ancient books glycosides can cause the apoptosis etc. of prostate gland cancer cell.Before flavone compound research and development are had extensively as new type antineoplastic medicine Scape.
Primary carcinoma of liver（primary carcinoma of the liver）It is one of most common malignant tumour, China Incidence be in the trend that increases year by year, the last correlation study China onset of liver cancer number accounts for about the 55% of the whole world.From Chinese medicine The antitumor active ingredient of middle extraction, develops that low toxicity, low side effect, that efficient new antitumoral natural drug has become this century is new Research direction.China's tradition herbal mixture, mostly using flavones as main component.
Flavones is extracted from feed with paper-mulberry leaf at present mostly uses traditional solvent extraction method, and related microwave radiation exaraction feed with paper-mulberry leaf The research of general flavone does not have but.
Recent studies indicate that the occurrence and development of liver cancer are not only related with cell Proliferation, also have extremely with Apoptosis It closes.Therefore, liver cancer apoptosis reducing has become the main new way of medicines resistant to liver cancer.
Invention content
It is an object of the invention to：The microwave auxiliary extracting method of total flavonoids of broussonetia papyrifera is provided.
Flavones is extracted from paper mulberry at present and mostly uses traditional solvent extraction method, it is solvent that the present invention, which selects ethyl alcohol, is used Orthogonal Experiment and Design microwave radiation exaraction total flavonoids of broussonetia papyrifera has been investigated the various factors for influencing Microwave Extraction, and has been carried out to it Optimization processing provides certain theoretical foundation for reasonable utilize of feed with paper-mulberry leaf resource.
It is another object of the present invention to：Open total flavonoids of broussonetia papyrifera extract application in preparation of anti-tumor drugs.
The present invention observes total flavonoids of broussonetia papyrifera to human liver cancer HepG-2 by Cell culture invitro and Protocols in Molecular Biology The proliferation and apoptotic effect of cell further study total flavonoids of broussonetia papyrifera to human liver cancer HepG-2 cells on this basis The expression of caspase-3, Bax, Bcl-2 albumen induces human liver cancer HepG-2 Apoptosis to inquire into total flavonoids of broussonetia papyrifera Molecule mechanism, the theoretical foundation of science is provided for the utilization of feed with paper-mulberry leaf.
Applicant has found that total flavonoids of broussonetia papyrifera has significant antitumor action for the first time, and furthers investigate paper mulberry by experiment Leaf flavonoids observe the inhibiting effect of human liver cancer cell HepG-2 cell strains using human liver cancer HepG-2 cells as research object Influence of the various concentration total flavonoids of broussonetia papyrifera under different time to human liver cancer HepG-2 cell strains proliferation and apoptotic effect.Experiment The result shows that total flavonoids of broussonetia papyrifera can inhibit the proliferation of human liver cancer HepG-2 cells, and it can induce human liver cancer HepG-2 cells Apoptosis presents time dependence and concentration dependent；Feed with paper-mulberry leaf is analyzed by RT-PCR and cell tissue immunochemical technique General flavone induces the apoptosis of human liver cancer HepG-2 cells, is the high expression for having adjusted pro apoptotic protein Bax, presses down apoptogene Bcl-2 low expressions have relationship.Caspase3 and Bcl-2 balances play an important role to the apoptosis regulation of cell.Therefore, it can incite somebody to action Total flavonoids of broussonetia papyrifera is applied to prepare anti-tumor drug.
The microwave auxiliary extracting method of total flavonoids of broussonetia papyrifera of the present invention, includes the following steps：
（1）Dry feed with paper-mulberry leaf powder 10g is weighed, is put into two mouthfuls of round-bottomed flasks, 60% ethyl alcohol is added, after impregnating 4h after shaking up；
（2）With the optimum process condition filtered out：Microwave power 350w, 45 DEG C of microwave temperature, microwave time 15min, solid-liquid ratio 1：10（g·mL-1）Carry out 3 microwave radiation exaractions；
（3）After Microwave Extraction, natural cooling, filtering；
（4）Filtrate is collected, through Rotary Evaporators ethanol evaporation to concentrate without alcohol taste；
（5）Concentrate is poured into pretreated polyamide column, 30min is stood, after solution to be concentrated is fully adsorbed, with a large amount of For distilled water flushing to being eluted with 70% ethyl alcohol of volume fraction after colourless, flow velocity is 0.8 mLmin-1, until efflux substantially without Color collects efflux；
（6）Efflux is concentrated through Rotary Evaporators recycling ethyl alcohol, concentrate is gone into 100mL volumetric flasks, constant volume shakes Even, extraction is completed.
Microwave radiation exaraction is a kind of technology that recovery rate is improved using microwave energy.With Microwave Heating extraction solvent, Target compound is segregated into solvent from sample matrices.During Microwave Extraction, due in system different material to microwave Absorbability is different, and heat production and its thermal energy for transmitting ambient enviroment are different, and certain active constituents is made to be entered by selectivity heating In solvent small to dielectric constant, microwave absorption capacity is poor.Compared with traditional soxhlet extraction, circumfluence method, Microwave Extraction because The high efficiency and strong selectivity for promoting reaction, with simple for process, the efficiency of heating surface is high, heating is uniform, saves solvent, selectivity Well, by-product is few, yield is high, can reduce the features such as target substance caused by high temperature decomposes, and is widely used in natural plant It is extracted in object in flavone compound reaction.
The present invention utilizes microwave radiation exaraction total flavonoids of broussonetia papyrifera, compared with traditional handicraft, has extracted amount height, accurately, Quickly, operating cost is low, and stability is good, and the features such as environmental sound.Therefore microwave radiation exaraction total flavonoids of broussonetia papyrifera is A kind of environmentally protective, energy-efficient new method, has broad application prospects.
Description of the drawings
Fig. 1 is rutin standard curve figure in experiment one.
Fig. 2 is influence signal of the total flavonoids of broussonetia papyrifera of various concentration in experiment two to human liver cancer HepG-2 cell Proliferations Figure.
Fig. 3 be in testing two total flavonoids of broussonetia papyrifera to the influence micrograph of human liver cancer HepG-2 Apoptosis.
Fig. 4 is influence fluorescent staining micrograph of the total flavonoids of broussonetia papyrifera to human liver cancer HepG-2 Apoptosis in experiment two.
Specific implementation mode
The content of present invention is further described with experimental example below in conjunction with the accompanying drawings.
It is prepared by experiment one, the extraction of total flavonoids of broussonetia papyrifera
1 materials and methods
1.1 key instrument
NJL07-3 types test special microwave oven (Nanjing Jie Quan microwave equipments Co., Ltd)；722 type visible spectrophotometers（On The close Science and Technology Ltd. of Nereid）；(it is public that Gongyi City gives magnificent instrument Limited Liability to DF-101S heat collecting types constant-temperature heating magnetic stirring apparatus Department)；SK5200H ultrasonic cleaners（Science and Technology of Shanghai ultrasonic instrument Co., Ltd）；SHZ-D circulating water type vacuum pumps (Gongyi City Ying Yuyuhua instrument plants).
1.2 drugs and reagent
Control substance of Rutin（Nat'l Pharmaceutical ＆ Biological Products Control Institute provides）；Experimental water is distilled water；Agents useful for same is point It analyses pure；Experiment feed with paper-mulberry leaf.
1.3 method
1.3.1 feed with paper-mulberry leaf sample preparation
The feed with paper-mulberry leaf being collected is screened, after the feed with paper-mulberry leaf sun dehydration of part no disease and pests harm, crosses 80 mesh after crushed Sieve, dry 3 h postcoolings are placed in room temperature in drying box in 40 DEG C of baking ovens of temperature, spare.
1.3.2 total flavonoids of broussonetia papyrifera assay
It is standard items that rutin is chosen in this experiment, and total flavonoids of broussonetia papyrifera content is measured using ultraviolet-visible spectrophotometry.
1.3.3 the preparation of reference substance solution
Precision, which is weighed, to be dried at 120 DEG C to 0.023 g of constant weight control substance of Rutin, is placed in 100mL volumetric flasks, is added volume fraction 30% ethyl alcohol 60mL, which is placed in supersonic wave cleaning machine, promotees its dissolving, adds ethyl alcohol to graduation mark, shakes up to get 0.23 mgmL-1 Control substance of Rutin solution.
1.3.4 the preparation of test sample solution
10 g of feed with paper-mulberry leaf powder is weighed, is placed in round-bottomed flask, is extracted using microwave radiation exaraction total flavonoids of broussonetia papyrifera（It extracts molten Agent volume fraction is 60% ethyl alcohol, solid-liquid ratio 1：10,60 DEG C of microwave temperature, 300 w of microwave power, 10 min of microwave time）.It carries It takes liquid to filter, recycles ethyl alcohol, cross polyamide column, collect filtrate, recycle ethanol solution, concentrate is gone into 100mL volumetric flasks, it is fixed Hold, shakes up, it is to be measured.
1.3.5 colorimetric principle and maximum absorption wavelength selection
Feed with paper-mulberry leaf flavone compound containing there are many, since it contains phenolic hydroxyl group and γ-pyrans copper ring in ambient condition appropriate Under can be generated with aluminium salt and yellow complex and have fluorescence.The certain density NaNO of this experiment2Solution, Al (NO3)3Solution, NaOH Solution colour developing is scanned at 400 ~ 600 nm wavelength, the results showed that, reference substance and solution to be measured have maximum at 510 nm It absorbs, therefore be determined as 510 nm by wavelength is measured.
1.3.6 the preparation of standard curve
Precision measures reference substance solution 0,1.0,1.5,2,2.5,3,3.5,4.0mL, is respectively placed in the volumetric flask of 10mL, uses body 30% ethanol solution fourth of fraction is held to 5mL, and the NaNO that mass fraction is 5.0% is added2Solution 0.3mL, mixing place 5 min, Al (the NO that mass fraction is 10.0% are added3)3Solution 0.3mL, mixing place 6 min, add 1.0 mol/LNaOH solution 2.0mL, then 30% ethyl alcohol is added to be settled to 10mL, place 15 min.Its absorbance A is measured at 510 nm wavelength, with absorbance A For ordinate, concentration C（mg·mL-1）For abscissa, standard curve is drawn, regression equation is obtainedA=0.8694X-0.0017（R 2 = 0.9992）.As a result visible Fig. 1, rutin standard items are in tried 0.25 ~ 0.95mgmL of concentration-1It is linear good in range.
1.3.7 repetitive test
It takes with a collection of 5 parts of feed with paper-mulberry leaf sample, is tested according to the preparation method of test sample solution, operated by standard curve item Colour developing, calculates the content of general flavone in every part of sample, and 5 average contents measured are 69.61mgg-1, RSD=1.87%（n= 5）, it can be seen that the assay method repeatability that this experiment is selected is good.
1.3.8 color developing agent stability experiment
Precision draws test solution 1mL, develops the color by being operated under standard curve item, is surveyed every 10 min in 0 ~ 60min Determine absorbance value.It calculatesRSD=0.99%（n=6）, show that sample is stablized at 27 DEG C of room temperature in 60 min, this experiment is herein It completes to measure in time.
1.3.9 sample recovery rate is tested
Accurate to weigh 9 parts of feed with paper-mulberry leaf sample, every part of each 10 g is accurate respectively that a certain amount of reference substance solution is added, by test sample Preparation tested, and take in 1mL10mL volumetric bottles, develop the color by being operated under normal term, measure trap；By regression equation General flavone amount is acquired, it is 98.31% to calculate average recovery rate, shows that the result that this method measures is accurate.It the results are shown in Table 1.
1 sample recovery rate measurement result of table
1.3.10 total flavonoids of broussonetia papyrifera sample size measurement takes each experimental group total flavonoids of broussonetia papyrifera prepare liquid 1mL, uses volume fraction It after 30% is diluted to 10mL, respectively takes 1mL to be put into volumetric flask and is handled by 2.6 method of standard curve item, measure absorbance and return Equation calculation each group total flavonoids of broussonetia papyrifera percentage composition, flavones content is returned to be calculated as follows：
Flavone compound extracted amount=, wherein C is sample solution mass concentration mgmL-1；V sample solution volumes mL；W sample qualities g.
Total flavonoids of broussonetia papyrifera extraction process
2.1 microwave―assisted extraction
Dry 10.0 g of feed with paper-mulberry leaf powder is accurately weighed, is put into two mouthfuls of round-bottomed flasks, 60% ethyl alcohol is added in different ratios, 4 h are impregnated after shaking up carries out microwave radiation exaraction, Microwave Extraction in different microwave temperatures, microwave time, microwave power respectively Afterwards, slightly cold, filtering.Filtrate is collected, through Rotary Evaporators ethanol evaporation to concentrate without alcohol taste.Concentrate is poured into pretreated In polyamide column, 30min is stood, after solution to be concentrated is fully adsorbed, is divided to after colourless with volume with a large amount of distilled water flushing Several 70% ethyl alcohol are eluted, and flow velocity is 0.8 mLmin-1, until efflux is substantially colorless, collect efflux.By efflux through rotation Turn evaporimeter recycling ethyl alcohol to be concentrated, concentrate is gone into 100mL volumetric flasks, constant volume shakes up, to be measured.
2.2 water-bath reflux extractions
Dry 10.0 g of feed with paper-mulberry leaf powder is accurately weighed, by 1：20 are added 60% ethyl alcohol, and 4 h are impregnated after shaking up.In extraction temperature 45 DEG C, under the conditions of extracting 15 min, extraction is three times, slightly cold by each filtrate, filtering.Filtrate three times is collected, is steamed through Rotary Evaporators Ethyl alcohol is sent out to concentrate without alcohol taste.Concentrate is poured into pretreated polyamide column, 30 min are stood, solution to be concentrated is abundant After absorption, with a large amount of distilled water flushing to being eluted with 70% ethyl alcohol of volume fraction after colourless, flow velocity 0.8mLmin-1, It is substantially colorless to efflux, collect efflux.Efflux is concentrated through Rotary Evaporators recycling ethyl alcohol, concentrate is gone to 100mL volumetric flasks, constant volume shake up, to be measured.
The orthogonal test of 2.3 microwave―assisted extraction optimize techniques
Due to broussonetia papyrifera leaf flavonoids extraction mainly by microwave power, microwave time, microwave temperature, solid-liquid ratio, extraction time etc. because The influence of element, makes a general survey of document, and when medicinal material selects ethyl alcohol to extract, extraction time is generally selected 2 or 3 times, therefore this experiment Extraction time is selected as 3 times, it is contemplated that safety and economy devise the experiment of 4 factor, 3 horizontal quadrature, that is, when choosing microwave Between, 4 microwave temperature, microwave power, solid-liquid ratio factors be investigation factor, using total flavonoids of broussonetia papyrifera content as index, application SPSS17.0 statistical analysis software tables are tested, and L is carried out9（34）Orthogonal Experiment and Design（Table 2）.
2 orthogonal test factor of table and level
3 results and analysis
The optimum process condition of 3.1 microwave radiation exaraction broussonetia papyrifera leaf flavonoids
The orthogonal experiments of table 3 show that four factors for influencing microwave radiation exaraction total flavonoids of broussonetia papyrifera content influence paper mulberry The sequence of leaf flavonoids content is：C（Microwave power）>A（Microwave temperature）>B（The microwave time）>D（Solid-liquid ratio）；But due to feed liquid The influence for comparing microwave radiation exaraction broussonetia papyrifera leaf flavonoids is minimum, therefore carries out variance analysis as error with it.
3 L of table9（34）The orthogonal experiment and result of microwave―assisted extraction
The results of analysis of variance, which is shown, is shown in Table 4,ACBecause being known as highly significant meaning,BThe significant meaning of factor；DThe shadow of factor Ring nonsignificance.The optimised process of Microwave Extraction total flavonoids of broussonetia papyrifera isC 3 A 2 B 3 D 2,I.e. optimal extraction conditionsC 3 A 2 B 3 D 1, i.e., In 350 w of microwave power, temperature 45 C, 15 min of microwave time, material ratio 1：10.
4 analysis of variance table of table
3.2 verification tests accurately weigh feed with paper-mulberry leaf powder 10g, after 60% ethyl alcohol impregnates 4h, with the optimised process filtered out Condition group, that is, microwave power 350w, temperature 45 C, microwave time 15min, solid-liquid ratio 1：10（g·mL-1）Carry out 3 microwave radiation technologies Extraction, measure each index the results are shown in Table 4.The microwave radiation exaraction paper mulberry that orthogonal test filters out can be seen that by 4 result of table Leaf flavonoids technique is reasonable.
5 verification test result of table
3.3 microwave―assisted extractions relatively take with traditional water bath circumfluence method and carry out confirmatory experiment with a batch of feed with paper-mulberry leaf sample.
Microwave method makees solvent by 1 with 60% ethyl alcohol：10（g·mL-1）Material ratio is impregnated four hours, in microwave power 350w, Temperature 45 C, 15 min of microwave time, is handled by 3.1.
Traditional water bath circumfluence method is solvent with 60% ethyl alcohol, by feed with paper-mulberry leaf powder with 1：20（g·mL-1）Solid-liquid ratio is added 60% ethyl alcohol impregnates 4 h after shaking up.In 45 DEG C of extraction temperature, under the conditions of extracting 15min, handled by 3.2 after extracting 3 times. Extraction the results are shown in Table 6.The result shows that microwave―assisted extraction is remarkably improved total flavonoids of broussonetia papyrifera content.
6 microwave―assisted extraction of table and water-bath circumfluence method results contrast
。
The influence that experiment two, total flavonoids of broussonetia papyrifera act on people's HepG-2 Cell apoptosis and proliferations
We have found that total flavonoids of broussonetia papyrifera has significant antitumor action for the first time.Total flavonoids of broussonetia papyrifera pair is furtherd investigate in this experiment The inhibiting effect of human liver cancer cell HepG-2 cell strains observes various concentration structure using human liver cancer HepG-2 cells as research object Influence of the leaf general flavone under different time to human liver cancer HepG-2 cell strains proliferation and apoptotic effect.
1 materials and methods
1.1 laboratory apparatus and main agents
1.1.1 cell strain
Human liver cancer HepG-2 cells（It is provided by Medical Colleges Of Guilin's scientific experiment center）.
1.1.2 laboratory apparatus
Inverted fluorescence microscope, CK40 types, Japanese Olympus companies；
Superclean bench, SW-CJ-1D types, Guangzhou Jia Xin scientific instrument Co., Ltd；
Congratulate Li Shi CO2 incubators, Thermo Fisher, the U.S.；
Ultra low temperature freezer, Thermo Fisher, the U.S.；
Electronic analytical balance, BP211D types, Germany；
Enzyme-linked immunosorbent assay instrument, DG3002 types, state-run East China Electronics Co., Ltd pipe factory；
Cell cryopreservation tube, 001015, upper SeaBird germinating object Science and Technology Ltd.；
96 orifice plates, Beijing Jino Lai Pu Bioisystech Co., Ltd；
6 orifice plates, Beijing Jino Lai Pu Bioisystech Co., Ltd.
1.1.3 drug and reagent
（1）Total flavonoids of broussonetia papyrifera（Total flzvonoids of broussonetia papyrifera, TFBP）, with paper mulberry Leaf is raw material, is extracted by this laboratory, up to 139.01 mg100g after optimized technique extraction-1.PH=7.8 TFBP PBS dissolves, and filtration sterilization saves backup in 4 DEG C of refrigerators, required concentration is diluted to before experiment；
（2）Streptomycin sulphate takes 10ml empty needles to extract 10ml distilled water（High pressure sterilization）Dissolve 1,000,000 units/bottle, sulfate chain One, mycin sodium, is sub-packed in EP pipes, and often 500 μ l of pipe are preserved in -20 DEG C of refrigerators；
(3) Benzylpenicillin sodium salt takes 20ml empty needle pipes to extract 16ml distilled water（High pressure sterilization）Dissolve 1,600,000 units/bottle penicillin One, sodium, is sub-packed in EP pipes, and often 500 μ l of pipe are preserved in -20 DEG C of refrigerators；
（4）DMSO (dimethyl sulfoxide (DMSO)), Sigma companies；
（5）MTT（3- (4,5- dimethylthiazole -2) -2,5- diphenyltetrazolium bromide bromides, tetrazolium bromide）, 100mg, Sigma companies. Advance autoclave sterilization 50ml centrifuge tubes one are taken, 20mlPBS is added, 1000 μ L PBS are drawn out of pipe and are put into MTT pipes, repeatedly 50ml centrifuge tubes are moved into after blowing and beating mixing, mixing is blown and beaten repeatedly, repeats five times, make small inside pipe wall noresidue MTT powder.MTT is complete After full mixing, after 0.22 μm of filtering with microporous membrane degerming, often pipe about 1.5ml is kept in dark place in -20 DEG C of refrigerators for packing；
（6）33342 fluorescent dyes of Hoechest, Kai Ji biological engineering materials Co., Ltd.
1.1.4 cell culture medium
（1）1640 culture mediums of RPMI, GOBCO Products；
（2）Super new fetal calf serum（FBS）, Sijiqing Bioengineering Material Inst., Hangzhou City.
The preparation of 1.2 main agents
（1）PBS：Accurately weigh NaCl 8g, Na2HPO4.7H2O 1.15g, KH2PO40.2g, KCl 0.2g are dissolved in distilled water In, it is settled to 1000ml, it is spare to be stored in 4 DEG C of refrigerators for high pressure sterilization after ultrasonic wave hydrotropy；
（2）Pancreatin：It accurately weighs pancreas enzyme powder 0.25g to be dissolved in 100ml PBS, is put into 4 DEG C of refrigerator overnights dissolvings, it is micro- with 0.22 μm After the membrane filtration degerming of hole, it is stored in 4 DEG C of refrigerators；
（3）Cell culture medium：1640 culture medium 200ml of fetal calf serum 30ml, RPMI, 100 μ l of Benzylpenicillin sodium salt, streptomycin sulphate 100μl；
（4）Paraformaldehyde：10.0g paraformaldehydes accurately are weighed in 90lPBS, is put into water-bath and 1MKOH is added, and are waited for completely With 1M HCl titration after dissolving, it is 7 to make pH value, and it is 100ml to adjust final volume.It stands to being completely dissolved, then configured with PBS At 4% fixer；
（5）Cells frozen storing liquid：According to DMSO：FBS：Complete medium=1：3：6 volumes are prepared.
1.3 cell cryopreservations, recovery and culture
1.3.1 cell cryopreservation
The cell of logarithmic growth phase freezes proxima luce (prox. luc) and replaces cell culture medium, observes cell growth condition, and estimation living cells reaches 95% or more.It will wait for freeze-stored cell, and discard former culture medium, PBS is washed three times, is added 0.25% pancreatin 1.5ml digestion 7min, is added 2ml complete Full culture medium terminates digestion, and 1000rpm centrifuges 10min.Supernatant is removed, frozen stock solution, mixing is added, adjustment cell concentration is 3 ×106/ ml is sub-packed in 1.5ml cryopreservation tubes, sealed membrane closing.4 DEG C of refrigerator 20min are set, 2h in -20 DEG C of refrigerators is transferred to, then - 80 DEG C of refrigerator overnights are transferred to, is put in liquid nitrogen and preserves after group.
1.3.2 cell recovery
Cryopreserved human HepG-2 cell line is quickly taken out from liquid nitrogen, and 37 DEG C of electric heating are put into rapidly after verification date, cellular informatics It is shaken rapidly in constant water bath box, until frozen stock solution melts completely.After 75% alcohol disinfecting, cells frozen storing liquid is moved into centrifuge tube It is interior, it is discarded supernatant after 1000rpm, 5min.4ml culture mediums are added into centrifuge tube, cell suspension moved on into training after blowing and beating mixing It supports in bottle, is placed in 37 DEG C, 5%CO2In the incubator of saturated humidity, next day changes the liquid observation adherent situation of cell.
1.3.3 cell culture
Cellular morphology, upgrowth situation are observed under inverted microscope, plating cells culture bottle area is that 70%-80% carries out passage point Bottle, concrete operations are as follows：
（1）The old culture solution in Tissue Culture Flask unexpectedly is abandoned, cell is rinsed 3 times with PBS；
（2）0.25% pancreatin 1.5ml is added, gently shakes up postposition incubator digestion 7min, a large amount of floatings are observed through inverted microscope When cell, digestion is terminated；
（3）The full culture medium of 2ml cells is added and terminates digestion, piping and druming cell face 15 times moves into centrifuge tube, 1000rpm, 5min After discard supernatant；
（4）The full culture medium of 3ml cells is added into centrifuge tube, cell suspension is moved on in culture bottle after blowing and beating mixing, is placed in 37 ℃、5%CO2In the incubator of saturated humidity, next day changes the liquid observation adherent situation of cell；
（5）Per changing the liquid once within 2-3 days, cell passage is carried out.
1.4 experiment packet
It sets up to positive controls（10 μ g/ml of cis-platinum）, negative control group（Add PBS）, experimental group（Total flavonoids of broussonetia papyrifera concentration 3,6,9 mg/ml）, each group sets three parallel holes.
1.5 mtt assay measure the inhibiting rate that total flavonoids of broussonetia papyrifera is proliferated human liver cancer HepG-2 cell strains
Adjust human liver cancer HepG-2 cell strains a concentration of 4 × 104A/ml is added in 96 well culture plates, per 100 μ l of hole, adds 80 μ l of culture medium.After inverted microscope observation cell is adherent, positive controls cis-platinum is added, makes 10 μ g/ of its final concentration of cis-platinum Ml is added the 20 μ l of total flavonoids of broussonetia papyrifera of various concentration, makes its final concentration of 3mg/ml, 6mg/ml, 9mg/ml, 12mg/ml, It is placed in 37 DEG C, 5%CO again224,48,72h are cultivated respectively in the incubator of saturated humidity.20 μ lMTT are added per hole after terminating culture Solution continues culture four hours in 37 DEG C of incubators.Supernatant is discarded, PBS is flushed three times, and 150 μ lDMSO are added, and oscillation is filled It is point dissolving crystallized, the absorbance in each hole at 490nm is measured with enzyme-linked immunosorbent assay instrument.Calculate cell inhibitory rate, inhibiting rate（%）= （Control group-experimental group）/ control group × 100%.
Influence of the 1.6 difference inverted microscope observation total flavonoids of broussonetia papyrifera to human liver cancer HepG-2 cell strain forms
Adjust human liver cancer HepG-2 cell strains a concentration of 4 × 105A/ml is added in 6 well culture plates, per 500 μ l of hole, adds 1300 μ l of culture medium.After inverted microscope observation cell is adherent, positive controls cis-platinum is added, makes 10 μ of its final concentration of cis-platinum G/ml is added the 200 μ l of total flavonoids of broussonetia papyrifera of various concentration, makes its final concentration of 3mg/ml, 6mg/ml, 9mg/ml, 12mg/ Ml, then it is placed in 37 DEG C, 5%CO248 are cultivated respectively in the incubator of saturated humidity.Culture is terminated, PBS is flushed three times, every time 3min replaces fresh culture, places observation cell adherent growth situation and cellular morphology variation under inverted microscope.
1.7 Hoechst33342 Fluorescent Staining Observation karyomorphisms change
Logarithmic growth phase cell, with every hole 2 × 105The density of a cells/well is inoculated in 6 well culture plates, pre- in 6 well culture plates It first puts the coverslip handled by high pressure sterilization well, after inverted microscope for 24 hours observes cell climbing sheet, it is suitable that positive controls is added Platinum makes 10 μ g/ml of its final concentration of cis-platinum, and the 200 μ l of total flavonoids of broussonetia papyrifera of various concentration are added, make its final concentration of 3mg/ Ml, 6mg/ml, 9mg/ml, 12mg/ml are placed in 37 DEG C, 5%CO again248h is cultivated respectively in the incubator of saturated humidity.Terminate training It supports, exhausts culture solution, PBS is rinsed 3 times；4% paraformaldehyde fixes cell 30min per hole 1ml；Fixer is removed, PBS rinses 3 It is secondary, each 3min.Hoechst33342 dye liquor 1ml are added per hole, are protected from light, mixing, 37 DEG C of incubation 30min.Dyeing liquor is removed, is used PBS is rinsed three times, each 3min, exhausts liquid.Cell climbing sheet is affixed on glass slide, avoids bubble as possible, it is aobvious to be inverted fluorescence Micro- microscopic observation karyomorphism.
1.8 statistical procedures
Experimental data carries out One-way ANOVA (one-way analysis of variance) check analysis with SPSS15.0 statistical softwares.
2 results
The inhibiting effect that 2.1 total flavonoids of broussonetia papyrifera are proliferated human liver cancer HepG-2 cell strains
Using cell inhibitory rate as the longitudinal axis, a concentration of horizontal axis of total flavonoids of broussonetia papyrifera makees Fig. 2.The total flavonoids of broussonetia papyrifera of various concentration is to people HepG-2 cell line strain has different inhibiting effect, with the raising of total flavonoids of broussonetia papyrifera concentration and prolonging for action time It is long, it is more and more significant to the inhibiting effect to human liver cancer HepG-2 cell strains.As shown in Fig. 2, right in 6~9mg/ml concentration HepG-2 inhibiting effect is the most apparent, and through one-way analysis of variance, difference is with conspicuousness compared with the control group（P<0.05）, group Between compare with statistical significance（P<0.05）.
2.2 inverted microscopes observe influence of the total flavonoids of broussonetia papyrifera to human liver cancer HepG-2 cell strain forms
As it can be seen that control group HepG-2 cells, adherent growth is good under inverted microscope, cell arrangement tight boundaries understand, cell Star or fusiformis is presented, nucleus is big, and cytoplasm is abundant and full.When drug concentration is 6mg/ml, cell number of adherent is reduced, Cell arrangement is disorderly, and cell outline is unintelligible, and cytoplasm is unevenly distributed, and mitosis figures are reduced, and cell fragment gradually increases, and sees figure 3。
After the effect of 2.3 fluorescence microscope total flavonoids of broussonetia papyrifera, the morphological change of HepG-2 nucleus
Human liver cancer HepG-2 cell strains are after the total flavonoids of broussonetia papyrifera of various concentration effect 48h, by hoechst33342 fluorescence After dyeing, observed under inverted microscope.Cellular control unit, coating is complete, and karyomorphism rule, caryoplasm color depth, kernel Big and complete, luminescence of cell is uniform.As a concentration of 6mg/ml of total flavonoids of broussonetia papyrifera, there is the fine and close dense dye of core, fluorescent staining is high Into being presented navy blue, the nucleus once fine and close dense dye of chunky shape, cell karyorrhexis, caryoplasm concentration, form and its irregular.
3 conclusions
The real mode that antitumor drug works is based on inducing apoptosis of tumour cell, supplemented by killing tumor cell.MTT（Thiophene Azoles is blue）Principle, which is it, to enter intracellular through cell membrane, and bluish violet is formed with the succinate dehydrogenase in living cells mitochondria Crystallization, therefore can reflect cell quantity indirectly.This experiment measures total flavonoids of broussonetia papyrifera to human liver cancer HepG-2 cell strains with mtt assay The inhibiting rate of proliferation, result of study show that total flavonoids of broussonetia papyrifera has inhibiting effect to human liver cancer HepG-2 cell strains proliferation, and Time and concentration-dependent relation is presented.After total flavonoids of broussonetia papyrifera concentration acts on 72h up to 12mg/ml, the substantially all death of cell.
The morphological change for observing nucleus, is the most basic method for judging Apoptosis.It is seen under inverted microscope Examining the morphological feature of Apoptosis is, cell volume becomes smaller, and loses original normal morphology, can although cell membrane is complete See foamed phenomenon.Hoechest33342 is the dyestuff that a kind of hypotoxicity may pass through after birth, and thin A-T is incorporated into so that A is non-embedded Base area.Hoechst33342 fluorescent stainings are to detect one of the common method of Apoptosis.When Apoptosis, because of chromatin Meeting pyknosis, Gu after Hoechest33342 is dyed, inverted fluorescence microscope visible cell core can be in fine and close dense dye, or in broken Blocky fine and close dense dye.This experiment carries out Hoechest33342 after choosing total flavonoids of broussonetia papyrifera effect human liver cancer HepG-2 cells 48h Dyeing, it has been observed that with the increase of drug concentration, the phenomenon that human liver cancer HepG-2 nucleus dense coalescence fragmentation, is all the more apparent, The petal-like cell fragment of visible likeness in form in 12mg/ml, this i.e. apoptotic body, is presented typical apoptosis morphological feature.With Upper morphologic observation shows that total flavonoids of broussonetia papyrifera can be with inducing cell apoptosis.
Experiment three, total flavonoids of broussonetia papyrifera express human hepatoma cell strain HepG-2 cells, Bax, Bcl-2 and Casepase-3 Influence
The initiative of apoptosis, that is, cell is dead, it plays irreplaceable important function in the development of tumour cell.Bcl-2 family Race shares two major classes albumen that is, pro apoptotic protein and suppression apoptotic proteins, are regulatory factors important in apoptosis process. It was found that the expression ratio of Bcl-2/Bax albumen, directly determines the destiny of oncocyte.The height of Bcl-2 albumen is expressed as cytoma Change provides opportunity, up-regulation mechanism mainly with Casepase-3 weak expressions or do not express closely related.This part is tested, and is passed through Casepase-3 fluorometric investigation kits detection various concentration total flavonoids of broussonetia papyrifera is to Casepase-3 in people's HepG-2 cells Variation；By Immunohistochemical technology, observation various concentration total flavonoids of broussonetia papyrifera is to Bcl-2, Bax albumen in people Change in HepG-2 cells；Pass through RT-PCR technology（Inverse transcription polymerase chain reaction）Observe various concentration total flavonoids of broussonetia papyrifera Casepase-3, Bcl-2, Bax albumen are changed in people's HepG-2 cells.
1 materials and methods
1.1 laboratory apparatus and main agents
1.1.1 laboratory apparatus
Inverted fluorescence microscope, CK40 types, Japanese Olympus companies；
Small-sized high speed centrifugal machine, 5415D, German Eppendbrf；
Desk centrifuge, TDL-2B, Town in Shanghai pavilion centrifuge；
PCR instrument, Tpersonal48, German Eppendbrf；
Electrophoresis apparatus, DYCP-310, Beijing Liuyi Instrument Factory；
Biological electrophoresis image analysis system, FR-980A；
6 well culture plates.
1.1.2 main agents
EDTA, one factory of Beijing analytical reagent；
Casepase-3 activity detection kits, green skies company；
Trizol（TotalRNAExtractor）Kit, raw work biology Shanghai Co., Ltd；
Phosphate buffer（DPBS）, Beijing Tai Zeruida Science and Technology Ltd.s；
Chloroform, raw work biology Shanghai Co., Ltd；
Absolute ethyl alcohol, raw work biology Shanghai Co., Ltd；
Isopropanol, raw work biology Shanghai Co., Ltd；
Agarose Agarose, Huamei Bio-Engrg Co.,；
Pyrocarbonic acid diethyl ester（DEPC）, the Shanghai bio tech ltd Yuan Ye；
2x Taq PCR Green Mix, Biomiga companies；
The anti-human Bcl-2 protein monoclonal antibodies of mouse, Zhong Shan Golden Bridge；
The anti-human Bax monoclonal antibodies of mouse, Zhong Shan Golden Bridge；
Concentrated type DAB kits, Zhong Shan Golden Bridge；
Qula is logical（Triton）X -100, Shanghai past bio tech ltd；
Two-step method immunohistochemistry detection reagent, middle mountain gold bridge；
Ethidium bromide（EB）, Fluka.
The preparation of 1.2 main agents
DEPC：It measures DEPC stostes 1ml to be dissolved in the distilled water of 1000ml, shake up, room temperature preserves overnight, and next day high pressure is cooling It is preserved afterwards in 4 DEG C of refrigerators；
RNA sample-loading buffers：30% glycerine water solution, 0.25% bromophenol blue, 0.25% dimethylbenzene ultramarine, mixing are stored in 4 DEG C of ice Case；
5 × TBE buffer solutions：Tris 54g, boric acid 27.5g, 0.5M EDTA (pH=8.0) 20ml, add distilled water to 1000ml It is spare.In use, being diluted to working concentration；
1.4% agarose：It weighs agarose 1.40g to be dissolved in the TBE of 100ml, microwave heating is cooled to 60 DEG C to dissolving, and is added It is spare after 10mg/mlEB is even；
10×TE Buffer：Measure 1 M Tris-HCl Buffer（pH7.4）100ml, 500 mM EDTA（pH8.0）20ml It is placed in 1L beakers, is slowly added to the deionized water of 800ml, be placed at room temperature for high pressure sterilization after 4h, room temperature preservation.
1.3 cell cryopreservations, recovery and culture
Operation is the same as experiment two
1.4 cell climbing sheet
Dripping a small amount of culture medium makes glass slide tightly be affixed on six hole board bottoms.Logarithmic growth phase HepG-2 cell line, 0.25% pancreatin Digestion separation is 4 × 10 with complete medium adjustment cell concentration5A/ml is added in six orifice plates, is adjusted with complete medium Cell concentration is 2 × 105/hole, is positioned in 37 DEG C of incubators and cultivates, and next day observes cell climbing sheet situation, is separately added into not With concentration total flavonoids of broussonetia papyrifera 6,9,12mg/ml, after 48 hours terminate culture, collect creep plate.
1.5 cellular immunity group methods（SP methods）
Immunocytochemical stain negative control is arranged：PBS is dyed instead of primary antibody.Positive staining：Nucleus or cytoplasm It is the positive brown yellow granule or lumps person inside occur.6 visuals field are observed continuously under light microscopic, calculates its positive rate, takes mean value.
1.6RNA is extracted and identification
1.6.1 cell total rna extracts
Logarithmic growth phase HepG-2 cell line, PBS are washed 3 times, and the digestion separation of 0.25% pancreatin is adjusted thin with complete medium Born of the same parents a concentration of 1 × 105A/ml takes 4ml cell suspensions to be added in culture bottle, is positioned in 37 DEG C of incubators and cultivates, and next day is seen Examine the adherent situation of cell.Be separately added into various concentration total flavonoids of broussonetia papyrifera 6,9,12mg/ml, after 48 hours terminate culture.
1.6.2RNA purity and quantitative
DEPC water of the 500 μ l without RNA is used to return to zero as blank tube.Take 2 μ l total serum IgE samples that 498 μ l DEPC water are added, in purple Absorbance of the RNA sample at 260 nm and 280nm is measured on outer spectrophotometer（OD）Value.The purity of RNA sample with its OD ratios at 260 nm and 280nm indicate.If ratio during 1.8-2.0, shows sample RNA without degradation, quality meets Later experiments requirement.If ratio ＜ 1.8, considers the pollution of extracting sample or need to be further purified.This test measurement each group sample In composite demand during product ratio 1.8-2.0.RNA concentration（μg/ml）=OD260× 40 × extension rate（μg/ml）.
1.6.3 RNA integralities are identified
It prepares 1% denaturation agar electrophoresis gel and the dipped gel liquid level 2mm of tbe buffer liquid in electrophoresis tank is taken into RNA sample, loading After each 2 μ l, 60V electrophoresis 30min of buffer solution, 18s and 28s band situations are observed under ultraviolet lamp, it is seen that the width of 28s and 18s Degree and brightness ratio are about 2：1, edge clear can be used for RT-PCR experiments.
1.7 reverse transcriptase polymerase chain reaction（RT-PCR）
1.7.1 primer and reaction amplification condition
Primer is：Caspase-3 、Bcl-2 、Bax ；
Amplification condition：
Caspase-3：37 DEG C of reverse transcription reverse transcriptions 60min, 94 DEG C of pre-degenerations 5min, 95 DEG C of denaturation 5s, 55 DEG C of 20s that anneal, 72 DEG C extend 20s, recycle 40 times, 72 DEG C be incubated 5min, product length 200bp；
Bcl-2：37 DEG C of reverse transcription reverse transcription 50min, 94 DEG C of pre-degeneration 1min, 95 DEG C of denaturation 30s, 60 DEG C of annealing 35s, 72 DEG C are prolonged 1min is stretched, is recycled 30 times, 72 DEG C are incubated 5min, product length 301bp；
Bax：37 DEG C of reverse transcription reverse transcription 50min, 94 DEG C of pre-degeneration 2min, 95 DEG C of denaturation 25s, 55 DEG C of annealing 40s, 72 DEG C extend 50s is recycled 32 times, and 72 DEG C are incubated 5min, product length 381bp.
1.7.2RT-PCR reaction system
Mix 10μl
TaqE 0.2μl
1.0 μ l of primer upstream
1.0 μ l of primer downstream
cDNA 3.0μl
ddH2O 4.8μl
It takes 2 μ g to prepare total serum IgE and carries out RT-PCR, carry out the amplification of target gene and internal reference respectively with reference to above-mentioned amplification system.
1.7.3 electrophoresis and gel analysis RT-PCR products
10 μ l of RT-PCR products are taken, 2 μ l, DNA Maker10 μ l of sample-loading buffer carry out 1.4% agargel electrophoresis, using FR- 980A biology electrophoresis image analysis systems carry out photometric scanning, and the gray scale for observing each band is strong and weak, as a result with the product of target gene Absorbance ratio is divided to indicate.
2 statistical procedures
Experimental data carries out One-way ANOVA (one-way analysis of variance) check analysis with SPSS17. statistical softwares, two-by-two Compare q inspections, P ＜ 0.05 are statistically significant, 0.01 significant differences of P ＜.
3 results
3.1 observe influence of the total flavonoids of broussonetia papyrifera to Bcl-2 albumen by SP methods
Bcl-2 is mainly cytoplasm receptor, is appeared on nuclear membrane on a small quantity.Positive cell is in the annular of brown color after dyeing.3、6、 After 9mg/ml total flavonoids of broussonetia papyrifera acts on HepG-2 cells 48 hours, it is observed that increase of the cytoplasm coloring with concentration And gradually become shallower as, as a concentration of 9mg/ml, nucleus is almost not colored.3,6,9mg/ml groups have statistics compared with the control group Meaning（P<0.05）.（It is shown in Table 3-1）
3.2 observe influence of the total flavonoids of broussonetia papyrifera to Bax albumen by SP methods
Bax protein expressions observe that cytoplasm stained yellow or brown yellow granule shape are the positive based on cytoplasm.3、6、9mg/ After ml total flavonoids of broussonetia papyrifera acts on HepG-2 cells 48 hours, it is observed that cytoplasm coloring with the increase of concentration and by Gradual change is deep, when a concentration of 9mg/ml, the visible compacted grains shape of subregion cytoplasm.3,6,9mg/ml groups have compared with the control group It is statistically significant（P<0.05）.（It is shown in Table 3-1）
Table 3-1
Bcl-2 Bax
0mg/ml 42.22±2.13 22.77±0.81
3 mg/ml 36.21± 2.05 27.27±0.32*
6 mg/ml 29.34±1.9* 30.5±0.5*
9 mg/ml 20.73±1.41* 36.17±0.05*
3.3 observe influence of the total flavonoids of broussonetia papyrifera to apoptosis-related genes by RT-PCR
This experiment by RT-PCR (inverse transcription polymerase chain reaction) detect through total flavonoids of broussonetia papyrifera act on 48h after Bcl-2, The expression of Bax, Caspase-3mRNA, testing result show occur Caspase-3 specific gene items on the position of 200bp Band occurs Bcl-2 specific gene bands on the position of 301bp, occurs Bax specific gene bands on the position of 381bp, Occur β-actin specific gene bands on the position of 313bp.By the analysis of electrophoretic band gray value as can be seen that people For HepG-2 after total flavonoids of broussonetia papyrifera acts on 48h, tumor suppressor gene Bcl-2 expression quantity is in the trend that is gradually reduced, 3,6,9mg/ml Group has significant difference compared with the control group（3.67±0.08vs4.3±0.078、3.19±0.18 vs4.3±0.078、 2.92 ± 0.09vs4.3 ± 0.0785, P<0.05）；Promote apoptogene Bax expression quantity to increase with the raising of concentration, through system Meter analysis 3,6, difference is significant compared with the control group for 9mg/ml groups；Caspase-3 gene levels are with total flavonoids of broussonetia papyrifera The increase of concentration, the statistical analysis although also showing trend, 3,6mg/ml groups compared with the control group, have statistical significance （0.807±0.067vs0.786±0.03、2.468± 0.169vs0.786±0.03）.
4 conclusions
This experiment acts on human liver cancer HepG-2 cells 48 hours by Immunohistochemical scientific discovery, total flavonoids of broussonetia papyrifera Afterwards, the Bcl-2 in cell presents low expression.And increase with concentration is presented, can significantly lower the expression quantity of Bcl-2 with Reduction, when concentration reach 12mg/ml when, the expression of Bcl-2 can be down to 27.95 ± 0.54.In conjunction with RT-PCR as a result, albumen It is horizontal consistent with the horizontal variation tendencies of Bcl-2 mRNA, as a concentration of 12mg/ml, gray value and the β-of Bcl-2 mRNA It is 2.316 ± 0.102 that the ratio between actin gray values, which fall below minimum point,.It is observed by immunohistochemical method, Bax albumen tables It is increased up to amount with the raising of drug concentration, and there is dose-dependent feature, RT-PCR is as a result, protein level for joint It is parallel with the horizontal variation tendencies of Bax mRNA, as a concentration of 12mg/ml, gray value and the β-actin gray values of Bax mRNA The ratio between be increased to 39.39 ± 0.70.Caspase-3 gene levels are detected by RT-PCR, total flavonoids of broussonetia papyrifera concentration gradually rises Height, the expression of caspase-3 gene levels gradually rise, 3,6,9, the gray level ratio of 12mg/ml be respectively 0.613 ± 0.12, 0.689±0.11、0.79±0.005、0.862±0.111、0.939±0.003 .In short, passing through common molecular biology Technology can find that total flavonoids of broussonetia papyrifera can inhibit the proliferation of human liver cancer HepG-2 cell strains in vitro, can be preliminary from experiment Speculate that total flavonoids of broussonetia papyrifera induces human liver cancer HepG-2 Apoptosis, it may be possible to which apoptogene Bax, caspase- are promoted by up-regulation 3 is horizontal, lowers and inhibits apoptogene Bcl-2 horizontal.It further infers that, total flavonoids of broussonetia papyrifera induces the mistake of HepG-2 Apoptosis Cheng Zhong, Bcl-2 and caspase-3 are mutually restricted.The regulation and control of certain Apoptosis are that numerous factors participate in, between the various factors There is the time order and functions that plays a role again, mutually restrict, mutually the relationships such as joint.Therefore total flavonoids of broussonetia papyrifera induces human liver cancer HepG-2 Apoptosis should also be from the further research such as death receptor mediated pathways, mitochondria mediated pathways, suppression of other approach Angiogenesis factor processed etc..We believe in laboratory, and as Apoptosis Mechanism gradually illustrates, experimental technique is gradually increased, Total flavonoids of broussonetia papyrifera anti-liver cancer and anti-mechanism can also define therewith, be that rational exploitation and utilization and the natural drug of feed with paper-mulberry leaf resource are anti-swollen The Mechanism Study of tumor provides sufficient scientific research theoretical foundation.

Claims (6)

1. the microwave auxiliary extracting method of total flavonoids of broussonetia papyrifera, which is characterized in that include the following steps：
（1）Dry feed with paper-mulberry leaf powder is weighed, is put into flask, 60% ethyl alcohol is added, is impregnated after shaking up；
（2）After immersion, microwave radiation exaraction is carried out；
After Microwave Extraction, natural cooling, filtering；
Filtrate is collected, through Rotary Evaporators ethanol evaporation to concentrate without alcohol taste；
（5）Concentrate is poured into pretreated polyamide column, stands, after solution to be concentrated is fully adsorbed, uses distilled water flushing It is eluted afterwards with 70% ethyl alcohol to colourless, until efflux is substantially colorless, collects efflux；
（6）Efflux is concentrated through Rotary Evaporators recycling ethyl alcohol, concentrate is gone into volumetric flask, constant volume shakes up, extraction It completes.
2. the microwave auxiliary extracting method of total flavonoids of broussonetia papyrifera described in claim 1, it is characterised in that：Step（2）It is described micro- The process conditions of wave assisted extraction are：Microwave power 350w, 45 DEG C of microwave temperature, microwave time 15min, solid-liquid ratio 1：10g· mL-1Carry out 3 Microwave Extractions.
3. the microwave auxiliary extracting method of total flavonoids of broussonetia papyrifera described in claim 1, it is characterised in that：Step（1）The leaching The bubble time is 4 h.
4. the microwave auxiliary extracting method of total flavonoids of broussonetia papyrifera described in claim 1, it is characterised in that：Step（5）It is described quiet It is 30min to set the time, and elution flow rate is 0.8 mLmin-1。
5. the total flavonoids of broussonetia papyrifera extract of any the method extraction in claim 1-4.
6. the total flavonoids of broussonetia papyrifera extract application in preparation of anti-tumor drugs described in claim 6.
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Publication number Priority date Publication date Assignee Title
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Title
朱开梅等: "构树叶中总黄酮微波提取工艺优化", 《中国实验方剂学杂志》 *
朱开梅等: "构树叶总黄酮对人肝癌细胞 HepG-2 增殖和凋亡的作用及其机制研究", 《中国实验方剂学杂志》 *

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