CN102552239A - Method for preparing anti-inflammatory and anti-tumor active ingredient group from liquorice dregs and application of anti-inflammatory and anti-tumor active ingredient group - Google Patents
Method for preparing anti-inflammatory and anti-tumor active ingredient group from liquorice dregs and application of anti-inflammatory and anti-tumor active ingredient group Download PDFInfo
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Abstract
The invention provides a method for preparing an anti-inflammatory and anti-tumor active ingredient group from liquorice dregs and an application of the anti-inflammatory and anti-tumor active ingredient group. The anti-inflammatory and anti-tumor active ingredient group prepared from the liquorice dregs comprises 13 flavone compounds; and the method for preparing the anti-inflammatory and anti-tumor active ingredient group from the liquorice dregs comprises the following steps: drying liquorice dregs; crushing the liquorice dregs; by using a solvent extracting method, extracting the liquorice dregs by ethanol or methanol; decompressing and recycling an extraction liquor so as to obtain a crude extract; suspending the crude extract by a distilled water so as to obtain an extract suspension; extracting the extract suspension by petroleum ether or cyclohexane and ethyl acetate in sequence; separating an ethyl acetate extract by silica gel column chromatography; eluting the separated ethyl acetate extract by petroleum ether/ethyl acetate or petroleum ether/acetone mixed solvent in a gradient way; separating an obtained fraction by polyamide column chromatography; and eluting the separated fraction by methanol/water or ethanol/water as mobile phase so as to obtain the active ingredient group. The anti-inflammatory and anti-tumor active ingredient group prepared by using the method provided by the invention, has the advantages of explicit composition, effects of suppressing the growth, proliferation and migration of tumor cell, quinone reductase inducing effect, tumor angiogenesis suppressing effect and anti-inflammatory effect.
Description
Technical field
The present invention relates to from the Radix Glycyrrhizae medicinal residues, prepare in the medical technical field method and the application thereof of antiinflammatory, antitumor effective ingredient group.
Background technology
Radix Glycyrrhizae (Licorice) has another name called Herba Hedyotis cantonensis, honeywort, MEICAO etc., medicinal part be pulse family (Leguminosae) Glycyrrhiza (
Glycyrrhiza) root and the root stock of plant.Radix Glycyrrhizae distributes very extensively in China, and reserves are big, and remarkable pharmacological action is one of the most frequently used medicinal plants of China, is the key object of domestic and international researcher research for many years always.Simultaneously, also just because of the remarkable pharmacological action of Radix Glycyrrhizae and of many uses causes the demand of Radix Glycyrrhizae to only increase all the time, therefore, Radix Glycyrrhizae shortage of resources problem is outstanding day by day, becomes a great problem gradually.At present, medical manufacturing enterprise often adopts that water is carried, moisture alcohol extraction, alkali are carried etc., and method is extracted glycyrrhizic acid from licorice medicinal materials, extracts the remaining Radix Glycyrrhizae medicinal residues in back and discards as industrial waste usually.For the medical value of this commercial production waste material of Radix Glycyrrhizae medicinal residues and further illustrating of economic worth, help solving the difficult problem that the production waste disposal is gone up in industry, more fully reasonable use Radix Glycyrrhizae plant resources and the ecological ring of protection.
We find to be rich in the Radix Glycyrrhizae medicinal residues a large amount of flavone compounds in research work, contained flavone compound is similar basically in its structure and the Radix Glycyrrhizae crude drug, in industrial process, also have the chemical compound of newtype to produce simultaneously.In order further to illustrate the difference of Radix Glycyrrhizae medicinal residues and former plant biological activity and material base, we are guidance with antitumor, antiinflammatory, quinone reductase induced activity, to the chemical constituent of the medicinal residues of Radix Glycyrrhizae and former plant carried out separating, analysis and contrast.The result shows; Radix Glycyrrhizae medicinal residues total flavones (being made up of 13 kinds of clear and definite flavone compounds of structure) has the effect of good restraining growth of tumour cell, inhibition tumor cell proliferation and migration, quinone reductase inducing action, tumor-blood-vessel growth inhibitory action, antiinflammatory action, can be used for the development and application of anti-inflammatory drug, antitumor drug, chemoprevention of cancer medicine.
Based on this we set up from the Radix Glycyrrhizae medicinal residues fast, enrichment effective site cheaply---the method for Radix Glycyrrhizae medicinal residues total flavones, analyze, detect in conjunction with HPLC, confirmed the content assaying method of 13 kinds of bonded structures of flavonoid and 3 kinds of main components; The clear and definite content requirement of main constituent: licochalcone A content>6%; Licoflavone B content>0.7%, α, 2'; 4,4'-tetrahydroxy chalcone derivative content>0.1%.Therefore, develop a kind of method and application thereof that from the Radix Glycyrrhizae medicinal residues, prepares antiinflammatory, antitumor effective ingredient group is the new problem that needs to be resolved hurrily always.
Summary of the invention
The object of the invention is to provide a kind of method and application thereof that from the Radix Glycyrrhizae medicinal residues, prepares antiinflammatory, antitumor effective ingredient group; This effective ingredient group has the effect of the growth of tumour cell of inhibition, inhibition tumor cell proliferation and migration, quinone reductase inducing action, tumor-blood-vessel growth inhibitory action, antiinflammatory action, can be used for the development and application of anti-inflammatory drug, antitumor drug, chemoprevention of cancer medicine.
The objective of the invention is to realize like this: a kind of antiinflammatory, antitumor effective ingredient group of from the Radix Glycyrrhizae medicinal residues, preparing; Form by 13 kinds of flavone compounds, have structure as shown in table 1 (wherein licochalcone A content>6%, licoflavone B content>0.7%; α; 2', 4,4'-tetrahydroxy chalcone derivative content>0.1%):
Table 1 Radix Glycyrrhizae medicinal residues effective ingredient group is formed
The method for preparing one that from the Radix Glycyrrhizae medicinal residues, prepares antiinflammatory, antitumor effective ingredient group, its preparation method comprise the steps,
(1) dry Radix Glycyrrhizae medicinal residues are pulverized after solvent extraction method, adopt ethanol or the methanol extraction of 60%-100%, and the reclaim under reduced pressure extracting solution gets crude extract;
(2) crude extract obtains extract suspendible solution with the distilled water suspendible, successively with petroleum ether or cyclohexane extraction, ethyl acetate extraction;
(3) step (2) gained acetic acid ethyl ester extract separates through silica gel column chromatography, with petrol ether/ethyl acetate or petroleum ether/acetone mixed solvent gradient elution;
(4) the gained flow point separates through polyamide column chromatography in the above-mentioned steps (3), is the mobile phase eluting with methanol or ethanol/water, obtains the effective ingredient group;
Extractant is 1:3-3:1 with extract suspendible liquor capacity ratio, and extraction times is 2-5 time; The silica gel column chromatography eluting solvent is petrol ether/ethyl acetate or the petroleum ether/acetone of 10:1-1:2; Polyamide column chromatography mobile phase is methanol or ethanol/water, is 1:9-9:1 from methanol or ethanol/water mixed solvent ratio, and methanol or ethanol/water mixed proportion are that 3:7-6:4 partly gets the grouping of target effective one-tenth;
The method for preparing two that from the Radix Glycyrrhizae medicinal residues, prepares antiinflammatory, antitumor effective ingredient group, its preparation method comprise the steps,
(1) dry Radix Glycyrrhizae medicinal residues are pulverized after solvent extraction method, adopt ethanol or the methanol extraction of 60%-100%, and the reclaim under reduced pressure extracting solution gets crude extract;
(2) crude extract is further purified through macroporous adsorbent resin, successively with methanol or ethanol/water eluting;
(3) step (2) gained eluate separates through polyamide column chromatography, is the mobile phase eluting with methanol or ethanol/water, obtains the effective ingredient group;
Macroporous adsorbent resin chromatography eluting solvent methanol or ethanol/water mixed solvent ratio are 1:9-9:1, methanol or ethanol/water mixed proportion be the eluate of 5:5-9:1 part merge thick product; Polyamide column chromatography separates, and mobile phase is methanol or ethanol/water, is 1:9-9:1 from methanol or ethanol/water mixed solvent ratio, and methanol or ethanol/water mixed proportion are that 3:7-6:4 partly gets the grouping of target effective one-tenth; Have the effect of the growth of tumour cell of inhibition, quinone reductase inducing action, antiinflammatory action, can be used for the development and application of anti-inflammatory drug, antitumor drug, chemoprevention of cancer medicine.
Main points of the present invention are its method for preparing and application thereof.Its principle is construction features and the physicochemical property according to flavones ingredient in the Radix Glycyrrhizae medicinal residues, chooses The suitable solvent and utilizes the solvent refluxing extraction method to prepare Radix Glycyrrhizae medicinal residues extract.Utilize flavone compound polarity and deliquescent difference in the Radix Glycyrrhizae medicinal residues, adopt system's solvent extraction binding silica gel column chromatography, or macroporous absorption resin chromatography carries out preliminary separation and purification; Make full use of in the Radix Glycyrrhizae medicinal residues difference of substituent group kind, phenolic hydroxyl group number in the flavone compound structure subsequently, utilize polyamide column chromatography to carry out further separation and purification, obtain Radix Glycyrrhizae medicinal residues total flavones.Utilize the HPLC technology, the composition of Radix Glycyrrhizae medicinal residues total flavones and the content of main component have been carried out assay determination, and test the final content requirement of having worked out product through different batches.For the practical application of the Radix Glycyrrhizae medicinal residues total flavones for preparing, we have adopted multiple external pharmacological evaluation model to illustrate and prove.Utilize mtt assay to test the GIA of Radix Glycyrrhizae medicinal residues total flavones to different tumor cells, and to the inhibitory action of tumor cell proliferation and migration; Utilize Hepa 1c1c7 cell screening model, tested the induced activity of Radix Glycyrrhizae medicinal residues total flavones quinone reductase; Utilize the RAW264.7 cell model, tested the effect of Radix Glycyrrhizae medicinal residues total flavones inhibition RAW264.7 cell activation.
From the Radix Glycyrrhizae medicinal residues, prepare the method for antiinflammatory, antitumor effective ingredient group and use compared with prior art; Having the effective ingredient group forms clear and definite; Main constituent content is clear; And have the effect of the growth of tumour cell of inhibition, inhibition tumor cell proliferation and migration, quinone reductase inducing action, tumor-blood-vessel growth inhibitory action, antiinflammatory action; Can be used for the advantages such as development and application of anti-inflammatory drug, antitumor drug, chemoprevention of cancer medicine, will be widely used in the medical technical field.
Description of drawings:
Below in conjunction with accompanying drawing and embodiment the present invention is elaborated.
The HPLC analysis chart of Fig. 1 gained Radix Glycyrrhizae medicinal residues effective ingredient group extract.
The specific embodiment:
Following embodiment will further explain the present invention, but therefore not limit the present invention.
Embodiment one
(1) dry Glycyrrhiza inflata Bat. medicinal residues (water carry glycyrrhizic acid after gained) 10kg is through 95% alcohol reflux three times, and 2 hours, 2 hours, 1.5 hours, the reclaim under reduced pressure extracting solution got the ethanol crude extract;
(2) ethanol extraction is suspended in the 5L distilled water, uses isopyknic petroleum ether, ethyl acetate extraction four times respectively;
(3) step (2) gained acetic acid ethyl ester extract separates through silica gel column chromatography, with petroleum ether: acetone 10:1,2:1, the 1:1 eluting,
(4) gained petroleum ether in the above-mentioned steps (3): acetone 2:1 flow point separates through polyamide column chromatography, with ethanol/water 2:8, and 6:4, the 9:1 gradient elution, ethanol/water 6:4 eluate is the effective ingredient group.
(5) analyze through HPLC, the method for standard substance comparison, clear and definite the composition of products therefrom (structure of S1-S13 is as shown in table 1):
Experimental technique: experimental apparatus: Hitachi Model D-2000 HPLC
Chromatographic condition acetonitrile-0.1% formic acid water
0-10?min?15-20%
10-12?min?20-23%
12-32?min?23-24%
32-63?min?24-55%
63-75?min?55-80%
Fig. 1 representes the HPLC analysis chart of gained Radix Glycyrrhizae medicinal residues effective ingredient group extract.
(6) adopt the HPLC method to measure licochalcone A content in the gained Radix Glycyrrhizae medicinal residues effective ingredient group: 7.1%, licoflavone B content: 1.7%, α, 2', 4,4'-tetrahydroxy chalcone derivative content: 0.9%.
Embodiment two
(1) dry Radix Glycyrrhizae medicinal residues (gained behind the aqueous alkali method extraction glycyrrhizic acid) 5kg is that solvent refluxing extracts with methanol, and the reclaim under reduced pressure extracting solution gets methanol crude extract;
(2) methanolic extract is suspended in the 1L distilled water, through the separation and purification of HPD100 macroporous adsorbent resin, uses the ethanol/water mixed solvent ratio to be 3:7 successively, 6:4, and the 9:1 eluting, the ethanol/water mixed proportion is that the eluate of 6:4 part gets thick product
(3) the thick product of step (2) gained separates through polyamide column chromatography, uses the ethanol/water mixed solvent ratio to be 2:8 successively, 4:6, and 6:4, the 8:2 eluting, the ethanol/water mixed proportion is that 4:6～6:4 part eluate must get the grouping of target effective one-tenth.
(4) analyze through HPLC, the method for standard substance comparison, the clear and definite composition of products therefrom, experimental technique is with embodiment one.
(5) adopt the HPLC method to measure licochalcone A content in the gained Radix Glycyrrhizae medicinal residues effective ingredient group: 6.7%, licoflavone B content: 0.7%, α, 2', 4,4'-tetrahydroxy chalcone derivative content: 0.3%.
Embodiment three
The quinone reductase induced activity test of effective ingredient group
1. experiment material:
(1) main agents and capital equipment: calf serum (available from Hyclone); Hyclone (TBD company);
Tetramethyl azo azoles salt (MTT) U.S. Sigma (St. Louis, MO); Glucose-6-phosphate dehydrogenase (G6PD) (worker is given birth in Shanghai); 96 porocyte culture plates (Costar company); Cell strain: Hepar Mus JEG-3 Hepa 1c1c7
2. experimental technique:
(1) cultivation of cell:
Every hole kind 10
4Individual cell, growth is 24 hours in the culture fluid that comprises 10% (v/v) hyclone, 0.01% benzylpenicillin, 0.15% sodium bicarbonate, 0.01% streptomycin sulfate, and environmental condition is 37 ℃, contain 5%CO
2Humid air.
(2) preparation of medicine
Testing compound uses the DMSO dissolving, is made into the mother solution that concentration is 50mmol/L, is stored in-20 ℃.
(3) the crystal violet method is confirmed cell survival rate
The testing compound of concentration known is dissolved in adds each hole among the DMSO and kept 24 hours.Should guarantee the final concentration of DMSO behind every hole adding DMSO
<0.5% (v/v).Discard culture fluid afterwards, every hole adds 200 μ L, 0.2 μ M crystal violet (2% alcoholic solution).Room temperature held dyeing about 10 minutes discards crystal violet solution, and water washs rapidly 3 times, and water is dried and use drier.Every hole adds 200 μ L, 0.5% SDS (50% alcoholic solution), and room temperature vibrated 5～10 minutes down, and 595 nm measure absorbance down.The absorbance that records is proofreaied and correct through blank control group (hole of gutless cell), and representes cell survival rate with the percentage rate that is equivalent to matched group (cell in the hole that does not have to handle) absorbance.Every group of experiment should independently at least repeat 3 times and average as end product.
Meansigma methods * 100% of the meansigma methods of cell survival rate %=administration group OD value/blank breast group OD value
(4) the quinone reductase induced activity is measured
It is that G-6-P can be reduced by glucose-6-phosphate dehydrogenase (G6PD) under the condition that cofactor NADP exists that QR induces the ultimate principle of mensuration; At this moment can produce NADPH; NADPH is in case formation just makes menadione be reduced to menadiol as a kind of electron donor; Menadiol can make MTT be reduced to the Jia Za, but last Dui Jia Za carries out the mensuration of absorbance.NADP and menadione are renewable in this catalytic cycle process, so need not replenish it in the experiment.The QR derivant can increase the generation of menadiol, thereby more Jia Za is formed.This experiment in, the compound concentrations that adopts to pass through Hepa 1c1c7 cell screening in advance, its concentration should satisfy makes cell survival rate
>55%.
Active being determined on 96 orifice plates of quinone reductase carried out with the Hepar Mus cancerous cell, and process is described below: behind the cell culture testing compound of concentration known is dissolved in and adds each hole among the DMSO and kept 24 hours.Should guarantee the final concentration of DMSO behind every hole adding DMSO
<0.5% (v/v).After this, culture fluid poured out and add contain 0.8% (w/v) digitonin and 2 mM EDTA, stir 10 minutes with peptic cell.Add " complete reaction mixes liquid " 200 μ l in every hole.This mixed liquid should face uses preceding configuration, and collocation method is following: the solution that Tris-Cl (pH=7.4), 100 mg calf serums, the polysorbas20 of 1 mL 1.5%, the FAD of 0.1 mL, 7.5 mM, the G-6-P of 1 mL, 150 mM, the NADP of 90 μ L, 50 mM, 300 unit glucose-6-phosphate dehydrogenase (G6PD)s, the 45 mg MTT of 7.5 mL, 0.5 M is configured to 150 mL with deionized water.Before adding mixed liquid, add 0.2 μ L menadione (50 mM are dissolved in the acetonitrile).96 orifice plates jolting 5 minutes gently after the completion to be added.0.3 mM dicoumarol is dissolved in 0.5%DMSO and the 5 mM potassium dihydrogen phosphates (pH=7.4) to be drawn 50 μ L and adds on the entering plate in every hole with cessation reaction.Under 590 nm wavelength, measure absorbance.Do not contain cell in the blank control group, matched group is a Hepa 1c1c7 cell and contain 0.5% DMSO substrate liquid but do not contain testing compound.The computational methods of the QR induced activity of testing compound: earlier the absorbance of administration group and matched group is deducted the absorbance of blank control group, refer to that with the absorbance of administration group absorbance (IR) than last matched group is as the index of QR induced activity then.Every group of experiment should independently at least repeat 3 times.
3. experimental result:
Guaranteeing that cell survival rate is higher than under 55% the situation, IR shows promptly that greater than 2 specimen has the QR inducing action, can be known by last table result, and Radix Glycyrrhizae medicinal residues effective ingredient group is than Radix Glycyrrhizae medicinal residues total extract, and the QR induced activity obviously increases, and toxicity is lower.
Embodiment four
Radix Glycyrrhizae medicinal residues effective ingredient group suppresses the test of RAW264.7 cell activation effect
1. experiment material:
(1) receives test product: Radix Glycyrrhizae medicinal residues total extract (sample 1), Radix Glycyrrhizae medicinal residues effective ingredient group (sample 2).
(2) positive drug: with NO release inhibitor minocycline (MINO) as positive control drug.
(3) experimental cell strain and source:
RAW264.7 macrophage: available from ATCC.
(4) experiment reagent and instrument:
DMEM culture medium Gibco BRL lot number: 1637269
Hyclone Gibco BRL lot number: 1339728
LPS (E5:055) U.S. Sigma lot number: 069k4005
Tetramethyl azo azoles salt (MTT) U.S. Sigma lot number: 201108
Dimethyl sulfoxide (DMSO) Shenyang chemical reagent factory
ELIASA Austria TECAN
96 porocyte culture plate Costar companies
2. experimental technique:
(1) cultivation of RAW264.7 macrophage system
All glass drying ovens that use in cell culture and the modelling and metallic weapon (culture bottle, pipet, solution bottle etc.) are all through 121 ℃ of autoclaving 30min, with the LPS of thorough removal pollution.Be mixed with the cell culture fluid that includes 10% hyclone, 100U/mL penicillin and 100U/mL streptomycin and 50 μ M 2 mercapto ethanols as basal medium with the DMEM culture medium.Macrophage is with about 4 * 10
5The concentration of cells/mL is at 5% CO
2, the cultivation of going down to posterity in 37 ℃ of culture bottles.
(2) method for preparation of drug
Testing sample is Powdered, dissolves with DMSO.Be made into mother solution, concentration is 100 mM or 100 μ g/mL, is stored in-20 ℃.Face the time spent and dilute with serum-free DMEM culture fluid, dilution is 100 μ g/mL, 10 μ g/mL, 1 μ g/mL and 0.1 μ g/mL successively.DMSO final concentration < 1 ‰.
(3) Griess method detection compound stimulates the inhibitory action of macrophage to LPS
[3]
The take the logarithm RAW264.7 macrophage of trophophase transfers to 5 * 10 with the fresh DMEM culture medium that contains 10% hyclone with cell density
5Cells/mL is inoculated in 96 orifice plates, 100 μ L/well, and in 37 ℃, 5%CO
2Incubator in cultivate.Cell attachment is cultivated the fresh medium that changes serum-free behind 24 h into, carries out dosing simultaneously and handles.Sample and LPS combined effect.The LPS final concentration is 10 ng/mL in each administration group and the positive controls.After cell dosing continued is cultivated 24 h, collect supernatant, the Griess colorimetry detects NO in the supernatant
2-Content.
(4) the mtt assay detection compound is to the influence of RAW264.7 macrophage survival rate
[4]
The RAW264.7 macrophage that the trophophase of taking the logarithm is cultivated transfers to 5 * 10 with the fresh DMEM culture medium that contains 10% hyclone with cell density
5Cells/mL is inoculated in 96 orifice plates, 100 μ L/well, and in 37 ℃, 5%CO
2Incubator in cultivate.Cell attachment changes fresh medium into after cultivating 24 h, carries out dosing simultaneously and handles.Sample number and LPS combined effect.Establish blank and positive control simultaneously.The LPS final concentration is 10 ng/mL in each administration group and the positive controls.Cell dosing continued is cultivated 24h, in Cell sap, adds MTT solution then, and 10 μ L/well are hatched 3 h jointly with cell and 0.25 mg/ml MTT under 37 ℃, absorb culture fluid, add isopyknic DMSO solution then, measure its optical density OD value.Date processing utilizes the ELIASA corresponding software to carry out date processing, calculates the meansigma methods of each three holes of sample OD value, utilize meansigma methods by following formula calculate cell survival rate (Cell Viability, CV%):
Meansigma methods * 100% of the meansigma methods of cell survival rate %=sample sets OD value/blank group OD value
(5) statistical method
All data adopt the analysis of testing of SPSS13.0 statistical packages.The result representes with Mean ± SE, estimates globality difference, and mean adopts One-Way ANOVA analytic process to carry out the homogeneity of variance analysis between group, and combine Dunnett ' stest analytical method organize between relatively.The Levene check is adopted in the multisample homogeneity test of variance, works as P>0.05, variance is a homogeneous, the difference of mean between the many groups of employing Dunnett ' s bilateral T check, < 0.05, heterogeneity of variance adopts Dunnett T3 to check the difference of mean between many groups as P.
(6) IC
50Computational methods
[5]
Parameters such as each dosage and suppression ratio are calculated IC with nonlinear regression and fitting
50
3. experimental result:
Sample stimulates RAW264.7 cell NO to discharge the influence (Mean ± SE) of (%) to LPS
Under the condition that does not influence cell survival rate, the RAW264.7 cellular inflammation factor NO that sample 2 (Radix Glycyrrhizae medicinal residues effective ingredient group) can reduce LPS to stimulate discharges, and this points out it to produce regulating action to inflammatory reaction.
Embodiment five
The test of Radix Glycyrrhizae medicinal residues effective ingredient group anti tumor activity in vitro
1. experiment material
(1) receives test product: Radix Glycyrrhizae medicinal residues total extract (sample 1), Radix Glycyrrhizae medicinal residues effective ingredient group (sample 2)
(2) experimental cell strain and source, human leukemia cell HL-60 (available from ATCC)
(3) experiment reagent
RPMI1640 culture medium: Gibco, lot number: 307964; Hyclone: Tianjin Hao ocean biological product company limited lot number: 20090525; Methyl-azoles salt (MTT): U.S. Sigma, lot number: 201108; Dimethyl sulfoxide (DMSO): Shenyang chemical reagent factory; NaCl, KCl, KH
2PO
3, Na
2HPO
3, NaHCO
3: Shenyang chemical reagent factory; ELIASA: Austrian TECAN; 96 porocyte culture plates: Costar company.
2, experimental technique
(1) drug treating
1) dissolving of medicine
Sample is Powdered, uses the DMSO dissolving.Be made into the mother solution that concentration is 100mg/mL, be stored in-20 ℃.Face the time spent and with corresponding culture fluid it is diluted, dilution is that 100 μ g/mL, 10 μ g/mL, 1 μ g/mL and 0.1 μ g/mL experimentize.When the sample of DMSO configuration experimentized, the final concentration of DMSO was 1 ‰.Positive drug etoposide concentration is 100 μ mol/L, 10 μ mol/L, 1 μ mol/L and 0.1 μ mol/L.
2) administration is handled
The take the logarithm cell of trophophase is adjusted suitable cell density, is inoculated in 96 orifice plates, and 100 μ l/well are incubated at 37 ℃, in the incubator of 5%CO2.Dosing behind the cultivation 24h, effect 48h.Set up blank control group, administration group and positive controls separately, establish 6 multiple holes for every group.
(2) MTT detects
1) ultimate principle of mtt assay
Cell survival rate is measured and is adopted the MTT analytic process, and ((4,5-dimethyl-2thiahiazoy1)-3,5-di-phenyl-tetrazolium bromide MTT) is the basis to 3-with living cells metabolism reduction tetramethyl azo azoles salt.MTT is a yellow compound; It is the hydrionic dyestuff of a kind of acceptance; Can act on the respiratory chain in the living cells mitochondrion; Tetrazole ring opening under the effect of succinate dehydrogenase and cytochrome C generates blue Formazan crystallization, and the crystalline growing amount of Formazan only is directly proportional with the living cells number.This enzyme disappears in the dead cell; Can not with the MTT lysate of 20% dodecyl sodium sulfate (pH4.7) in dissolve; Utilize ELIASA to measure the optical density OD value that 492 nm go out; The size of OD value is directly proportional with the crystalline amount of the Formazan that is generated, thereby reflects the influence of medicine pair cell survival rate.
2) assay method of mtt assay
Behind the drug effect 48h, cell and 0.25mg/ml MTT are hatched 3-4h jointly under 37 ℃, centrifugal, carefully absorb culture fluid after every hole add 100 μ l dimethyl sulfoxide (DMSO), dissolving back uses ELIASA to measure its optical density OD value in 492nm fully.Be 100% with blank control group OD value at last, calculate and respectively organize cell inhibitory rate.
Cell inhibitory rate %=1-
(3) statistical method
All data adopt the analysis of testing of SPSS (16.0) statistical packages.Each organize data with the mean standard error (expression of Mean ± S.E.) adopts One-Way ANOVA to estimate globality difference, and carry out Dunnett or Dunnett ' s T3 check organize between relatively.
(4) computational methods of IC50
Parameters such as each dosage and suppression ratio are calculated IC50 with nonlinear regression and fitting.
3, experimental result
Sample is to the survival suppression ratio (%) of HL-60 cell strain (Mean ± SE)
The result shows that Radix Glycyrrhizae medicinal residues total extract and Radix Glycyrrhizae medicinal residues effective ingredient group all have anti tumor activity in vitro, and the activity of Radix Glycyrrhizae medicinal residues effective ingredient group is significantly higher than Radix Glycyrrhizae medicinal residues total extract.
Claims (9)
1. one kind prepares antiinflammatory, antitumor effective ingredient group from the Radix Glycyrrhizae medicinal residues; It is characterized in that: form by 13 kinds of flavone compounds, have structure as shown in table 1 (wherein licochalcone A content>6%, licoflavone B content>0.7%; α; 2', 4,4'-tetrahydroxy chalcone derivative content>0.1%):
Table 1 Radix Glycyrrhizae medicinal residues effective ingredient group is formed
2. the method for preparing one that from the Radix Glycyrrhizae medicinal residues, prepares antiinflammatory, antitumor effective ingredient group according to claim 1 is characterized in that: its preparation method comprises the steps,
(1) dry Radix Glycyrrhizae medicinal residues are pulverized after solvent extraction method, adopt ethanol or the methanol extraction of 60%-100%, and the reclaim under reduced pressure extracting solution gets crude extract;
(2) crude extract obtains extract suspendible solution with the distilled water suspendible, successively with petroleum ether or cyclohexane extraction, ethyl acetate extraction;
(3) step (2) gained acetic acid ethyl ester extract separates through silica gel column chromatography, with petrol ether/ethyl acetate or petroleum ether/acetone mixed solvent gradient elution;
(4) the gained flow point separates through polyamide column chromatography in the above-mentioned steps (3), is the mobile phase eluting with methanol or ethanol/water, obtains the effective ingredient group.
3. the method for preparing that from the Radix Glycyrrhizae medicinal residues, prepares antiinflammatory, antitumor effective ingredient group according to claim 2 is characterized in that: extractant is 1:3-3:1 with extract suspendible liquor capacity ratio, and extraction times is 2-5 time.
4. the method for preparing that from the Radix Glycyrrhizae medicinal residues, prepares antiinflammatory, antitumor effective ingredient group according to claim 2 is characterized in that: the silica gel column chromatography eluting solvent is petrol ether/ethyl acetate or the petroleum ether/acetone of 10:1-1:2.
5. the method for preparing that from the Radix Glycyrrhizae medicinal residues, prepares antiinflammatory, antitumor effective ingredient group according to claim 2; It is characterized in that: polyamide column chromatography mobile phase is methanol or ethanol/water; From methanol or ethanol/water mixed solvent ratio is 1:9-9:1, and methanol or ethanol/water mixed proportion are that 3:7-6:4 partly gets the grouping of target effective one-tenth.
6. the method for preparing two that from the Radix Glycyrrhizae medicinal residues, prepares antiinflammatory, antitumor effective ingredient group according to claim 1 is characterized in that: its preparation method comprises the steps,
(1) dry Radix Glycyrrhizae medicinal residues are pulverized after solvent extraction method, adopt ethanol or the methanol extraction of 60%-100%, and the reclaim under reduced pressure extracting solution gets crude extract;
(2) crude extract is further purified through macroporous adsorbent resin, successively with methanol or ethanol/water eluting;
(3) step (2) gained eluate separates through polyamide column chromatography, is the mobile phase eluting with methanol or ethanol/water, obtains the effective ingredient group.
7. the method for preparing that from the Radix Glycyrrhizae medicinal residues, prepares antiinflammatory, antitumor effective ingredient group according to claim 6; It is characterized in that: macroporous adsorbent resin chromatography eluting solvent methanol or ethanol/water mixed solvent ratio are 1:9-9:1, methanol or ethanol/water mixed proportion be the eluate of 5:5-9:1 part merge thick product.
8. the method for preparing that from the Radix Glycyrrhizae medicinal residues, prepares antiinflammatory, antitumor effective ingredient group according to claim 6; It is characterized in that: polyamide column chromatography separates; Mobile phase is methanol or ethanol/water; From methanol or ethanol/water mixed solvent ratio is 1:9-9:1, and methanol or ethanol/water mixed proportion are that 3:7-6:4 partly gets the grouping of target effective one-tenth.
9. antiinflammatory, the antitumor effective ingredient group of from the Radix Glycyrrhizae medicinal residues, preparing according to claim 1; It is characterized in that: have the effect of the growth of tumour cell of inhibition, quinone reductase inducing action, antiinflammatory action, can be used for the development and application of anti-inflammatory drug, antitumor drug, chemoprevention of cancer medicine.
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104013668A (en) * | 2014-06-27 | 2014-09-03 | 新疆维吾尔自治区中药民族药研究所 | Application of licoflavone extract in preparation of medicine for treating ulcerative colitis |
| CN105012289A (en) * | 2014-04-29 | 2015-11-04 | 上海鑫昊生物科技有限公司 | Anti-AIDS application of licoricone or derivative of licoricone |
| CN105232501A (en) * | 2015-11-17 | 2016-01-13 | 北京大学 | Novel application of echinatin |
| CN105777521A (en) * | 2016-05-11 | 2016-07-20 | 西安兴博凯生物科技有限责任公司 | Industrial separating and purifying method for licoflavone series monomer |
| CN105997990A (en) * | 2015-11-10 | 2016-10-12 | 邢丽丽 | Medicine composition for treating infantile diarrhea |
| CN107510710A (en) * | 2017-10-12 | 2017-12-26 | 沈阳药科大学 | A kind of method and medical usage that diabetes B target spot inhibitor is enriched with from Glycyrrhiza uralensisFisch residue |
| CN110772504A (en) * | 2019-02-27 | 2020-02-11 | 中国人民解放军总医院第五医学中心 | Application of echinocandin as inhibitor |
| CN112494478A (en) * | 2020-12-04 | 2021-03-16 | 新疆维吾尔自治区中药民族药研究所 | Composition with anti-inflammatory synergistic effect and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1752081A (en) * | 2005-11-16 | 2006-03-29 | 李秉� | Method of extracting licorice flavone from licorice slag |
| CN102151296A (en) * | 2010-02-11 | 2011-08-17 | 兰州大学 | Method for extracting active ingredients for reducing sugar blood from waste residues of liquorice |
-
2011
- 2011-12-31 CN CN 201110456688 patent/CN102552239B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1752081A (en) * | 2005-11-16 | 2006-03-29 | 李秉� | Method of extracting licorice flavone from licorice slag |
| CN102151296A (en) * | 2010-02-11 | 2011-08-17 | 兰州大学 | Method for extracting active ingredients for reducing sugar blood from waste residues of liquorice |
Non-Patent Citations (2)
| Title |
|---|
| 刘芬等: "甘草药渣化学成分的分离与鉴定", 《中国药物化学杂志》 * |
| 刘芬等: "甘草药渣的研究进展", 《中国现代中药》 * |
Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105012289A (en) * | 2014-04-29 | 2015-11-04 | 上海鑫昊生物科技有限公司 | Anti-AIDS application of licoricone or derivative of licoricone |
| CN104013668A (en) * | 2014-06-27 | 2014-09-03 | 新疆维吾尔自治区中药民族药研究所 | Application of licoflavone extract in preparation of medicine for treating ulcerative colitis |
| CN104013668B (en) * | 2014-06-27 | 2017-10-24 | 新疆维吾尔自治区中药民族药研究所 | Licoflavone class extract is used to prepare to be applied in treatment ulcerative colitis medicine |
| CN105997990A (en) * | 2015-11-10 | 2016-10-12 | 邢丽丽 | Medicine composition for treating infantile diarrhea |
| CN105232501A (en) * | 2015-11-17 | 2016-01-13 | 北京大学 | Novel application of echinatin |
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| CN110772504A (en) * | 2019-02-27 | 2020-02-11 | 中国人民解放军总医院第五医学中心 | Application of echinocandin as inhibitor |
| WO2020173486A1 (en) * | 2019-02-27 | 2020-09-03 | 中国人民解放军总医院第五医学中心 | Use of echinatin as inflammasome inhibitor |
| CN112494478A (en) * | 2020-12-04 | 2021-03-16 | 新疆维吾尔自治区中药民族药研究所 | Composition with anti-inflammatory synergistic effect and application thereof |
| CN112494478B (en) * | 2020-12-04 | 2022-06-07 | 新疆维吾尔自治区中药民族药研究所 | Composition with anti-inflammatory synergistic effect and application thereof |
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