CN104922268B - A kind of method that active material is extracted from natural plants - Google Patents
A kind of method that active material is extracted from natural plants Download PDFInfo
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Abstract
The present invention relates to the methods that active material is extracted from natural plants, can effectively solve the problems, such as oncotherapy, and the technical solution solved is to include the following steps：1）The crushing and extraction of raw material；2）Flash concentration；3）Decoloration removal of impurities；4）Ether extracts the preparation at position；5）The preparation of Ethyl acetate fraction；6）The preparation at extracting n-butyl alcohol position；7）The preparation of active material；The active material extracted in le ＆-Floor Chinese prickly ashes of the present invention, is used to prepare treatment anti-tumor drug, so as to solve the problems, such as the prevention of tumour, opens the new application for having warded off le ＆-Floor Chinese prickly ashes for treating tumour, this is the big innovation on Chinese medicine.
Description
Technical field
The present invention relates to extracted form natural plant field, particularly a kind of method that active material is extracted from natural plants.
Background technology
Le ＆-Floor Chinese prickly ash Zanthoxylum avicennae are Rutaceae xanthoxylum, be distributed mainly on about 25 ° of north latitude with
Southern area, is grown on low altitude area level land, hillside fields or valley floor.China is distributed in Hainan, Fujian, Guangdong, Taiwan, Guangxi, Yunnan etc.
Southern area.It is enriched in Sanya, Hainan , le ＆-Floor Chinese prickly ash resource distributions.It mainly contains and waves in document report Rutaceae xanthoxylum
The ingredients such as hair oil, flavones, alkaloid, cumarin, amide.Fresh leaf, root skin and pericarp have Chinese prickly ash smell, and that chews has cement, taste
Bitter and numb tongue, pericarp and root skin taste are denseer.It is civil to be used as herbal medicine.There are dispelling wind and eliminating dampness, promoting the circulation of qi resolving sputum, analgesic and other effects, Zhiduo County's class
Pain, and make ascarifuge agent.Anti-inflammatory analgetic clinically is carried out with Chinese prickly ash ether extract or pepper volatile oil, obtains good treatment
Effect.According to another document report, xanthoxylum has 4 kinds of deep fungals and 11 kinds of dermatophytes certain antibacterial and sterilization to make
With.Le ＆-Floor Chinese prickly ashes have the report of antitumor activity as a kind of natural plants from le ＆-Floor pepper extracts are found no.
Invention content
For the above situation, to solve the defect of the prior art, the purpose of the present invention is just to provide one kind from natural plants
The method of middle extraction active material, can effectively solve the problems, such as oncotherapy.
The technical solution that the present invention solves is to include the following steps：
1) crushing and extraction of raw material：The leaf of le ＆-Floor Chinese prickly ashes is dried, is ground into coarse powder, 20-40 mesh sieve is crossed, is placed in appearance
The hydrous ethanol infiltration 4-6h that the mass concentration of , Yong le ＆-Floor Chinese prickly ashes 1 times of weight of coarse powder is 50-90% in device, is fitted into percolator,
Seepage pressure effects are carried out Yong the 50-90% hydrous ethanols of le ＆-Floor Chinese prickly ashes 10-15 times of weight of coarse powder, are oozed with the collection of 3-10mL/min flow velocitys
It filters extracting solution；
2) flash concentration：By obtained extracting solution, flash concentration to the 1/10-1/15 of original volume, must concentrate below 70 DEG C
Liquid；
3) decoloration removal of impurities：Concentrate is distributed in water, the dosage of water is 1/3rd of the volume of the concentrated liquid, uses petroleum ether
Extraction 2-5 time, each dosage are measured for 1-2 times of the volume of the concentrated liquid, to remove pigment and grease type impurity, after static layering, are divided
Petroleum ether layer is removed, merges petroleum ether extraction liquid, vacuum concentration steams petroleum ether；
4) preparation at ether extraction position：To decolourize concentrate ether extraction 3-6 times after cleaning, and each dosage is dense
1-3 times of contracting liquid product is measured, and after static layering, is divided and is removed ether layer, merges ether extraction liquid, and normal pressure concentration steams ether, obtains
Ether extract position concentrate, continue concentrate being concentrated in vacuo, then be dried in vacuo, with mortar it is finely ground after, obtain second
Ether extract dry powder；
5) preparation of Ethyl acetate fraction：The water layer concentrate separated after ether is extracted, is extracted with ethyl acetate
3-6 times, each dosage is 1-3 times of the volume of the concentrated liquid and measures, and after static layering, divides and removes ethyl acetate layer, combined ethyl acetate extraction
After liquid, less than 60 DEG C vacuum-concentrcteds is taken to steam ethyl acetate, the concentrate of Ethyl acetate fraction is obtained, vacuum is done
It is dry, with mortar it is finely ground after, obtain ethyl acetate extract dry powder；
6) preparation at extracting n-butyl alcohol position：The water layer concentrate separated after ethyl acetate is extracted, uses extracting n-butyl alcohol
2-5 times, each dosage is 1-2 times of the volume of the concentrated liquid and measures, and after static layering, divides and removes n-butanol layer, merges butanol extraction liquid,
Vacuum-concentrcted is carried out in flash concentration device below 80 DEG C, n-butanol portion concentrate is obtained after steaming n-butanol,
Vacuum drying, with mortar it is finely ground after, obtain n-butanol extract dry powder；
7) preparation of active material：By ethyl acetate extract dry powder and n-butanol extract dry powder according to weight ratio 1:1
Mixing, obtains anti-tumor active substance.
The active material extracted in le ＆-Floor Chinese prickly ashes of the present invention, is used to prepare treatment anti-tumor drug, so as to solve tumour
Prevention problem, Kai Pi le ＆-Floor Chinese prickly ashes are used to treat the new application of tumour, this is the big innovation on Chinese medicine.
Specific embodiment
The specific embodiment of the present invention is described in further detail with reference to embodiments.
Embodiment 1
1) crushing and extraction of raw material：The leaf of le ＆-Floor Chinese prickly ashes is dried, is ground into coarse powder, 20 mesh sieve is crossed, is placed in container
The hydrous ethanol that the mass concentration of middle , le ＆-Floor Chinese prickly ashes 1 times of weight of coarse powder is 60% infiltrates 4 hours, is fitted into , le in percolator
The hydrous ethanol that the mass concentration of ＆-Floor Chinese prickly ashes 10 times of weight of coarse powder is 60% carries out seepage pressure effects, is oozed with the collection of 7mL/min flow velocitys
It filters extracting solution；
2) flash concentration：Obtained extracting solution is subjected to flash concentration extremely below 70 DEG C using Reduced Pressure Concentration Device
The 1/10 of original volume；
3) decoloration removal of impurities：Concentrate is distributed in water, the dosage of water is 1/3rd of the volume of the concentrated liquid, uses petroleum ether
Extraction 2 times, each dosage are 2 times of volume of the concentrated liquid amounts, to remove pigment and grease type impurity, after static layering, point remove stone
Oily ether layer merges petroleum ether extraction liquid, is concentrated in vacuo in Rotary Evaporators and steams petroleum ether；
4) preparation at ether extraction position：It by the concentrate after cleaning that decolourizes, is extracted 6 times with ether, each dosage is dense
1 times of contracting liquid product is measured, and after static layering, is divided and is removed ether layer, merges ether extraction liquid, and normal pressure, which concentrates, in Rotary Evaporators steams
Go out ether, obtain the concentrate at ether extraction position, continue concentrate being concentrated in vacuo, then be dried in vacuo into dry powder, use
After mortar is finely ground, ether extract dry powder is obtained；
5) preparation of Ethyl acetate fraction：The water layer concentrate separated after ether is extracted, is extracted with ethyl acetate 6
Secondary, each dosage is 1 times of the volume of the concentrated liquid and measures, and after static layering, divides and removes ethyl acetate layer, combined ethyl acetate extract liquor,
In flash concentration device, after less than 60 DEG C vacuum-concentrcteds steam ethyl acetate, the dense of Ethyl acetate fraction is obtained
Contracting liquid, vacuum drying, with mortar it is finely ground after, obtain ethyl acetate extract dry powder；
6) preparation at extracting n-butyl alcohol position：The water layer concentrate separated after ethyl acetate is extracted, with extracting n-butyl alcohol 5
Secondary, each dosage is 1 times of the volume of the concentrated liquid and measures, and after static layering, divides and removes n-butanol layer, merges butanol extraction liquid, 80
Vacuum-concentrcted is carried out below DEG C in flash concentration device, n-butanol portion concentrate, vacuum are obtained after steaming n-butanol
It is dry, with mortar it is finely ground after, obtain n-butanol extract dry powder；
7) preparation of active material：By ethyl acetate extract dry powder and n-butanol extract dry powder according to 1:1 combination, obtains
To anti-tumor active substance.
Embodiment 2
1) crushing and extraction of raw material：The leaf of le ＆-Floor Chinese prickly ashes is dried, is ground into coarse powder, 20 mesh sieve is crossed, is placed in container
The hydrous ethanol that the mass concentration of middle , le ＆-Floor Chinese prickly ashes 1 times of weight of coarse powder is 70% infiltrates 5 hours, is fitted into , le in percolator
The hydrous ethanol that the mass concentration of ＆-Floor Chinese prickly ashes 12 times of weight of coarse powder is 70% carries out seepage pressure effects, is oozed with the collection of 6mL/min flow velocitys
It filters extracting solution；
2) flash concentration：Obtained extracting solution is subjected to flash concentration extremely below 70 DEG C using Reduced Pressure Concentration Device
The 1/12 of original volume, obtains concentrate；
3) decoloration removal of impurities：Concentrate is distributed in water, the dosage of water is 1/3rd of the volume of the concentrated liquid, uses petroleum ether
Extraction 4 times, each dosage are 1.5 times of volume of the concentrated liquid amounts, to remove pigment and grease type impurity, after static layering, point are gone
Petroleum ether layer merges petroleum ether extraction liquid, is concentrated in vacuo in Rotary Evaporators and steams petroleum ether；
4) preparation at ether extraction position：It by the concentrate after cleaning that decolourizes, is extracted 5 times with ether, each dosage is dense
1.5 times of contracting liquid product are measured, and after static layering, are divided and are removed ether layer, merge ether extraction liquid, normal pressure concentrates in Rotary Evaporators
Ether is steamed, the concentrate at ether extraction position is obtained, continues concentrate being concentrated in vacuo, then be dried in vacuo, use mortar
After finely ground, ether extract dry powder is obtained；
5) preparation of Ethyl acetate fraction：The water layer concentrate separated after ether is extracted, is extracted with ethyl acetate 5
Secondary, each dosage is 1.5 times of the volume of the concentrated liquid and measures, and after static layering, divides and removes ethyl acetate layer, combined ethyl acetate extraction
Liquid in flash concentration device, after less than 60 DEG C vacuum-concentrcteds steam ethyl acetate, obtains Ethyl acetate fraction
Concentrate, vacuum drying, with mortar it is finely ground after, obtain ethyl acetate extract dry powder；
6) preparation at extracting n-butyl alcohol position：The water layer concentrate separated after ethyl acetate is extracted, with extracting n-butyl alcohol 4
Secondary, each dosage is 1.5 times of the volume of the concentrated liquid and measures, and after static layering, divides and removes n-butanol layer, merges butanol extraction liquid,
Less than 80 DEG C carry out vacuum-concentrcted in flash concentration device, after steaming n-butanol, obtain n-butanol portion concentrate, very
Sky is dry, with mortar it is finely ground after, obtain n-butanol extract dry powder；
7) preparation of active material：By ethyl acetate extract dry powder and n-butanol extract dry powder according to 1:1 combination, obtains
To anti-tumor active substance.
Embodiment 3
1) crushing and extraction of raw material：The leaf of le ＆-Floor Chinese prickly ashes is dried, is ground into coarse powder, 40 mesh sieve is crossed, is placed in container
The hydrous ethanol that the mass concentration of middle , le ＆-Floor Chinese prickly ashes 1 times of weight of coarse powder is 80% infiltrates 6 hours, is fitted into , le in percolator
The hydrous ethanol that the mass concentration of ＆-Floor Chinese prickly ashes 13 times of weight of coarse powder is 80% carries out seepage pressure effects, is oozed with the collection of 8mL/min flow velocitys
It filters extracting solution；
2) flash concentration：Obtained extracting solution is subjected to flash concentration extremely below 70 DEG C using Reduced Pressure Concentration Device
The 1/13 of original volume；
3) decoloration removal of impurities：Concentrate is distributed in water, the dosage of water is 1/3rd of the volume of the concentrated liquid, uses petroleum ether
Extraction 5 times, each dosage are 1 times of volume of the concentrated liquid amount, to remove pigment and grease type impurity, after static layering, point remove stone
Oily ether layer merges petroleum ether extraction liquid, is concentrated in vacuo in Rotary Evaporators and steams petroleum ether；
4) preparation at ether extraction position：It by the concentrate after the removal of impurities that will decolourize, is extracted 4 times with ether, each dosage is
2 times of the volume of the concentrated liquid are measured, and after static layering, are divided and are removed ether layer, merge ether extraction liquid, normal pressure concentrates in Rotary Evaporators
Ether is steamed, the concentrate at ether extraction position is obtained, continues concentrate being concentrated in vacuo, then be dried in vacuo, use mortar
Ether extract dry powder is obtained after finely ground；
5) preparation of Ethyl acetate fraction：The water layer concentrate separated after ether is extracted, is extracted with ethyl acetate 4
Secondary, each dosage is 2 times of the volume of the concentrated liquid and measures, and after static layering, divides and removes ethyl acetate layer, combined ethyl acetate extract liquor,
In flash concentration device, after less than 60 DEG C vacuum-concentrcteds steam ethyl acetate, the dense of Ethyl acetate fraction is obtained
Contracting liquid, vacuum drying, with mortar it is finely ground after, obtain ethyl acetate extract dry powder；
6) preparation at extracting n-butyl alcohol position：The water layer concentrate separated after ethyl acetate is extracted, with extracting n-butyl alcohol 3
Secondary, each dosage is 2 times of the volume of the concentrated liquid and measures, and after static layering, divides and removes n-butanol layer, merges butanol extraction liquid, 80
Vacuum-concentrcted is carried out below DEG C in flash concentration device, after steaming n-butanol, obtains n-butanol portion concentrate, vacuum
It is dry, with mortar it is finely ground after, obtain n-butanol extract dry powder；
7) preparation of active material：By ethyl acetate extract dry powder and n-butanol extract dry powder according to 1:1 combination, obtains
To anti-tumor active substance.
The active material extracted in le ＆-Floor Chinese prickly ashes of the present invention is treated available for preparing in antitumor drug through testing and analyzing
Application, effectively solves the problems, such as the prevention of tumour, and satisfied technique effect is achieved through experiment, it is as follows in relation to testing data：
(1) anti tumor activity in vitro experiment (mtt assay of cell Proliferation detection)
Mtt assay is also known as MTT colorimetric methods, is a kind of method for detecting cell survival and growth, due to cell viability and cell
Number is proportionate, therefore is also usually used to the proliferative conditions of detection cell.This method is widely used in some bioactie agents
Activity determination, large-scale screening anti-tumor medicine, cell toxicity test and tumor radiosensitivity measure etc..With spirit
The characteristics of sensitivity is high, quick and economic.
Testing principle：Entitled 3- (4,5- dimethylthiazole -2) -2, the 5- diphenyltetrazolium bromide bromide (commodity of chemistry of MTT
Name：Tetrazolium bromide), it is a kind of dyestuff of yellow color.Succinate dehydrogenase in living cells mitochondria can restore exogenous MTT
Bluish violet for water-insoluble crystallizes first a ceremonial jade-ladle, used in libation and is deposited in cell, and dead cell is without this function.Dimethyl sulfoxide (DMSO) (DMSO) energy
The first a ceremonial jade-ladle, used in libation in cell is dissolved, its absorbance value is measured at 490nm (or 570nm) wavelength with enzyme-linked immunosorbent assay instrument, it can be indirect
Reflect living cells quantity.In the range of certain cell number, it is directly proportional to cell number that MTT crystallizes the amount to be formed.
Reagent and instrument：Calf serum；Phosphate buffer PBS:(NaCl8g, KCl0.2g, Na2HPO41.44g
KH2PO40.24g adjusts pH7.4, constant volume 1L)；Culture solution (DMEN, RPMI1640, McCoys5A)；0.25% trypsase；Tire ox
Serum；MTT kits (Sigma Co., USA)；Dimethyl sulfoxide (DMSO)；Other reagents are pure to analyze.Microcomputerized electric Water Tank with Temp.-controlled；
Biohazard Safety Equipment；Cell incubator；Microplate reader；96 porocyte culture plates；
The human liver cancer cell strain of in vitro culture：Human liver cancer H22, human liver cancer PLC/PRF/5, human liver cancer QGY-7701, people
Liver cancer BEL-7404；PBS references《Cell culture》It prepares；Rotary Evaporators.
Experimental procedure：The cell of in vitro culture, up to 50% to 90% fusion, sucks culture solution, adds in one in culture dish bottom
Quantitative PBS sucks PBS to clean cell after gently rinsing cell.Add in 0.5 milliliter of 0.4% trypsase EDTA liquid, covering
Entire culture dish bottom, allows all cells to contact trypsase.Place it in 37 degree of 5%CO2Tryptose is carried out in incubator
The digestion of enzyme.Culture solution is added in, cell is blown and beaten with 1 milliliter of micropipettor head, into individual cells suspension, it is dense to count cell
Degree.With the cell of every hole certain amount, by cell inoculation in 96 porocyte culture plates, per 200 μ L of pore volume, 37 DEG C, 5%
CO2Culture is administered, and set positive control and negative control after 124 hours.Using extract of the present invention as drug sample, DMSO is used
Dissolving, drug set 100,50,25,12.5 μ gmL-14 concentration gradients, action time be 60 hours, each concentration gradient
Four multiple holes administrations,.
MTT colorimetric determinations：The culture medium for the sample containing various concentration that experimental group more renews, control group, which is then replaced, to be contained
The culture medium of isometric solvent, 37 DEG C, 5% CO2After continuing culture 48 hours, supernatant is removed, MTT solution is added in per hole
(5mg/mL is prepared with PBS, pH=7.4)) 20 μ L, under the conditions of 37 DEG C after insulating box culture 4 hours, culture is terminated, it is careful to inhale
Culture supernatant in hole is abandoned, 150 μ LDMSO, decolorization swinging table concussion dissolving 10min, with tested wavelength in microplate reader are added in per hole
For 570nm, reference wavelength measures OD values for 450nm, calculates the inhibiting rate of tumor cell proliferation, growth of tumour cell inhibiting rate %
=(1-OD experiments/OD controls) × 100%.The mapping of growth of tumour cell inhibiting rate can be obtained with the various concentration of same sample
To dose-effect curve, the IC of sample is therefrom obtained50。IC50To inhibit drug concentration (μ gmL during growth of cancer cells 50%-1).Synthesize the IC of compound or plant extract sterling5010 μ gmL of ＜-1Or the IC of Plant crude extract5020 μ gmL of ＜-1When,
Then judgement sample has tumour cell lethal effect, i.e., with active anticancer.Difference extraction position and combinations thereof is external anti-swollen
The IC of tumor activity50Value is shown in Table 1.
The IC of the different extraction positions of table 1 and combinations thereof anti tumor activity in vitro50(μg·mL-1)-mtt assay
As can be seen from Table 1, Ethyl acetate fraction of the invention, extracting n-butyl alcohol position and combinations thereof drug are to people
Murine Hepatoma22, human liver cancer PLC, human liver cancer QGY-7701, human liver cancer BEL-7404 IC50Respectively less than 100 μ gmL-1, show this
The drug of invention has significant inhibiting effect to the proliferation of human liver cancer cell.
As it can be seen from table 1 to ethereal extract, three positions of acetic acid ethyl ester extract and n-butyl alcohol extract and 2
The combination of a different parts carries out anti tumor activity in vitro screening using mtt assay, finds ethyl acetate extract and n-butanol respectively
Position is according to weight ratio 1:1 mixed mixture, to the proliferation of human liver cancer cell, inhibiting effect is more notable, illustrates second
The mixture of acetoacetic ester position and n-butanol portion has the effect of collaboration and synergy, and half killing cancer cell concentration is lower, resists
Function of tumor is more obvious.
(2) antitumor experiment in Mice Body
With ethyl acetate extract and n-butanol portion according to weight ratio 1:1 mixed mixture is trial drug sample,
With physiological saline solution, height is configured to, in, the suspension sample liquid of low three kinds of concentration, 4 DEG C are stored refrigerated, resist as in Mice Body
The drug of tumor activity test.Appropriate cyclophosphamide is taken to be made into the solution of 20mg/mL with normal saline dilution, 4 DEG C of preservations are standby
With.
Animal：Male SPF grades of mouse of ICR kinds, 3~4 weeks, 18~22g, H22 tumor-bearing mices (protected by regular intraperitoneal inoculation passage
Kind).
Drug and reagent：Syklofosfamid ampoule (CTX), AST kits, ALT kits, TNF-α kit and IL-2
Kit, other reagents are that analysis is pure.
Instrument：InfiniteM200 microplate reader；KQ-250B type ultrasonic cleaners；R201B Rotary Evaporators.
H22 tumor-bearing mices solid tumor models and test method：Mouse is randomly divided into Normal group, model group, ring phosphorus
High, medium and low (0.8g/kg, the 0.4g/kg, 0.2g/kg) dosage group of amide positive group (20mg/kg), drug of the present invention, every group 10
Only.It will pass on 10 days, grow lotus H22 ascites tumor mouse cervical dislocation execution vigorous and without diabrosis, and put superclean bench,
It extracts the knurl liquid of milky or milk yellow under aseptic condition out from abdominal cavity, l is pressed with sterile saline:H22 is made in 7 concentration dilution
Tumor cell suspension, and concentration is adjusted to 5.0 × 107A/mL is spare.In addition to blank group, in every mouse right fore armpit
Under with 75% ethanol disinfection, be subcutaneously injected prepared tumor cell suspension 0.2mL, it is interior in 30min to complete operation.Modeling
After for 24 hours, it is administered by the following method：Cyclophosphamide positive group be injected intraperitoneally (ip) dosage 20mg/kg, the next day 1 time, continuous gavage
(ig) it is administered 15 days；High, medium and low (0.8g/kg, the 0.4g/kg, 0.2g/kg) dosage group of drug one time a day, continuous gavage (ig)
Administration 15 days.Blank control group and model group give the physiological saline of equivalent respectively, once a day, successive administration 2 weeks.It sees daily
Phenomena such as surveying the weight after mouse administration, hair color, situation of ingesting, stool state, mobility, the state of mind.Last dose for 24 hours after
(being deprived of food but not water), weighs, and after etherization, plucks eyeball and takes blood, stands 30min, and 3000r/min centrifugation 10min take supernatant
Liquid, using in microplate reader and kit measurement mice serum glutamic-pyruvic transaminase ALT, glutamic-oxalacetic transaminease AST, tumor necrosis factor
TNF-α, the content of interleukins IL-2 require to be operated and measured according to kit specification.Utilize cervical dislocation
Put to death mouse, it is sterile it is lower completely strip solid tumor tumor mass, hepatic tissue, spleen tissue, thymus gland rapidly, claim respectively after normal saline flushing
Weight, knurl weight and tumour inhibiting rate and each shoot formation are calculated by formula.Tumour inhibiting rate (%)=(model control group average knurl weight-administration
Group average knurl weight)/model control group average knurl weight × 100%, shoot formation (%)=organ weight/mice weights × 100%.
H22 tumor-bearing mice ascites tumor models and test method：Mouse is randomly divided into Normal group, model group, ring phosphorus
High, medium and low (0.8g/kg, the 0.4g/kg, 0.2g/kg) dosage group of amide positive group (20mg/kg), drug of the present invention, every group 10
Only.It will pass on 10 days, grow Murine Hepatoma22 tumor-bearing mice cervical dislocation execution vigorous and without diabrosis, and put superclean bench,
It extracts the knurl liquid of milky or milk yellow under aseptic condition out from abdominal cavity, l is pressed with sterile saline:7 concentration dilution is made swollen
Oncocyte suspension, and concentration is adjusted to 5.0 × 107A/mL is spare.In addition to blank group, in every mouse peritoneal (in abdomen
Centre) injection inoculation 0.2mL tumor cell suspension, interior completion in 30min.It is administered by the following method：Cyclophosphamide positive group
Be injected intraperitoneally (ip) dosage 20mg/kg, the next day 1 time, continuous gavage (ig) be administered 15 days；Drug it is high, medium and low (0.8g/kg,
0.4g/kg, 0.2g/kg) dosage group one time a day, continuous gavage (ig) be administered 15 days.Blank control group and model group are given respectively
The physiological saline of equivalent, once a day, successive administration 2 weeks.Start observation after tumor cell inoculation until death.Experimentation
In, the variations such as weight, diet, the state of mind and build after mouse administration are observed daily, and draw mouse changes of body mass song
Line.After natural death, mouse is dissected, observes ascites and internal organ situation.And life cycle curve is drawn using Kaplan-Meier methods.
After dead mouse, mouse survival number of days is recorded, calculates each group mean survival time and median survival time, calculates and gives birth to by formula
Order rate elongation L.Every group of life span is evaluated using median survival time MST.Calculation formula：The mediant of every group of mouse number-
Dead mouse number before intermediate survival day.The mouse number of MST=(intermediate survival day -0.5)+centre survival day death.Life
The calculation formula of rate elongation is：L (%)=(N-No)/No × 100%.Wherein L is increase in life span, and N is put down for medicine group mouse
Equal survival day, No are model control group mouse the average survival time number of days.Data processing uses SPSS18.0 processing.Using pairing t
Inspection analyzed between group, with p<0.05 is used as significant difference, with p<0.01 conduct has pole significant difference to be marked to examine
It is accurate.
The drug of the present invention is to the influence result of lotus H22 solid tumor mouse models：Test each group mouse liver index, thymus gland
Index, index and spleen index, tamor index and tumour inhibiting rate the results are shown in Table 2.From table 2 it can be seen that compared with blank control group, model group
Mouse liver index, Thymus and Spleen index are remarkably decreased, and have pole significant difference (P<0.01).Cyclophosphamide is positive
Group and model group ratio, liver index, Thymus and Spleen index are significantly increased, but without difference (P>0.05).Medicine
The high, medium and low dosage group mouse liver index of object, Thymus and Spleen index are above model group, wherein drug height, middle dosage
Group is with model group than significant difference (P<0.05).Drug height, middle low dose group mouse tumor index are respectively less than model group, and
With pole significant difference (P<0.01), knurl is easily peelable, and tumor weight is substantially reduced.Document report inhibition rate of tumor growth<
40% thinks invalid, inhibition rate of tumor growth >=40%, and is statistically analyzed P<0.05 is effective.Drug high dose group presses down
Ratio of outflow reaches 72.28%, and drug middle dose group inhibitory rate has significant difference (P to 69.94%<0.05), ring phosphinylidyne
The tumour inhibiting rate of amine is 76.57%, shows that drug is high, middle dose group tumor killing effect is suitable with cyclophosphamide.And between each dosage group
Tumor killing effect be proportionate with concentration.Show drug height and middle dose group can significantly inhibit mice bearing H_ 22 tumour growth and
Improve each shoot formation of mouse.
The each shoot formation variation of 2 solid tumor mouse of table and tumor control rate
To experiment each group solid tumor mouse upon administration, mouse weight is weighed daily, draws extension Mice Body at any time
The curve changed again.Changes of weight is shown in Table 3 before and after the mouse administration of experiment each group solid tumor.It can be seen from change curve and table 3
Start within the 5th day after administration, the weight of blank control group mouse has increased slightly, but overall tendency keeps stablizing；Model group mouse
Weight gain it is apparent, and the overall apparent abruptly increase of tendency；But the mouse of cyclophosphamide group and the high, medium and low dosage group of drug to
Changes of weight unobvious after medicine 3 days, body mass index totality tendency keep stablizing.
Changes of weight before and after the administration of 5 lotus H22 solid tumors mouse of table
| Group | Average mice body weight (g) before administration | Average mice body weight (g) after administration |
| Blank control group | 21.32 | 23.32 |
| Model group | 21.69 | 34.62 |
| CTX positive groups | 20.94 | 26.97 |
| Drug high dose group | 21.37 | 25.51 |
| Drug middle dose group | 21.68 | 28.38 |
| Drug low dose group | 20.75 | 31.74 |
TNF-α in each group mice serum, IL-2, AST are tested, ALT levels are shown in Table 4.ALT and AST is that liver cell is damaged most
Sensitive index.TNF-α is inflammatory factor, participates in systemic inflammatory response, is a kind of endogenous pyrogen, can cause fever,
Inducing cell apoptosis, and inflammation can be caused by generating the factors such as IL-1, IL-2.TNF-α can also stimulate monocyte core macronucleus
Hemapoiesis and release IL-1, IL-8, cause inflammation further to develop expansion.Document points out that TNF-α is between inflammation and cancer
Indispensable powerful link, and TNF-α is not only involved in transformation and proliferation, and also becomes in the transfer of tumour
The crucial chronic inflammatory factor.Compared with Normal group, AST, ALT, TNF-α and IL-2 contents in model group mice serum
Significantly raising, and with pole significant difference (P<0.01), illustrate that liver region lesion is extremely serious in Mice Body.With model group
It compares, medicine group AST, ALT, TNF-α and IL-2 contents significantly reduce, wherein drug height and middle dose group significantly (P<
0.05), show that medicine group has apparent reduction tumor-bearing mice in-vivo tumour inflammatory factor, interleukins and transaminase
Effect.CTX positive groups are compared with model group, and AST, ALT, TNF-α and IL-2 contents reduce significantly, but otherness is not notable, table
Bright positive drug CTX has apparent tumor killing effect, but to being damaged to a certain extent property of liver.Above the experimental results showed that, this hair
Bright medicine group sample can directly act on tumour cell, significantly improve H22 solid tumor mouse liver damage situations.With apparent
Antitumor (liver cancer) effect.
TNF-α, the content of IL-2, AST, ALT in 4 lotus H22 solid tumor mice serums of table
| Group | TNF-α(μg·L-1) | IL-2(pg·L-1) | AST (millet straw) | ALT (paddy the third) |
| Control group | 83.34±1.89 | 314.32±0.37 | 37.45±16.17 | 42.58±4.28 |
| Model group | 168.84±4.93** | 485.54±1.36** | 146.36±1.38** | 163.47±8.63** |
| CTX positive groups | 97.27±12.37 | 412.35±5.92* | 62.43±0.58 | 78.46±3.65 |
| Drug high dose group | 95.71±0.83** | 323.58±4.65* | 68.36±2.74* | 82.34±1.35* |
| Drug middle dose group | 104.92±1.84** | 346.57±1.68** | 76.37±1.54** | 96.46±3.76** |
| Drug low dose group | 126.51±14.94 | 387.34±0.37** | 89.43±11.34* | 107.361.58* |
The drug of the present invention is to the influence result of lotus H22 ascites tumor mouse models：Each group mouse is tested to lotus H22 ascites tumors
The influence result of mouse weight index is：The body mass index totality tendency of blank control group mouse keeps increasing, lotus H22 ascites tumors
The body mass index totality tendency growth of mouse model group is slowed by, and the body mass index totality tendency of cyclophosphamide group becomes smaller, drug
The body mass index totality tendency of senior middle school's low dose group keeps stablizing.
Changes of weight before and after the administration of 5 Murine Hepatoma22 ascites tumor mouse of table
| Group | Average mice body weight (g) before administration | Average mice body weight (g) after administration |
| Blank control group | 20.57 | 23.67 |
| Model group | 21.18 | 32.43 |
| CTX positive groups | 21.72 | 21.72 |
| Drug high dose group | 20.36 | 22.51 |
| Drug middle dose group | 20.89 | 24.38 |
| Drug low dose group | 21.46 | 27.93 |
To experiment each group H22 ascites tumors mouse upon administration, mouse weight is weighed daily, draws extension mouse at any time
The curve of changes of weight.Changes of weight is shown in Table 5 before and after the mouse administration of experiment each group H22 ascites tumors.It can by change curve and table 5
Know, compared with blank control group, model group mouse weight after being administered the 5th day increases sharply, and abdomen gradually swells.But with model
Group is compared, and CTX and each dosage group weight gain of drug are slow.Model group mouse is extremely swelled to late period mouse web portion.Treat death
Dissection is found afterwards, and mouse ascites are in cerise, intracorporeal organ extreme degradation.The life span of mouse is obviously prolonged after administration, CTX
Group and each medicine group mouse just gradually appeared that apathetic, few dynamic, delay of response, water inlet diet be few, excrement since the 14th day
Phenomena such as dry.
It tests influence of each group mouse to lotus H22 ascites tumor Bearing Mice Life Prolongation rates and the results are shown in Table 6.As can be seen from Table 6,
The each sample administration group mouse survival time is all remarkably higher than model control group, and significant difference (P<0.05), CTX groups, medicine
The mean survival time of the high, medium and low dosage group of object extends than model control group.Compared with model control group, CTX and drug are each
Dosage group is respectively provided with statistical significance, and wherein high dose group life cycle and increase in life span significantly increases.
The life span and increase in life span of 6 ascites tumor mouse of table
| Group | Mean survival time (my god) | Increase in life span (%) |
| Model control group | 14.25±1.26 | -- |
| CTX positive groups | 21.50±0.78* | 50.8 |
| Drug high dose group | 22.25±1.36* | 56.1 |
| Drug middle dose group | 20.75±4.71* | 45.6 |
| Drug low dose group | 16.50±2.35* | 15.8 |
It is apparent that above-mentioned result of the test shows that the proliferation of natural active products confrontation human liver cancer cell prepared by the present invention has
Inhibiting effect, and can significantly inhibit the tumour growth of H22 mouse, extend the life span of mouse, to H22 mouse liver solid tumors
Apparent tumor-inhibiting action is respectively provided with ascites tumor.
Compared with prior art, the present invention it has the following advantages：
(1) present invention strangles party's Chinese prickly ash using percolation extraction method extraction natural plants.Since the extracting method remains
Certain concentration difference can be carried out continuously extraction operation, and having this method, extraction efficiency is high, extraction is fast and convenient, solvent is used
Amount is smaller, operates at room temperature, is conducive to protect heat-sensitive ingredients therein the advantages such as not to be damaged.It is concentrated using vacuum film
Device is concentrated, and has that solution to be concentrated heating temperature is relatively low, heated time is extremely short, the thermal sensitivity in effective protection concentrate into
Divide and be not destroyed, concentration speed is fast, efficient, the advantages such as easy to operate, easy to use, energy conservation and environmental protection.Party's Chinese prickly ash is strangled as day
Right plant has apparent antitumor activity.Excavating Liao Le parties Chinese prickly ash by the present invention has the new activity of antitumor action.
The plant resources for having new bioactivity as one, strangling party's Chinese prickly ash has wide development prospect.
(2) drug activity substance prepared by the present invention is using petroleum ether extraction first, and the purpose is to eliminate extract
In pigment and the impurity such as grease type, ether, ethyl acetate, extracting n-butyl alcohol is then respectively adopted, is concentrated by air-distillation
Or it is concentrated in vacuo and obtains each extraction position.And as the impurity of the big polarity such as polysaccharide, protein, polypeptide, amino acid, then it is retained in water
In solution, it is not extracted and is transferred out.In this way so that the ethereal extract, acetic acid ethyl ester extract and the n-butanol that are obtained by extraction
Drug effect active constituent is more concentrated in extract, and drug effect is more notable.
(3) present invention by ethyl acetate extract and n-butanol portion according to weight ratio 1:1 mixing, the sample being mixed with
There is more significant antitumor action than pure ethyl acetate extract and n-butanol portion.Illustrate the present invention using ethyl acetate
Position and n-butanol portion mixing, have played anti-tumor synergetic and synergistic effect, to the proliferation of liver cancer cells, inhibiting effect is more
Add significantly.
(4) present invention by ethyl acetate extract and n-butanol portion according to weight ratio 1:1 sample being mixed with, particularly
High dose group sample is respectively provided with apparent tumor-inhibiting action to H22 mouse livers solid tumor and ascites tumor, can significantly inhibit H22 small
The tumour growth of mouse substantially prolongs the time-to-live of mouse.
Claims (4)
- A kind of 1. method that active material is extracted from natural plants, which is characterized in that include the following steps：1）The crushing and extraction of raw material：The leaf of le ＆-Floor Chinese prickly ashes is dried, is ground into coarse powder, 20-40 mesh sieve is crossed, is placed in container The hydrous ethanol infiltration 4-6h that the mass concentration of middle , Yong le ＆-Floor Chinese prickly ashes 1 times of weight of coarse powder is 50-90%, is fitted into percolator, uses The 50-90% hydrous ethanols of le ＆-Floor Chinese prickly ashes 10-15 times of weight of coarse powder carry out seepage pressure effects, and diacolation is collected with 3-10mL/min flow velocitys Extracting solution；2）Flash concentration：By obtained extracting solution, flash concentration to the 1/10-1/15 of original volume, obtains concentrate below 70 DEG C；3）Decoloration removal of impurities：Concentrate is distributed in water, the dosage of water is 1/3rd of the volume of the concentrated liquid, uses petroleum ether extraction 2-5 times, each dosage is measured for 1-2 times of the volume of the concentrated liquid, to remove pigment and grease type impurity, after static layering, point removes stone Oily ether layer, merges petroleum ether extraction liquid, and vacuum concentration steams petroleum ether；4）Ether extracts the preparation at position：To decolourize concentrate ether extraction 3-6 times after cleaning, and each dosage is concentrate 1-3 times of volume is measured, and after static layering, is divided and is removed ether layer, merges ether extraction liquid, and normal pressure concentration steams ether, obtains ether Extract position concentrate, continue concentrate being concentrated in vacuo, then be dried in vacuo, with mortar it is finely ground after, obtain ether and carry Take object dry powder；5）The preparation of Ethyl acetate fraction：The water layer concentrate separated after ether is extracted, is extracted with ethyl acetate 3-6 Secondary, each dosage is 1-3 times of the volume of the concentrated liquid and measures, and after static layering, divides and removes ethyl acetate layer, combined ethyl acetate extraction Liquid after less than 60 DEG C vacuum-concentrcteds steam ethyl acetate, obtains the concentrate of Ethyl acetate fraction, vacuum drying, With mortar it is finely ground after, obtain ethyl acetate extract dry powder；6）The preparation at extracting n-butyl alcohol position：The water layer concentrate separated after ethyl acetate is extracted, with extracting n-butyl alcohol 2-5 Secondary, each dosage is 1-2 times of the volume of the concentrated liquid and measures, and after static layering, divides and removes n-butanol layer, merges butanol extraction liquid, Less than 80 DEG C carry out vacuum-concentrcted in flash concentration device, obtain n-butanol portion concentrate after steaming n-butanol, very Sky is dry, with mortar it is finely ground after, obtain n-butanol extract dry powder；7）The preparation of active material：By ethyl acetate extract dry powder and n-butanol extract dry powder according to weight ratio 1:1 is mixed It closes, obtains anti-tumor active substance.
- 2. the method according to claim 1 that active material is extracted from natural plants, which is characterized in that including following step Suddenly：1）The crushing and extraction of raw material：The leaf of le ＆-Floor Chinese prickly ashes is dried, is ground into coarse powder, 20 mesh sieve is crossed, is placed in container, use The hydrous ethanol that the mass concentration of le ＆-Floor Chinese prickly ashes 1 times of weight of coarse powder is 60% infiltrates 4 hours, is fitted into , Yong le ＆-Floor in percolator and spends The hydrous ethanol that the mass concentration of 10 times of weight of green pepper coarse powder is 60% carries out seepage pressure effects, and collecting diacolation with 7 mL/min flow velocitys carries Take liquid；2）Flash concentration：Obtained extracting solution is subjected to flash concentration to substance below 70 DEG C using Reduced Pressure Concentration Device Long-pending 1/10；3）Decoloration removal of impurities：Concentrate is distributed in water, the dosage of water is 1/3rd of the volume of the concentrated liquid, uses petroleum ether extraction 2 times, each dosage is 2 times of volume of the concentrated liquid amounts, to remove pigment and grease type impurity, after static layering, point removes petroleum ether Layer merges petroleum ether extraction liquid, is concentrated in vacuo in Rotary Evaporators and steams petroleum ether；4）Ether extracts the preparation at position：It by the concentrate after cleaning that decolourizes, is extracted 6 times with ether, each dosage is concentrate 1 times of volume is measured, and after static layering, is divided and is removed ether layer, merge ether extraction liquid, normal pressure concentrates and steams second in Rotary Evaporators Ether obtains the concentrate at ether extraction position, continues concentrate being concentrated in vacuo, then be dried in vacuo into dry powder, use mortar After finely ground, ether extract dry powder is obtained；5）The preparation of Ethyl acetate fraction：The water layer concentrate separated after ether is extracted is extracted with ethyl acetate 6 times, Each dosage is 1 times of the volume of the concentrated liquid and measures, and after static layering, divides and removes ethyl acetate layer, combined ethyl acetate extract liquor is dodging In inspissation compression apparatus, after less than 60 DEG C vacuum-concentrcteds steam ethyl acetate, the concentrate of Ethyl acetate fraction is obtained, Vacuum drying, with mortar it is finely ground after, obtain ethyl acetate extract dry powder；6）The preparation at extracting n-butyl alcohol position：The water layer concentrate separated after ethyl acetate is extracted, with extracting n-butyl alcohol 5 times, 1 times amount of each dosage for the volume of the concentrated liquid after static layering, point removes n-butanol layer, merges butanol extraction liquid, 80 DEG C with Under vacuum-concentrcted is carried out in flash concentration device, obtain n-butanol portion concentrate after steaming n-butanol, be dried in vacuo, With mortar it is finely ground after, obtain n-butanol extract dry powder；7）The preparation of active material：By ethyl acetate extract dry powder and n-butanol extract dry powder according to 1:1 combination, is resisted Tumor promotion substance.
- 3. the method according to claim 1 that active material is extracted from natural plants, which is characterized in that including following step Suddenly：1）The crushing and extraction of raw material：The leaf of le ＆-Floor Chinese prickly ashes is dried, is ground into coarse powder, 20 mesh sieve is crossed, is placed in container, use The hydrous ethanol that the mass concentration of le ＆-Floor Chinese prickly ashes 1 times of weight of coarse powder is 70% infiltrates 5 hours, is fitted into , Yong le ＆-Floor in percolator and spends The hydrous ethanol that the mass concentration of 12 times of weight of green pepper coarse powder is 70% carries out seepage pressure effects, and collecting diacolation with 6 mL/min flow velocitys carries Take liquid；2）Flash concentration：Obtained extracting solution is subjected to flash concentration to substance below 70 DEG C using Reduced Pressure Concentration Device Long-pending 1/12, obtains concentrate；3）Decoloration removal of impurities：Concentrate is distributed in water, the dosage of water is 1/3rd of the volume of the concentrated liquid, uses petroleum ether extraction 4 times, each dosage is 1.5 times of volume of the concentrated liquid amounts, to remove pigment and grease type impurity, after static layering, point removes oil Ether layer merges petroleum ether extraction liquid, is concentrated in vacuo in Rotary Evaporators and steams petroleum ether；4）Ether extracts the preparation at position：It by the concentrate after cleaning that decolourizes, is extracted 5 times with ether, each dosage is concentrate 1.5 times of volume are measured, and after static layering, are divided and are removed ether layer, merge ether extraction liquid, normal pressure is concentrated and steamed in Rotary Evaporators Ether obtains the concentrate at ether extraction position, continues concentrate being concentrated in vacuo, then be dried in vacuo, finely ground with mortar Afterwards, ether extract dry powder is obtained；5）The preparation of Ethyl acetate fraction：The water layer concentrate separated after ether is extracted is extracted with ethyl acetate 5 times, Each dosage is 1.5 times of the volume of the concentrated liquid and measures, and after static layering, divides and removes ethyl acetate layer, combined ethyl acetate extract liquor, In flash concentration device, after less than 60 DEG C vacuum-concentrcteds steam ethyl acetate, the concentration of Ethyl acetate fraction is obtained Liquid, vacuum drying, with mortar it is finely ground after, obtain ethyl acetate extract dry powder；6）The preparation at extracting n-butyl alcohol position：The water layer concentrate separated after ethyl acetate is extracted, with extracting n-butyl alcohol 4 times, Each dosage is 1.5 times of the volume of the concentrated liquid and measures, and after static layering, divides and removes n-butanol layer, merges butanol extraction liquid, at 80 DEG C Vacuum-concentrcted is carried out in flash concentration device below, after steaming n-butanol, obtains n-butanol portion concentrate, vacuum is done It is dry, with mortar it is finely ground after, obtain n-butanol extract dry powder；7）The preparation of active material：By ethyl acetate extract dry powder and n-butanol extract dry powder according to 1:1 combination, is resisted Tumor promotion substance.
- 4. the method according to claim 1 that active material is extracted from natural plants, which is characterized in that including following step Suddenly：1）The crushing and extraction of raw material：The leaf of le ＆-Floor Chinese prickly ashes is dried, is ground into coarse powder, 40 mesh sieve is crossed, is placed in container, use The hydrous ethanol that the mass concentration of le ＆-Floor Chinese prickly ashes 1 times of weight of coarse powder is 80% infiltrates 6 hours, is fitted into , Yong le ＆-Floor in percolator and spends The hydrous ethanol that the mass concentration of 13 times of weight of green pepper coarse powder is 80% carries out seepage pressure effects, and collecting diacolation with 8 mL/min flow velocitys carries Take liquid；2）Flash concentration：Obtained extracting solution is subjected to flash concentration to substance below 70 DEG C using Reduced Pressure Concentration Device Long-pending 1/13；3）Decoloration removal of impurities：Concentrate is distributed in water, the dosage of water is 1/3rd of the volume of the concentrated liquid, uses petroleum ether extraction 5 times, each dosage is 1 times of volume of the concentrated liquid amount, to remove pigment and grease type impurity, after static layering, point removes petroleum ether Layer merges petroleum ether extraction liquid, is concentrated in vacuo in Rotary Evaporators and steams petroleum ether；4）Ether extracts the preparation at position：It by the concentrate after the removal of impurities that will decolourize, is extracted 4 times with ether, each dosage is concentration 2 times of liquid product are measured, and after static layering, are divided and are removed ether layer, merge ether extraction liquid, normal pressure is concentrated and steamed in Rotary Evaporators Ether obtains the concentrate at ether extraction position, continues concentrate being concentrated in vacuo, then be dried in vacuo, finely ground with mortar After obtain ether extract dry powder；5）The preparation of Ethyl acetate fraction：The water layer concentrate separated after ether is extracted is extracted with ethyl acetate 4 times, Each dosage is 2 times of the volume of the concentrated liquid and measures, and after static layering, divides and removes ethyl acetate layer, combined ethyl acetate extract liquor is dodging In inspissation compression apparatus, after less than 60 DEG C vacuum-concentrcteds steam ethyl acetate, the concentrate of Ethyl acetate fraction is obtained, Vacuum drying, with mortar it is finely ground after, obtain ethyl acetate extract dry powder；6）The preparation at extracting n-butyl alcohol position：The water layer concentrate separated after ethyl acetate is extracted, with extracting n-butyl alcohol 3 times, 2 times amounts of each dosage for the volume of the concentrated liquid after static layering, point remove n-butanol layer, merge butanol extraction liquid, 80 DEG C with Under vacuum-concentrcted is carried out in flash concentration device, after steaming n-butanol, obtain n-butanol portion concentrate, vacuum is done It is dry, with mortar it is finely ground after, obtain n-butanol extract dry powder；7）The preparation of active material：By ethyl acetate extract dry powder and n-butanol extract dry powder according to 1:1 combination, is resisted Tumor promotion substance.
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