CN101926844B - Stellera chamaejasme L extract and anti-tumor action thereof - Google Patents
Stellera chamaejasme L extract and anti-tumor action thereof Download PDFInfo
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Abstract
The invention relates to a Stellera chamaejasme L extract and an anti-tumor action thereof. The Stellera chamaejasme L extract of the invention is extracted by absolute ethyl alcohol to obtain a primary extract; the primary extract is further treated by macroporous resin column chromatography or polyamide column chromatography and then eluted by water and ethyl alcohol to obtain a secondary extract; and in the secondary extract, the secondary extract separated by a macroporous resin column is further treated by polyamide column chromatography and then eluted by water and ethyl alcohol to obtain a tertiary extract.
Description
Technical field:
The present invention relates to new extract and extracting method and the purposes of Chinese medicine Stellera chamaejasme L..
Background technology:
Stellera chamaejasme L. (Stellera chamaejasme L.) is the root of thymelaeceae stellera plant, it is the certified products in the Radix Euphorbiae Fischerianae (Radix Euphorbiae Ebracteolatae), Shennong's Herbal begins to be stated from, it is hot, bitter to call its nature and flavor, flat, poisonous, enter lung, liver, spleen channel, function with " expectorant powder junction of relieving oedema or abdominal distension through diuresis or purgation " cures mainly " gathering diet, the cold and heat aqueous vapor ".Clear Zhang Zhi clever " herbal high former " annotates and says: " removing mass is poly-, and then diet is stopped up stagnant and is the disease of cold and heat, also can control." the Sun Simiao notes: " accumulator, so air flue is not all right ear.Diet cold and heat person, gas loses it and controls, and stagnation of QI water holds, and this medicine suffering can be opened it." Li Shizhen (1518-1593 A.D.) because of its " poisonous " so in Compendium of Material Medica, classify them as low-grades.Fixed its nature and flavor of " property of medicine opinion " of the beam Zhen Quan of the Northern and Southern Dynasties be " hardship, suffering, poisonous." the beam TAO Hong-Jing puts down in writing the Radix Euphorbiae Fischerianae (Radix Euphorbiae Ebracteolatae) " except long-pending addiction under the breast in " Mingyi Bielu "." Holy Benevolent Prescriptions controls with Radix Euphorbiae Fischerianae (Radix Euphorbiae Ebracteolatae) ball and gather, abdominal distnetion is principal agent such as drum person with the Radix Euphorbiae Fischerianae (Radix Euphorbiae Ebracteolatae).It is hard that " mending the fault Handbook of Prescriptions for Emergencies " controls soldier Xin Fu Disorder, under two sides of body depressed person arranged, and is principal agent with the Radix Euphorbiae Fischerianae (Radix Euphorbiae Ebracteolatae).
Statistics comprises about 109 altogether of the prescriptions of the Radix Euphorbiae Fischerianae (Radix Euphorbiae Ebracteolatae) Christian era between 618-1911, wherein with the treatment phlegm syndrome, the difficult miscellaneous diseases person such as to gather in the majority.Among the people in China, also have and much utilize the Radix Euphorbiae Fischerianae (Radix Euphorbiae Ebracteolatae) to treat the proved recipe of the malignant tumor such as gastric cancer, hepatocarcinoma, the esophageal carcinoma, breast cancer tumour, thyroid carcinoma.Many pieces of bibliographical informations are also arranged clinically, and stellera chamaejasme L extract treatment malignant tumor obtains good effect.Modern medicine study finds that it has good antitumor action, and numerous scholars place high hopes to the potential important medical value of this Chinese medicine in the native land.
In recent years, domestic external in the ascendant with pharmaceutical research to the Stellera chamaejasme L. chemistry.Stellera chamaejasme L. mainly contains Diterpenes, Coumarins, lignanoids and flavonoids, wherein the terpenoid such as daphnane compounds is that this Chinese medicine is distinctive and possess preferably anti-tumor biological, not yet obtain at present the effective site of this compounds
Extract in the existing document belongs to a bit cures the symptoms, not the disease, and some uses expensive composition, and some is on stream because uncertain therapeutic efficacy is cut interrupts developing.
The invention provides a kind of determined curative effect, safe ready, little, the cheap pure Chinese medicine extract of side effect.
Summary of the invention:
The invention provides a kind of Radix Euphorbiae Fischerianae (Radix Euphorbiae Ebracteolatae) extract, described extract adopts dehydrated alcohol extraction, obtain the one-level extract, the one-level extract is further adopted macroporous resin column or polyamide column chromatography, water and ethanol elution obtain the secondary extract, the secondary extract that separates with macroporous resin column in the secondary extract further adopts polyamide column chromatography, and water and ethanol elution obtain three grades of extracts
Whole three other extracts of level all belong to content of the present invention.
One-level method for preparing extractive of the present invention is as follows:
Radix Euphorbiae Fischerianae (Radix Euphorbiae Ebracteolatae) medical material adds ethanol, reflux, extract,, extracting solution decompression recycling ethanol, the concentrated extract 2 that obtains.
Secondary method for preparing extractive of the present invention is as follows:
Macroporous resin HPD100 on the extract 2, successively water, 40% ethanol, 80% ethanol elution are collected respectively and the concentrated extract 3,4,5 that obtains respectively.
Polyamide column on the extract 2, successively water, 30% ethanol, 60% ethanol, 100% ethanol elution are collected respectively and the concentrated extract 15,16,17,18 that obtains respectively.
Three grades of method for preparing extractive of the present invention are as follows:
Get polyamide column on the extract 5, successively water, 30% ethanol, 60% ethanol, 100% ethanol elution are collected respectively and the concentrated extract 11,12,13,14 that obtains respectively.
The concrete preparation method of one-level extract of the present invention is as follows:
Radix Euphorbiae Fischerianae (Radix Euphorbiae Ebracteolatae) medical material 500g adds dehydrated alcohol 2L, reflux, extract, 3 times, and each 2h, merge extractive liquid,, decompression recycling ethanol, behind the drying with water bath, drying under reduced pressure (70 ℃) gets extract 2.
The concrete preparation method of secondary extract of the present invention is as follows:
Radix Euphorbiae Fischerianae (Radix Euphorbiae Ebracteolatae) medical material 2000g adds dehydrated alcohol 7L, reflux, extract, 3 times, each 2h, merge extractive liquid, is evaporated to 1/5 volume, adds isopyknic water, evaporate into without the alcohol flavor, upper macroporous resin HPD100 (3.3kg), each 2.5 times of column volume eluting of water, 40% ethanol, 80% ethanol successively are behind the drying with water bath, drying under reduced pressure (70 ℃) obtains respectively extract 3,4,5.
Upper polyamide column (the column volume 900ml of extract 2 (10.0g), about 280g, polyamide prepolymer is processed), water, 30% ethanol, 60% ethanol, 100% ethanol are washed respectively 3 column volumes successively, decompression recycling ethanol, water bath method, the vacuum normal temperature drying obtains respectively extract 15,16,17,18.
Three grades of concrete preparation methoies of extract of the present invention are as follows:
Get upper polyamide column (the column volume 900ml of extract 5 (10.0g), about 280g, polyamide prepolymer is processed), water, 30% ethanol, 60% ethanol, 100% ethanol are washed respectively 3 column volumes successively, decompression recycling ethanol, water bath method, the vacuum normal temperature drying obtains respectively extract 11,12,13,14.
Therefore extract of the present invention comprises extract 2, extract 3, extract 4, extract 5, extract 15, extract 16, extract 17, extract 18, extract 11, extract 12, extract 13, extract 14.
More than extract of the present invention have good anti-tumor activity through evidence,
The present invention also provides the pharmaceutical composition that is prepared into as active constituents of medicine with extract of the present invention, and pharmaceutical composition of the present invention comprises active component, and said composition can also add the medicine acceptable carrier as required.
Compositions of the present invention is the pharmaceutical dosage forms of unit dose, and described unit dosage form refers to the unit of preparation, such as every of tablet, and every capsules of capsule, every bottle of oral liquid, every bag of granule etc.
Compositions of the present invention active component wherein, its shared percentage by weight in preparation can be 0.1-99.9%, all the other are the medicine acceptable carrier.
Compositions of the present invention obtains by above-mentioned active component and medicine acceptable carrier are mixed with.
Compositions of the present invention, its pharmaceutical dosage forms can be any pharmaceutically useful dosage form, and these dosage forms comprise: tablet, sugar coated tablet, film coated tablet, enteric coated tablet, capsule, hard capsule, soft capsule, oral liquid, suck agent, granule, electuary, pill, powder, unguentum, sublimed preparation, suspensoid, powder, solution, injection, suppository, ointment, plaster, cream, spray, drop, patch.Preparation of the present invention, peroral dosage form preferably, as: capsule, tablet, oral liquid, granule, pill, powder, sublimed preparation, unguentum etc.
Compositions of the present invention, the preparation of its oral administration can contain excipient commonly used, such as binding agent, filler, diluent, tablet agent, lubricant, disintegrating agent, coloring agent, flavoring agent and wetting agent, can carry out coating to tablet in case of necessity.
Applicable filler comprises cellulose, mannitol, lactose and other similar filler.Suitable disintegrating agent comprises starch, polyvinylpyrrolidone and starch derivatives, for example sodium starch glycollate.Suitable lubricant comprises, for example magnesium stearate.The suitable acceptable wetting agent of medicine comprises sodium lauryl sulphate.
Can fill by mixing, the method that tabletting etc. are commonly used prepares solid oral composition.Repeatedly mix active substance is distributed in those compositionss of a large amount of filleies of whole use.
The form of oral liquid for example can be aqueous or oily suspensions, solution, Emulsion, syrup or elixir, perhaps can be a kind of before use available water or other suitable composite dry products of carrier.This liquid preparation can contain conventional additive, such as suspending agent, for example sorbitol, syrup, methylcellulose, gelatin, hydroxyethyl-cellulose, carboxymethyl cellulose, aluminium stearate gel or hydrogenation edible fat, emulsifying agent, for example lecithin, anhydro sorbitol monooleate or arabic gum; Non-aqueous carrier (they can comprise edible oil), for example almond oil, fractionated coconut oil, such as oily ester, propylene glycol or the ethanol of the ester of glycerol; Antiseptic, for example para hydroxybenzene methyl ester or propyl p-hydroxybenzoate or sorbic acid, and if necessary, can contain conventional flavouring agent or coloring agent.
For injection, the liquid unit dosage forms of preparation contains active substance of the present invention and sterile carrier.According to carrier and concentration, this chemical compound can be suspended or dissolving.The preparation of solution is normally by being dissolved in active substance in a kind of carrier filter-sterilized before it is packed into a kind of suitable bottle or ampoule, then sealing.Adjuvant for example a kind of local anesthetic, antiseptic and buffer agent also can be dissolved in this carrier.In order to improve its stability, can be after the bottle of packing into that this compositions is freezing, and under vacuum, water is removed.
Compositions of the present invention, when being prepared into medicament, optionally add suitable medicine acceptable carrier, described medicine acceptable carrier is selected from: mannitol, sorbitol, sodium pyrosulfite, sodium sulfite, sodium thiosulfate, cysteine hydrochloride, TGA, methionine, vitamin C, EDETATE SODIUM, Ethylenediaminetetraacetic Acid Calcium Salt, the alkali-metal carbonate of monovalence, acetate, phosphate or its aqueous solution, hydrochloric acid, acetic acid, sulphuric acid, phosphoric acid, aminoacid, sodium chloride, potassium chloride, sodium lactate, xylitol, maltose, glucose, fructose, dextran, glycine, starch, sucrose, lactose, mannitol, silicon derivative, cellulose and derivant thereof, alginate, gelatin, polyvinylpyrrolidone, glycerol, POLYSORBATE 80, agar, calcium carbonate, calcium bicarbonate, surfactant, Polyethylene Glycol, cyclodextrin, beta-schardinger dextrin-, the phospholipid material, Kaolin, Pulvis Talci, calcium stearate, magnesium stearate etc.
Compositions of the present invention is determined usage and dosage according to patient's situation in use, but takes every day three times, each 1-20 agent, as: 1-20 bag or grain or sheet.
Following data declaration beneficial effect of the present invention by experiment.
Drug effect Functional observation in stellera chamaejasme L extract ( extract 2,5,13, the 17) body
1. experiment material
1.1 laboratory animal
SPF level Kunming mouse, male, age in 6-8 week, body weight 18-22g, Test Animal Centre, Academy of Military Medical Sciences, P.L.A provides, credit number: SCXK (army) 2007-004.Raising is in the clean laboratory animal room, 20 ℃ of room temperatures, relative humidity 60%.
1.2 transplanted tumor strain:
Mice H22 hepatoma carcinoma cell is available from Chinese Academy of Sciences tumor cell storehouse, by the preservation of going down to posterity of Institute Of Chinese Materia Medica Of China Academy of Chinese Medical Sciences's Experimental Animal Center.
1.3 experiment reagent
Cyclophosphamide (CTX) sheet: Tianjin Jinshi Pharmaceutical Co., Ltd.'s product, lot number: 20070201;
Fluorouracil (5-Fu) injection: Shanghai Xudong Hipu Medicine Co., Ltd's product, lot number: 080401;
Sodium carboxymethyl cellulose: east, Beijing ring amalgamation plant produced, lot number: 971230;
Tween 80: Guangzhou southization glass company packing product, lot number: 021204;
1.4 tested medicine
During the administration by gavage administration, be subjected to reagent rear with 0.5% carboxymethylcellulose sodium solution suspendible with an amount of tween 80 dissolving, the tween 80 final concentration is 0.5%; The solvent control group is for containing 0.5% tween 80 and 0.5% carboxymethylcellulose sodium solution.When extract 5,13,17 intraperitoneal injection administration, be subjected to reagent to be settled to desired concn so that an amount of tween 80 dissolving is rear with physiological saline solution, the tween 80 final concentration is 0.5%; The solvent control group is the normal saline solution that contains 0.5% tween 80.The cyclophosphamide sheet is the Gastric lavage positive control, behind the pulverize with 0.5% carboxymethylcellulose sodium solution suspendible; Fluorouracil Injection is intraperitoneal injection method positive control, and original packing concentration 0.25g/10ml is diluted to desired concn with physiological saline solution.
2. experimental technique
2.1 subcutaneous transplantation tumor model is set up
By sterile working's program, get the Kunming mouse of intraperitoneal inoculation H22 (7-9 days), after the abdominal part fur takes off iodine with iodine disinfection, medical alcohol successively, from the abdominal cavity, extract the tumor cell suspension with aseptic syringe, cell suspension calculates viable count＞95% through 0.2% after expecting blue dyeing, adding physiological saline solution adjustment tumor cell suspension born of the same parents concentration is 1 * 107/ml, and the right axil that is seeded to experiment mice is subcutaneous, every 0.2ml.
2.3 subcutaneous transplantation tumor H22 mouse model Gastric lavage tumor inhibition effect is observed
Behind the subcutaneous transplantation tumor H22 Establishment of mouse model, extract 2 high, medium and low dosage group dosage are respectively 2g/kg, 1g/kg, 0.5g/kg (in the crude drug amount, lower same); Extract 5,13,17 high dose group are all got 16g/kg, in, low dose group is respectively 8g/kg, 4g/kg.Gastric infusion, every day 1 time, successive administration 10d, mice is weighed, put to death to next day in the last administration, strips tumor, thymus and spleen and weigh calculating suppression ratio, tumor heavy coefficient, spleen and thymus index.Computing formula is as follows:
Tumour inhibiting rate (%)=(the average tumor of the average tumor weight-administration of matched group group is heavy)/average tumor of matched group heavy * 100%
The heavy coefficient of tumor=tumor weight/Mouse Weight; Spleen (thymus) index=spleen (thymus) weight/Mouse Weight
2.3 subcutaneous transplantation tumor H22 mouse model abdomen is annotated the dose regimen tumor inhibition effect and is observed
Behind the subcutaneous transplantation tumor H22 Establishment of mouse model, extract 5,13,17 high dose group are got 250mg/kg, in, low dose group is respectively 125mg/kg, 62.5mg/kg.Intraperitoneal injection, every day 1 time, successive administration 10d, mice is weighed, put to death to next day in the last administration, strips tumor, thymus and spleen and weigh calculating suppression ratio, tumor heavy coefficient, spleen and thymus index.
3. experimental result
3.1 drug effect Functional observation in stellera chamaejasme L extract 2 bodies
Experimental result shows: the high dose group of extract 2 has remarkable inhibitory action (p＜0.01) to mouse subcutaneous transplanting tumor H22, and tumour inhibiting rate is 36.24%; Middle dosage group also has statistical significance (p＜0.05) to the H22 inhibitory action.See Table 1.
Table 1 stellera chamaejasme L extract extract 2 gastric infusions are on the impact of subcutaneous transplantation tumor H22 (x ± s)
Annotate: compare with the solvent control group,
*P＜0.05,
*P＜0.01
3.2 the inhibitory action of Gastric lavage extract 5,13,17 couples of transplanted tumor H22
Table 2 stellera chamaejasme L extract 5,13,17 gastric infusions are on the impact of subcutaneous transplantation tumor H22 (x ± s)
Annotate: compare with the solvent control group,
*P＜0.05,
*P＜0.01
3.3 abdomen is annotated the effect that 5,13,17 couples of transplanted tumor H22 of dose regimen extract suppress
Table 3 stellera chamaejasme L extract 5,13,17 intraperitoneal injections are on the impact of subcutaneous transplantation tumor H22 (x ± s)
Annotate: compare with the solvent control group,
*P＜0.05,
*P＜0.01.
Stellera chamaejasme L extract suppresses the experimentation of Human Hepatic Carcinoma Cell Line BEL-7402 nude mice by subcutaneous transplanted tumor
1. experiment material
1.1 cell strain
Human liver cancer cell BEL-7402 is for being so kind as to give at China Medicine University new medicament screen center.
1.2 laboratory animal
SPF level BALB/C nude mouse, female, age in days 35-40 days, body weight 18-22g was provided by Shanghai Slac Experimental Animal Co., Ltd..Quality certification numbering: No. 122, Shanghai dynamic circuit connector card word.Raising is at ten thousand grades of barrier systems (SPF level), 25 ℃ of room temperatures, relative humidity 60%.
1.3 medicine
Be subjected to reagent: extract 17.
Preparation: to be settled to desired concn with physiological saline solution after an amount of tween 80 dissolving, the tween 80 final concentration is 0.5%; The solvent control group is the normal saline solution that contains 0.5% tween 80.The positive drug Fluorouracil Injection, Shanghai Xudong Hipu Medicine Co., Ltd produces, lot number: 080401, original packing concentration 0.25g/10ml is diluted to desired concn with physiological saline solution.
2. experimental technique
2.1 subcutaneous transplantation tumor BEL-7402 nude mouse model is set up
It is subcutaneous and set up to be inoculated in nude mouse by human hepatocellular carcinoma BEL-7402 cell's strain.The cell inoculum concentration is 3 * 10
6, inoculation is used after forming and passing for 3 generations again in the nude mouse body after the transplanted tumor.
2.2 subcutaneous transplantation tumor model tumor is observed in the stellera chamaejasme L extract body the originate inhibitory action of hepatocarcinoma BEL-7402 to the people
List of references
[19]The BEL-7402 tumor tissue of getting the growth animated period cuts into 1.5mm
3About, under aseptic condition, it is subcutaneous to be inoculated in nude mouse right side axillary fossa.Nude Mice is treated tumor growth to 100～300mm with vernier caliper measurement transplanted tumor diameter
3After divide at random 5 groups, 12 of solvent control treated animals, each 6 of positive controls and extract 17 administration groups after the weight of animals and gross tumor volume matched.Use the method for measuring the tumor footpath, dynamically observe tested thing antineoplastic effect.The measurement number of times of diameter of tumor is that each measurement claims Mus heavy simultaneously 3 times weekly.Extract 17 dosages are respectively 250mg/kg (high dose is amounted to into the crude drug amount, and is lower same), 187.5mg/kg (middle dosage), 125mg/kg (low dosage); Positive controls 5-Fu injection 20mg/kg; The solvent control group contains the solution of cosolvent simultaneously to equivalent, be intraperitoneal injection, gives 3 times weekly, and nude mice is dissected in administration the 21st day, strips tumor and spleen, weighs, and leaves and takes tumor specimen by the subsequent experimental requirement.
Attached: as to detect index and computational methods
1) gross tumor volume (tumor volume, TV), computing formula is: TV=1/2 * a * b
2(wherein a, b represent respectively length and width);
2) relative tumour volume (relative tumor volume, RTV), computing formula is: RTV=TVt/TV
0(wherein TV0 is when dividing the cage administration, i.e. d
0Gross tumor volume, the TVt gross tumor volume when measuring each time).
3) relative tumor proliferation rate T/C (%), computing formula is: T/C (%)=T
RTV/ C
RTV* 100 (T
RTV: treatment group RTV; C
RTV: negative control group RTV).
Result of the test is with the evaluation index (with reference to " cell toxicant series antineoplastic medicament non-clinical study technological guidance principle ") of relative tumor proliferation rate T/C (%) as anti-tumor activity.
3. experimental result
Extract 17 intraperitoneal injections are high, middle dosage has stronger growth inhibited effect to people's hepatocarcinoma BEL-7402 Nude Mice, and T/C is respectively 52.65%, 59.94%; Low dosage to people's hepatocarcinoma BEL-7402 Nude Mice inhibitory action a little less than, T/C is 71.60%.Positive controls 5-Fu chamber drug administration by injection 20mg/kg has stronger growth inhibited effect to people's hepatocarcinoma BEL-7402 Nude Mice, and T/C is 46.11%.
Extract 17 is very large on the body weight impact of lotus tumor nude mouse, the high dose group weight loss is very obvious, about the low 2.5g of body weight and solvent control group (average weight is respectively 17.47g, 19.94g), afterwards, the body weight that takes by weighing before each administration all fluctuates about 17.5g after for the first time administration; In, low dose group also sees obvious reduction in administration initial stage body weight, slightly go up afterwards, generally every administration in 2 days the time, weighs and find that body weight gos up obviously, and the Mouse Weight Loss reappears after the double every other day administration, thus according to the time the meta-body weight curve drawn present wave form varies trend.See Table 4,5, Fig. 5,6 and Fig. 7.
The relative rate of increase of people's hepatocarcinoma BEL-7402 Nude Mice changes before and after 17 medications of table 4 extract
Annotate: d0 is a minute cage administration time, and d21 is administration the 21st day;
Compare with the solvent control group,
*P＜0.05,
*P＜0.01; Compare with positive controls,
△ △P＜0.01.
Table 5. extract 17 is on the inhibitory action of people's hepatocarcinoma BEL-7402 Nude Mice and the impact of index and spleen index
Annotate: compare with the solvent control group,
*P＜0.05,
*P＜0.01
Stellera chamaejasme L extract is to the inhibiting medium effective concentration of vitro culture of human source hepatoma carcinoma cell
Hepatoma carcinoma cell HepG2, the BEL-7402 in the 2 couples of people of stellera chamaejasme L extract source, the inhibitory action of SMMC-7721 a little less than, suppression ratio can reach about 80% when 200 μ g/ml, but the following drug level of 100 μ g/ml sharply descends to the suppression ratio of tumor cell, and linear regression method calculates IC
50＞100 μ g/ml; Extract 5,13,17 couples of hepatoma carcinoma cell HepG2, BEL-7402, SMMC-7721 all have preferably inhibitory action, during 100 μ g/m drug level its suppression ratio all more than 80%, IC
50All below 40 μ g/ml, wherein extract 17 inhibitory action are the strongest.See Table 6.
Table 6 stellera chamaejasme L extract is external to the originate inhibiting IC of hepatoma carcinoma cell of people
50(unit: μ g/ml)
Stellera chamaejasme L extract is on the impact of TGF-signal beta path
Experiment one:
1. experiment material
1.1 cell strain
HepG2 cell line (stable cell line of transfection TGF-β gene) is set up by Yale's medical college pharmacology department; Human liver cancer cell BEL-7402 is so kind as to give by China Medicine University new medicament screen center.
1.2 reagent instrument
Luciferase test kit: U.S. Promega products C at.#4550;
The passive lysate of reporter gene: U.S. Promega products C at.#E153A;
The multi-functional microplate reader of TECAN Safire-2: the Switzerland Supreme Being agree company.
1.3 medicine
Be subjected to reagent: extract 2,5,13,17.
Preparation: be dissolved among the DMSO behind the sample accurate weighing, then be made into 10 * mother solution with PBS liquid, DMSO final concentration＜0.1%, now with the current.Become variable concentrations with the cell culture solution dilution during dosing.
2. experimental technique
Fluorescence report gene approach screening stellera chamaejasme L extract is to the target gene of tumor cell effect
1) bed board: in 96 orifice plates, plant HepG2 cell (transfection the stable cell line of TGF-β), 5 * 10
4/ hole, the training base adopts without phenol red RPMI and 10%FBS;
2) constant incubator (37 ℃, 4%CO
2) hatch 48h, cell fusion;
3) change liquid, dosing: being subjected to the reagent final concentration is 0,0.375 μ g/ml, 1.25 μ g/ml, 3.75 μ g/ml;
4) continue to hatch 1h;
5) add 2ng/mlTGF-β in the cell after medicine is processed;
6) hatch 4h;
7) add the passive lysate of 20 μ l, 5 * reporter gene;
8) culture plate-70 ℃ freeze overnight;
9) carefully scrape all cell lysis, with pipettor 30 μ l cracking supernatants are moved into 96 hole ELISA Plate;
10) add 30 μ l luciferases under excitation wavelength 430nm, emission wavelength 550nm arrange, use the multi-functional microplate reader fluorescence intensity of TECAN company.
3. experimental result:
The TGF-β of extract 5,13,17 pairs of high concentrations has remarkable downward modulation effect, the results are shown in Figure 8.
Experiment two:
1. extract 17 is on the impact of BEL-7402 cell Smad4 and the p-Akt protein expression of In vitro culture
Extract 17 can significantly suppress the interior TGF-signal beta passage downstream factor p-Akt of hepatoma carcinoma cell BEL-7402 and the Smad4 protein expression of In vitro culture.The p-Akt protein expression descends extremely significantly after the medication, expresses to compare with matched group when 12.5 μ g/ml and has just descended about 1/2, and it is maximum that drug level p-Akt when 25 μ g/ml expresses fall, is about 1/7 of matched group.Descend after the Smad4 medication and have obvious dose-dependence: it is not obvious that Smad4 expresses decline when 12.5 μ g/ml drug level, is down to about 1/3 of matched group during 25 μ g/ml, and the Smad4 protein content is extremely low during 50 μ g/ml.As seen, p-Akt and Smad4 protein expression all present downward trend after the medication, and p-Akt the obviously change of inhibition namely occurs at low drug level; During drug level 25 μ g/ml, p-Akt and Smad4 relative amount all significantly reduce and the former is low than the latter, may be that extract 17 survives with death cell by TGF-signal beta path network and regulates very crucial drug level.See Fig. 9,10 and table 7.
In vitro culture BEL-7402 cell Smad4 and p-Akt albumen relative amount before and after 17 medications of table 7 extract
Experiment three:
1. extract 17 is on the impact of BEL-7402 subcutaneous transplantation tumor tissue Smad4 and T β R-I positive expression
In the subcutaneous people's hepatocarcinoma of the nude mouse BEL-7402 transplanted tumor tissue, the positive expression classification of Smad4 and T β R-I is descended by each dosage group positive expression of reagent between " +～++ " refers to, and 1/6～1/3 feminine gender occurs and express.See Table 8,9 and Figure 11,12.
Table 8 extract 17 is organized the impact (n=6) of Smad4 positive expression on BEL-7402 transplanted tumor
Table 9 extract 17 is on the impact (n=6) of BEL-7402 transplanted tumor tissue T β R-I positive expression
2. extract 17 is on the impact of BEL-7402 subcutaneous transplantation tumor tissue Smad4mRNA expression
The ratio of nude mouse subcutaneous transplantation tumor BEL-7402Smad4mRNA and GAPDH mRNA is 0.92 ± 0.09, the expression that extract 17 each dosage group all can be reduced Smad4mRNA, low dose group ratio is down to 0.77 ± 0.14, and significant difference (P＜0.05) is arranged.The expression that medicine may have been reduced Smad4mRNA by the factor that acts on signal path Smad4 upstream, but Smad4mRNA reaches a plateau value under doses, continues to improve drug dose and no longer continues to descend; And the amplitude that mrna expression descends does not have the cultured cell in vitro protein expression level obvious, and as seen being subjected to reagent is not mainly to transcribe link to the inhibition of Smad4, may produce material impact with degraded to the synthetic of albumen.See Table 10.
BEL-7402 transplanted tumor is organized the variation (n=6) of Smad4/GAPDH ratio before and after 17 medications of table 10 extract
Annotate: compare with the solvent control group,
*P＜0.05
" other extract to the tumour inhibiting rate of mouse subcutaneous transplanting tumor H22 about 30% ".
Beneficial effect of the present invention is:
1. effectively remove impurity in the extraction and separation process of the present invention, improved the content of effective ingredient, can obtain fast and accurately effective ingredient.
2. extract chemical constituent provided by the invention is simply clear and definite, is easier to illustrate its mechanism of action at pharmacological research, is easier to aborning the quality control of medicine.
Method provided by the invention obtains active component first from medical material, and first it is carried out medicine efficacy screening in various tumor cell strains, because composition is definite, content is clear and definite, and preparation technology is convenient, and is active good, the suitable anti-tumor Chinese medicine new drug that is developed to.
Description of drawings:
Fig. 1 extract 2 chemical fingerprints
Fig. 2 extract 5 chemical fingerprints
Fig. 3 extract 13 chemical fingerprints
Fig. 4 extract 17 chemical fingerprints
The experimental therapy effect of 17 pairs of people's hepatocarcinoma of Fig. 5 extract BEL-7402 Nude Mice
The experimental therapy effect of 17 pairs of people's hepatocarcinoma of Fig. 6 extract BEL-7402 Nude Mice
Fig. 7 extract 17 is on the impact of lotus tumor nude mouse body weight gain
The TGF-β of Fig. 8 extract 2,5,13,17 pairs of high concentrations has remarkable downward modulation effect
In vitro culture BEL-7402 cell Smad4 and p-Akt protein expression before and after 17 medications of Fig. 9 extract
BEL-7402 cell Smad4 and p-Akt albumen relative amount are relatively before and after 17 medications of Figure 10 extract
Figure 11 BEL-7402 transplanted tumor is organized Samd4 SABC picture (* 200)
Figure 12 BEL-7402 transplanted tumor tissue T β R-I SABC picture (* 200)
The specific embodiment:
Further describe flesh and blood of the present invention below in conjunction with embodiments of the invention, this embodiment only is used for explanation the present invention and the present invention is not limited.
Radix Euphorbiae Fischerianae (Radix Euphorbiae Ebracteolatae) medical material 500g adds dehydrated alcohol 2L, reflux, extract, 3 times, and each 2h, merge extractive liquid,, decompression recycling ethanol, behind the drying with water bath, drying under reduced pressure (70 ℃) gets extract 2.
Radix Euphorbiae Fischerianae (Radix Euphorbiae Ebracteolatae) medical material 2000g adds dehydrated alcohol 7L, reflux, extract, 3 times, each 2h, merge extractive liquid, is evaporated to 1/5 volume, adds isopyknic water, evaporate into without the alcohol flavor, upper macroporous resin HPD100 (3.3kg), each 2.5 times of column volume eluting of water, 40% ethanol, 80% ethanol successively are behind the drying with water bath, drying under reduced pressure (70 ℃) obtains respectively extract 3,4,5.
Upper polyamide column (the column volume 900ml of extract 2 (10.0g), about 280g, polyamide prepolymer is processed), water, 30% ethanol, 60% ethanol, 100% ethanol are washed respectively 3 column volumes successively, decompression recycling ethanol, water bath method, the vacuum normal temperature drying obtains respectively extract 15,16,17,18.
Get upper polyamide column (the column volume 900ml of extract 5 (10.0g), about 280g, polyamide prepolymer is processed), water, 30% ethanol, 60% ethanol, 100% ethanol are washed respectively 3 column volumes successively, decompression recycling ethanol, water bath method, the vacuum normal temperature drying obtains respectively extract 11,12,13,14.
Claims (7)
1. the extract of a Stellera chamaejasme L., formed by one-level, secondary and three grades of extracts, it is characterized in that, the Stellera chamaejasme L. medical material adopts dehydrated alcohol extraction, obtains the one-level extract, and the one-level extract is further adopted macroporous resin column or polyamide column chromatography, water and ethanol elution obtain the secondary extract, the secondary extract that separates with macroporous resin column chromatography in the secondary extract further adopts polyamide column chromatography, and water and ethanol elution obtain three grades of extracts
Wherein, the one-level method for preparing extractive is as follows:
Radix Euphorbiae Fischerianae (Radix Euphorbiae Ebracteolatae) medical material adds ethanol, reflux, extract,, extracting solution decompression recycling ethanol, the concentrated extract 2 that obtains;
Wherein, the secondary method for preparing extractive is as follows:
Macroporous resin HPD100 on the extract 2, successively water, 40% ethanol, 80% ethanol elution are collected respectively and the concentrated extract 3,4,5 that obtains respectively;
Polyamide column on the extract 2, successively water, 30% ethanol, 60% ethanol, 100% ethanol elution are collected respectively and the concentrated extract 15,16,17,18 that obtains respectively;
Wherein, three grades of method for preparing extractive are as follows:
Get polyamide column on the extract 5, successively water, 30% ethanol, 60% ethanol, 100% ethanol elution are collected respectively and the concentrated extract 11,12,13,14 that obtains respectively.
2. the extract of a Stellera chamaejasme L. is comprised of one-level, secondary and three grades of extracts, it is characterized in that the one-level method for preparing extractive is as follows:
Radix Euphorbiae Fischerianae (Radix Euphorbiae Ebracteolatae) medical material 500g adds dehydrated alcohol 2L, reflux, extract, 3 times, and each 2h, merge extractive liquid,, decompression recycling ethanol, behind the drying with water bath, 70 ℃ of drying under reduced pressure get extract 2;
The secondary method for preparing extractive is as follows:
Radix Euphorbiae Fischerianae (Radix Euphorbiae Ebracteolatae) medical material 2000g adds dehydrated alcohol 7L, reflux, extract, 3 times, each 2h, merge extractive liquid, is evaporated to 1/5 volume, adds isopyknic water, evaporate into without the alcohol flavor, the macroporous resin HPD100 of upper 3.3kg, each 2.5 times of column volume eluting of water, 40% ethanol, 80% ethanol successively are behind the drying with water bath, 70 ℃ of drying under reduced pressure obtain respectively extract 3,4,5;
10.0g polyamide column on the extract 2, polyamide column volume 900ml, process with the 280g polyamide prepolymer, water, 30% ethanol, 60% ethanol, 100% ethanol are washed respectively 3 column volumes successively, decompression recycling ethanol, water bath method, the vacuum normal temperature drying obtains respectively extract 15,16,17,18;
Three grades of concrete preparation methoies of extract are as follows:
10.0g get polyamide column on the extract 5, polyamide column volume 900ml, process with the 280g polyamide prepolymer, water, 30% ethanol, 60% ethanol, 100% ethanol are washed respectively 3 column volumes successively, decompression recycling ethanol, water bath method, the vacuum normal temperature drying obtains respectively extract 11,12,13,14.
3. with claim 1 or the 2 described extracts pharmaceutical composition as active component.
4. the pharmaceutical composition of claim 3 is characterized in that, is any pharmaceutically useful dosage form.
5. the pharmaceutical composition of claim 4 is characterized in that, is injection.
6. claim 1 or 2 the extract application in the preparation antitumor drug.
7. claim 1 or 2 extract have application in the medicine of downward modulation effect in preparation to TGF-β.
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| CN102078444A (en) * | 2010-12-30 | 2011-06-01 | 苏州瑞蓝博中药技术开发有限公司 | Preparation method of Chinese stellera root total flavonoids |
| CN103933027B (en) * | 2014-04-09 | 2016-08-17 | 中国中医科学院中药研究所 | Stellera chamaejasme L extract application in antitumor |
| CN106866601B (en) * | 2017-03-01 | 2019-02-19 | 中日友好医院 | A kind of preparation method of the peaceful B bulk pharmaceutical chemicals of radix euphorbiae lantu |
| CN110302380B (en) * | 2019-07-19 | 2021-11-16 | 漯河医学高等专科学校 | Application of litchis water extract radiotherapy sensitizer |
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