CN103623066A - Corydalis impatiens total alkaloid extractive for preparing anti-cancer drugs and application thereof - Google Patents
Corydalis impatiens total alkaloid extractive for preparing anti-cancer drugs and application thereof Download PDFInfo
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Abstract
The invention discloses corydalis impatiens total alkaloid extractive for preparing anti-cancer drugs. The content of total alkaloids in the discloses corydalis impatiens total alkaloid extractive is not less than 50wt percent; the disclosed corydalis impatiens total alkaloid extractive comprises 10 to 12wt percent of protopine, 8 to 10wt percent of alpha-bicuculline and 10 to 15wt percent of ochotensimine; the extracting steps of the extractive comprise grinding corydalis impatiens drugs, performing reflux extraction to the corydalis impatiens drugs through saturated alkaline ethyl alcohol or saturated alkaline methyl alcohol, and concentrating and trying the corydalis impatiens drugs, so as to obtain corydalis impatiens extractive, then dissolving and filtering the extractive through acid solution, adding alkali solution into filter liquor, and adjusting the pH value of the solution to be 9 to 10, so as to obtain acid and alkali treated fluid, adding organic solvent into the acid and alkali treated fluid, so as to perform extraction, obtaining corydalis impatiens total alkaloid crude extract, and then purifying the corydalis impatiens total alkaloid crude extract, so as to obtain the corydalis impatiens total alkaloid extractive. The corydalis impatiens total alkaloid extractive obtained through the method has effects of breast cancer resistance, ovarian cancer resistance, stomach cancer resistance and lung cancer resistance.
Description
Technical field
The present invention relates to a kind of application of extract of Chinese herbal medicine, specifically, relate to a kind of total alkaloids extract of Tibetan medicine material Corydalis impatiens (Pall.) Fisch. for the preparation of the application of cancer therapy drug.
Background technology
The dry herb of Tibetan medicine Corydalis impatiens (Pall.) Fisch. Wei Ying section Genus Corydalis Corydalis impatiens (Pall.) Fisch. (another name Corydalisimpatiens(Pall.)Fisch), be distributed in the ground such as Qinghai, Tibet, Gansu, Sichuan, for the common medicine in tibetan traditional medicine, data show has in 25% tibetan traditional medicine and contains this medical material, and in its medical material, ingredient is mainly various alkaloids.The all herbal medicine of multiplex Genus Corydalis in Tibetan medicine, some is also used as medicine separately with different parts such as the root of this plant, stem, leaves, be mainly used in treating various bleedings, the diseases associated with inflammation of fever, influenza, gastrointestinal and liver and gall, also can be used for treating traumatic injury or other wound etc.At present, the different kind organism alkali composition that separation obtains from this plant has had many, and these alkaloids have biologic activity widely, as antioxidation, antiinflammatory, arrhythmia and protection liver wound etc.In addition, the alkaloid in this plant can acetylcholine esterase inhibition activity, can improve memory, so be also used for treating senile dementia.In the progress > > of the < < Yunnan Genus Corydalis that Li Jichang, Wang Jun etc. deliver, the current effect of Genus Corydalis is summarized, wherein clearly show Corydalisimpatiens(Pall.)Fisch all herbal medicine, there is the effect for the treatment of scabies sore, gastritis, gastric ulcer, traumatic injury.The < < Corydalisimpatiens(Pall.)Fisch total alkaloids antiinflammatory action research > > (hubei agricultural science that Song Xiaonan, Wu's instruction etc. are delivered, 2013,52(6)) the antiinflammatory performance of Corydalisimpatiens(Pall.)Fisch total alkaloids has been carried out to systematic study.
Alkaloid is the nitrogenous alkaline organic compound of a class that is present in occurring in nature, has the character like alkali, is called again counterfeit alkali, great majority have complicated circulus, nitrogen is included in ring more, has significant biological activity, is one of effective ingredient important in Chinese herbal medicine.Have optical activity, some alkali-free and derive from the organic compounds containing nitrogen of plant, has obvious biological activity, is also included within alkaloidal scope.At present, known alkaloid kind is a lot, about 10,000 kinds of left and right, has some structural formulas also completely not definite, and their complicated structure, can be divided into 59 types.Along with new alkaloidal discovery, classification also will be upgraded thereupon, because alkaloidal kind is a lot, respectively has different structural formulas, and therefore, character is to each other difference to some extent.
In prior art, the method of extracting total alkaloids in plant is two kinds of organic solvent extractionprocess and chromatographys, conventional in chromatography is three kinds, silica gel column chromatography, aluminum oxide column chromatography and ion exchange resin, although said method can be prepared total alkaloids, but exist efficiency of pcr product low, the problem that impurity content is high, and the pharmaceutical characteristic of the total alkaloids directly obtaining by the method is slightly poor, can not meet clinical research and application.
Summary of the invention
The technical problem that the application solves is that the research for Tibetan medicine Corydalis impatiens (Pall.) Fisch. in prior art only concentrates on antioxidation, antiinflammatory, arrhythmia and protection liver wound, and then expands its application, and a kind of Corydalis impatiens (Pall.) Fisch. total alkaloids with antitumaous effect is provided.
For this reason, the technical scheme that the present invention takes is:
A kind of Corydalis impatiens (Pall.) Fisch. total alkaloids extract for the preparation of cancer therapy drug, in described Corydalis impatiens (Pall.) Fisch. total alkaloids extract, the content of total alkaloids is not less than 50wt%, the protopine that comprises β-Bicuculline, β-hydrystine, 10-12wt%, α-Bicuculline of 8-10wt% and the bright alkali of the Herba Corydalis ochotensis of 10-15wt%.
Preferably, in the above-mentioned Corydalis impatiens (Pall.) Fisch. total alkaloids extract for the preparation of cancer therapy drug, in described Corydalis impatiens (Pall.) Fisch. total alkaloids extract, the content of total alkaloids is not less than 50wt%, comprise β-Bicuculline, β-hydrystine, 11wt% protopine, 9wt% α-Bicuculline and the bright alkali of 13wt% Herba Corydalis ochotensis.
The above-mentioned Corydalis impatiens (Pall.) Fisch. total alkaloids extract for the preparation of cancer therapy drug, wherein said cancer therapy drug is the medicine of anti-breast cancer, ovarian cancer, gastric cancer or pulmonary carcinoma.
The present invention also provides the application of a kind of extract of Corydalis impatiens (Pall.) Fisch. total alkaloids for the preparation of cancer therapy drug, and wherein, described extract is extracted by following steps,
(1) by Corydalis impatiens (Pall.) Fisch. pulverizing medicinal materials, and utilize the saturated alkaline ethanol of 50-95% volumetric concentration or saturated alkaline methanol, reflux, extract, at 40-80 ℃, obtain Corydalis impatiens (Pall.) Fisch. extracting solution, the 7-10 of Corydalis impatiens (Pall.) Fisch. quality of medicinal material doubly described in the consumption of wherein said saturated alkaline ethanol or described saturated alkaline methanol;
(2) by concentrated, the dry Corydalis impatiens (Pall.) Fisch. Extract that obtains of described Corydalis impatiens (Pall.) Fisch. extracting solution;
(3) to adding volumetric concentration in described Corydalis impatiens (Pall.) Fisch. Extract, be that the acid solution of 0.5-4% dissolves, wherein, the addition of acid solution and the mass ratio of described Corydalis impatiens (Pall.) Fisch. Extract are (1-6): 1, filtration obtains acid liquid, to adding volumetric concentration in described acid liquid, it is the aqueous slkali of 4-15%, regulator solution pH value, to 9-10, obtains acid-alkali treatment liquid;
(4) in described acid-alkali treatment liquid, add organic solvent extraction, reclaim described organic solvent and obtain Corydalis impatiens (Pall.) Fisch. total alkaloids crude extract, wherein the each addition of organic solvent is 2-4 times of long-pending volume of described acid-alkali treatment liquid.
(5) get silica gel ethyl acetate wet method dress post, and the described Corydalis impatiens (Pall.) Fisch. total alkaloids crude extract obtaining is added to loading after acetic acid ethyl dissolution, and adopt ethyl acetate eluting, dry, obtain required Corydalis impatiens (Pall.) Fisch. total alkaloids, wherein for dissolving the 1-3 that the consumption of the ethyl acetate of total alkaloids crude extract is its quality, doubly measure, silica gel consumption be described Corydalis impatiens (Pall.) Fisch. total alkaloids crude extract quality 5-10 doubly, for the consumption of the ethyl acetate of eluting be described Corydalis impatiens (Pall.) Fisch. total alkaloids crude extract quality 50-100 doubly.
The extract of above-mentioned Corydalis impatiens (Pall.) Fisch. total alkaloids is for the preparation of the application of cancer therapy drug, and its extraction step is as follows,
(1) by Corydalis impatiens (Pall.) Fisch. pulverizing medicinal materials to 5-50 order, and utilize the saturated alkaline ethanol of 50-95% volumetric concentration or saturated alkaline methanol, at 40-80 ℃, reflux, extract, is 3-8 time, obtain Corydalis impatiens (Pall.) Fisch. extracting solution, the 7-10 of Corydalis impatiens (Pall.) Fisch. quality of medicinal material doubly described in the consumption of wherein said saturated alkaline ethanol or described saturated alkaline methanol;
(2) described Corydalis impatiens (Pall.) Fisch. extracting solution is concentrated into clear paste, the dry Corydalis impatiens (Pall.) Fisch. Extract that obtains;
(3) to adding volumetric concentration in described Corydalis impatiens (Pall.) Fisch. Extract, be the acid solution dissolution filter several times of 0.5-4%, in each dissolution filter step, the addition of acid solution and the mass ratio of described Corydalis impatiens (Pall.) Fisch. Extract are (1-6): 1, collect to filter and obtain all filtrate formation acid liquids, to adding volumetric concentration in described acid liquid, it is the aqueous slkali of 4-15%, regulator solution pH value, to 9-10, obtains acid-alkali treatment liquid;
(4) in described acid-alkali treatment liquid, add organic solvent extraction 3-6 time, reclaim described organic solvent and obtain Corydalis impatiens (Pall.) Fisch. total alkaloids crude extract, wherein the each addition of organic solvent is 2-4 times of long-pending volume of described acid-alkali treatment liquid.
(5) get silica gel ethyl acetate wet method dress post, and the described Corydalis impatiens (Pall.) Fisch. total alkaloids crude extract obtaining is added to loading after acetic acid ethyl dissolution, and adopt ethyl acetate to carry out eluting with the elution flow rate of a 1-2 per hour column volume, dry, obtain required Corydalis impatiens (Pall.) Fisch. total alkaloids, wherein for dissolving the 1-3 that the consumption of the ethyl acetate of total alkaloids crude extract is its quality, doubly measure, silica gel consumption is 5-10 times of described Corydalis impatiens (Pall.) Fisch. total alkaloids crude extract quality, the consumption that is used for the ethyl acetate of eluting is 50-100 times of described Corydalis impatiens (Pall.) Fisch. total alkaloids crude extract quality, described drying steps is the first reclaim under reduced pressure ethyl acetate of eluent, then at 60-70 ℃ of drying under reduced pressure.
In the application of the extract of above-mentioned Corydalis impatiens (Pall.) Fisch. total alkaloids for the preparation of cancer therapy drug,
Described acid solution is hydrochloric acid, sulphuric acid, phosphoric acid or vinegar aqueous acid;
Described aqueous slkali is the aqueous solution of sodium hydroxide, sodium carbonate, sodium bicarbonate;
Described organic solvent is dichloromethane, chloroform or ethyl acetate.
In the application of the extract of above-mentioned Corydalis impatiens (Pall.) Fisch. total alkaloids for the preparation of cancer therapy drug, the extraction step of its extract is,
(1) by Corydalis impatiens (Pall.) Fisch. pulverizing medicinal materials to 5-50 order, and the saturated alkaline ethanol that utilizes 80% volumetric concentration reflux, extract, 4 times at 40-80 ℃, mention 3 hours at every turn, obtain Corydalis impatiens (Pall.) Fisch. extracting solution, wherein the addition of saturated alkaline ethanol is 10 times of Corydalis impatiens (Pall.) Fisch. quality of medicinal material;
(2) by described Corydalis impatiens (Pall.) Fisch. extracting solution at 60 ℃ of decompression recycling ethanols, reconcentration is to clear paste shape, the dry Corydalis impatiens (Pall.) Fisch. Extract that obtains of spraying;
(3) to adding volumetric concentration in described Corydalis impatiens (Pall.) Fisch. Extract, be 0.5% dissolve with hydrochloric acid solution filter 23, wherein, each addition of acid solution and the mass ratio of solid are 5:1, merge the filtrate of filtering for 3 times and obtain acid liquid, to adding volumetric concentration in described acid liquid, it is 10% sodium hydrate aqueous solution, regulator solution pH value to 10, obtains acid-alkali treatment liquid;
(4) in described acid-alkali treatment liquid, add organic solvent extraction 3-6 time, reclaim described organic solvent and obtain Corydalis impatiens (Pall.) Fisch. total alkaloids crude extract, wherein the each addition of organic solvent is 3 times of long-pending volumes of acid-alkali treatment liquid.
(5) get silica gel ethyl acetate wet method dress post, and the described Corydalis impatiens (Pall.) Fisch. total alkaloids crude extract obtaining is added to loading after acetic acid ethyl dissolution, and adopt ethyl acetate to carry out eluting with the elution flow rate of a 1-2 per hour column volume, dry, obtain required Corydalis impatiens (Pall.) Fisch. total alkaloids, wherein for dissolving the 1-3 that the consumption of the ethyl acetate of total alkaloids crude extract is its quality, doubly measure, silica gel consumption is 10 times of described Corydalis impatiens (Pall.) Fisch. total alkaloids crude extract quality, the consumption of eluent ethyl acetate agent is 80 times of described Corydalis impatiens (Pall.) Fisch. total alkaloids crude extract quality, described drying steps is the first reclaim under reduced pressure ethyl acetate of eluent, then at 60-70 ℃ of drying under reduced pressure.
In the application of the extract of above-mentioned Corydalis impatiens (Pall.) Fisch. total alkaloids for the preparation of cancer therapy drug, described cancer therapy drug is the medicine of anti-breast cancer, ovarian cancer, gastric cancer or pulmonary carcinoma.
The preparation method of the saturated alkaline ethanol of the 50-95% volumetric concentration adopting in the present invention or saturated alkaline methanol is, ethanol or the methanol aqueous solution of first preparing 50-95% volumetric concentration, then in this aqueous solution, add sodium hydroxide or potassium hydroxide to saturated, obtain the saturated saturated alkaline ethanol of required 50-95% volumetric concentration or saturated alkaline methanol.
Compared with prior art, tool of the present invention has the following advantages,
(1) in the method for extraction Corydalis impatiens (Pall.) Fisch. total alkaloids provided by the invention, adopt the saturated alkaline ethanol of 50-95% and saturated alkaline methanol, alkaloid in medical material, especially protopine, the bright alkali of α-Bicuculline and Herba Corydalis ochotensis changes drifting alkaloids into by alkaloid salt and is then dissolved in ethanol or methanol and is extracted under alkali condition, add again sour water, drifting alkaloids salify is dissolved in sour water, thereby separated with liposoluble constituent, the pH that further controls acid-alkali treatment liquid is 9-10, alkaloid salt is dissociated out again, with organic solvent extraction, thereby obtain total alkaloids extract of the present invention, in the described Corydalis impatiens (Pall.) Fisch. total alkaloids extract that employing the method for the invention obtains, the content of total alkaloids is not less than 50wt%, the protopine that comprises 10-12wt%, α-Bicuculline of 8-10wt% and the bright alkali of the Herba Corydalis ochotensis of 10-15wt%, research shows this alkaloid extract tool anti-breast cancer, ovarian cancer, the effect of gastric cancer and pulmonary carcinoma.
(2) in the method for extraction Corydalis impatiens (Pall.) Fisch. total alkaloids provided by the invention, total alkaloids crude extract finally carries out purification with silica gel column chromatography technology, and eluant used only has ethyl acetate, when obtaining target product, the recyclable recycling of solvent, easy and simple to handle, economize on resources.
(3) in the method for extraction Corydalis impatiens (Pall.) Fisch. total alkaloids provided by the invention, the extraction times of organic solvent is 3-6 time, not only can effectively extract the total alkaloids residuing in mother solution, can also reduce extraction times as far as possible and increase the energy waste bringing.
(4) method of extraction Corydalis impatiens (Pall.) Fisch. total alkaloids provided by the invention, the productive rate of total alkaloids is high, and total alkaloid content is high, and impurity is few, can be directly medicinal.Simultaneously simple to operate, organic solvent can be reused, and is conducive to promote on producing.
Experimental example
Following experimental example is used for proving technique effect of the present invention.
The Corydalis impatiens (Pall.) Fisch. total alkaloids extract obtaining in the embodiment of the present invention 1 of take is example, and details are as follows,
The mensuration of Corydalis impatiens (Pall.) Fisch. total alkaloids extract yield and content
Get the about 50mg of Corydalis impatiens (Pall.) Fisch. total alkaloids extract fine powder in embodiment 1, accurately weighed, put in tool plug conical flask, add volume parts than chloroform-ammonia mixed solution 50ml of 100:1, weighed weight, ultrasonic 30min, placement is spent the night, filter, residue adds volume parts than chloroform-ammonia mixed solution 50ml of 100:1, jolting 1 hour, filter, residue is used volume parts than chloroform-ammonia mixed solution washing of 100:1 3 times, and each 15m, filters; Merge above-mentioned filtrate and washing liquid, low-temperature reduced-pressure (40 ℃ following) evaporate to dryness, residue adds saturated alkaline ethanol 10ml to make to dissolve, add active carbon 1g, jolting decolouring 10min, filters, and precision measures in subsequent filtrate 5ml to 250ml conical flask, precision adds 3 of sulphuric acid volumetric solution (0.01mol/l) 15ml, water 15ml and C.I. 13020. indicator solutions, uses sodium hydroxide volumetric solution (0.02mol/l) to be titrated to yellow.Every 1ml sulphuric acid volumetric solution (0.01mol/l) is equivalent to the protopine (C20H19NO5) of 7.07mg
The sulphuric acid cumulative volume of V1-add, unit: ml
V2-in and the sulphuric acid volume that consumes of sodium hydroxide solution, unit: ml
The concentration of sulphuric acid for C-titration, unit: mol/L
7.07/0.01 the sulfuric acid solution of-1ml0.01mol/L is equivalent to the protopine of 7.07mg
The mensuration of monomer component content in embodiment 1 Corydalis impatiens (Pall.) Fisch. total alkaloids extract
According to high performance liquid chromatography (appendix VI D of < < Chinese Pharmacopoeia > > version in 2010), measure;
Chromatographic condition and system suitability be take octadecylsilane chemically bonded silica as filler; Take acetonitrile as mobile phase A, and take containing volume parts is Mobile phase B than 0.2% glacial acetic acid and volume parts than the aqueous solution of 0.5% triethylamine (pH value is 6.0), and according to the form below carries out gradient elution; Detection wavelength is 285nm; Number of theoretical plate calculates and should be not less than 3000 by protopine peak;
| Time (minute) | Mobility A(%) | Mobile phase B (%) |
| 0～60 | 20→40 | 80→60 |
| 60～90 | 40 | 60 |
Protopine, the bright alkali of Ochotensimine(Herba Corydalis ochotensis are got in the preparation of reference substance solution), α-Bicuculline(α-Bicuculline) reference substance is appropriate, add methanol and make every 1ml containing the solution of protopine 20 μ g, Ochotensimine30 μ g, α-Bicuculline10 μ g, obtain;
The about 20mg of Corydalis impatiens (Pall.) Fisch. total alkaloids fine powder is got in the preparation of need testing solution, accurately weighed, adds methanol 25ml reflux, extract, 2 hours, filter, get subsequent filtrate 25ml, reclaim under reduced pressure is to dry, residue is used 15ml sulphuric acid (1 → 10) solution to dissolve, and filtrate is used petroleum ether 2 times, each 20ml, discard petroleum ether part, water layer is used ammonia to regulate pH value to 9-10, chloroform extraction 3 times, each 20ml, reclaim under reduced pressure is to dry, with dissolve with methanol solution and be settled to 10ml.
Algoscopy is accurate object of reference solution and each 10ul of need testing solution of drawing respectively, and injection liquid chromatography, records chromatogram, according to corresponding alkaloidal go out peak area and concentration ratio, and calculating alkaloid.
The high-efficient liquid phase chromatogram of need testing solution as shown in Figure 1 in this step, in its spectrogram, also include the peak of β-Bicuculline and β-hydrystine, show also to contain in total alkaloids extract a certain amount of β-Bicuculline, β-hydrystine, in accompanying drawing 1, monomer 1 is α-Bicuculline, and monomer 2 is protopine, and monomer 3 is the bright alkali of Herba Corydalis ochotensis, monomer 4 is β-hydrystine, and monomer 5 is β-Bicuculline.
Corydalis impatiens (Pall.) Fisch. total alkaloids extract pharmacological experimental data
90 of the healthy Kunming mouses of 1 laboratory animal, SPF level, male, body weight 30 ± 2g.By Lukang Medical Co., Ltd., Shandong, provided the animal quality certification number: SCXK Shandong 20130001.
2 experimental cell cell line people's carcinoma of prostate (pc3) cell, breast carcinoma (MCF-7) cell, stomach cancer cell, adenocarcinoma of lung (A549) cell, ovarian cancer (skov3) cell provides by Shandong Prov. Hospital.
3 Experimental agents
Experimental agents: adopt the Corydalis impatiens (Pall.) Fisch. total alkaloids extract that described in embodiment 1 and comparative example 1-3, method prepares;
Positive control medicine: cisplatin, lot number: 1010041DC;
Reagent: 1640 cell culture mediums (Hyclone, Sai Mo flies generation that biochemistry goods Beijing company limited); Hyclone (Hyclone, Sai Mo flies generation that biochemistry goods Beijing company limited); Pancreatin cell dissociation buffer (the green skies), penicillin streptomycin mixed liquor is dual anti-, cisplatin for inj (Qilu Pharmaceutical Co., Ltd.).
4 experimental techniques
4.1 cell culture
From ultra cold storage freezer, get frozen human prostata cancer (pc3) cell, breast carcinoma (MCF-7) cell, stomach cancer cell, adenocarcinoma of lung (A549) cell, ovarian cancer (skov3) cell, rapidly input is heated in the aquesterilisa of 37 ℃, after cryopreserving liquid dissolves in advance, centrifugal three minutes of 2000r/min, collecting cell.Then to cultivate in 37 ℃, 5%CO2, saturated humidity incubator containing 1640 fresh cultures of 10% hyclone, the trophophase cell of taking the logarithm is tested.
The preparation of 4.2 Contained Serums
Kunming kind is little 90, is divided into 6 groups, and blank group, cisplatin group (10 μ g/ml), experimental group (extracting the total alkaloids obtaining in embodiment 1), matched group 1-3(are respectively the extract that in comparative example 1-3, extraction obtains), 15 every group.15 mices are divided into 5 groups, corresponding 5 tumor cell lines, three every group.The equal gavage of total alkaloids and matched group gives mice 3mg/ml suspension.Blank group gavage gives equivalent normal saline.The continuous gavage of mice 3 days, every day 0.5ml.After last day gavage 1 hour, 10% chloral hydrate anesthesia, abdominal aortic blood, 3000 turn/min are centrifugal, get upper serum ,-80 ℃ of preservations.Serum mixes on the same group, and 0.22 μ m filtering with microporous membrane degerming is in standby.
4.3MTT method is measured the impact of Contained Serum on different activity of tumor cells
By CO
2the different tumor cells of cultivating in incubator, the pancreatin cell dissociation buffer with 0.25% (containing 0.02%EDTA) digests, with 1 * 10
4the every hole of individual cell is inoculated in 96 orifice plates, reserved three zeroing holes, and zeroing hole only adds not containing 1640 culture medium of hyclone, does not add cell.Cell in 96 orifice plates covers with after monolayer, discard culture medium, zeroing group, blank group add the blank serum of 100 μ L, and cisplatin group adds containing cisplatin serum 100 μ l, the every hole of test group adds respectively corresponding Contained Serum 100 μ l, and every kind of cancerous cell adds Contained Serum 12 holes.Then cell is at 37 ℃, 5%CO
2in incubator, continue to cultivate 24h.Add 5mg/mlMTT solution 20 μ l lucifuge reaction 4h in incubator.Then add 100 μ L DMSO in 492nm, to measure OD value by microplate reader, calculate suppression ratio: inhibitory rate of cell growth=(matched group absorbance meansigma methods-experimental group absorbance meansigma methods)/matched group absorbance meansigma methods * 100%.
4.4 statistical method
All data all show with (X ± SD), adopt GraphPad Prism5 software analysis deal with data, utilize each group of t survey and between blank group, whether have significant difference, and P<0.05 is significant difference.
5, mtt assay is measured cell inhibitory rate result (n=12)
Experimental result is in Table 1.
Between the total alkaloids extract preparing in table 1 embodiment 1 and blank group, whether there is significant difference tables of data
It is active that table 1 result shows that the total alkaloids extract of embodiment 1 can significantly reduce the breast carcinoma of In vitro culture, gastric cancer, pulmonary carcinoma and ovarian cancer cell.And comparative example group can not suppress the activity of above-mentioned tumor cell.
The Corydalis impatiens (Pall.) Fisch. total alkaloids extract that adopts method of the present invention to extract to obtain is as the minimum onset dosimetry of cancer therapy drug
Experimental cell cell line: gastric cancer, breast carcinoma (MCF-7), lung carcinoma cell (A549) and ovarian cancer cell (SKOV3) cell strain, provide by Shandong Prov. Hospital.
Experimental agents: the embodiment of the present application 1 is extracted the Corydalis impatiens (Pall.) Fisch. total alkaloids obtaining.
Positive control medicine: cisplatin, lot number: 1010041DC
Reagent: 1640 cell culture mediums (Hyclone, Sai Mo flies generation that biochemistry goods Beijing company limited); Hyclone (Hyclone, Sai Mo flies generation that biochemistry goods Beijing company limited); Pancreatin cell dissociation buffer (the green skies), penicillin streptomycin mixed liquor is dual anti-, cisplatin for inj (Qilu Pharmaceutical Co., Ltd.).
Experimental apparatus
Vertical pressure steam sterilization pan (LDZX-50FBS, Shen, Shanghai peace); Double one side clean work station (SW-CJ-1C, Suzhou purifies); CO2 gas incubator (BB16UV/BB5060UV, HERAEUS); Desk centrifuge (H3, Sigma); Microplate reader (Multiskan MK3, Thermo Scientific); Electric heating constant-temperature blowing drying box (101, Shanghai Peng Shun scientific instrument company limited); Inverted microscope (XDS-1B, optical instrument factory, Chongqing); Water-bath constant temperature oscillator (SHZ-82, Jintan City Medical Instruments factory); Pipettor (Gilson, Eppendorf); Culture bottle (Corning); 96 orifice plates (Costar, USA)
Experimental technique
1, cell recovery:
From ultra cold storage freezer, get frozen cancerous cell, rapidly input is heated in the aquesterilisa of 37 ℃ in advance, after cryopreserving liquid dissolves, and centrifugal three minutes of 2000r/min, collecting cell., then to cultivate in 37 ℃, 5%CO2 incubator containing 1640 fresh cultures of 10% hyclone.
2, inoculation:
When Growth of Cells to 80% fusion, pancreatin cell dissociation buffer with 0.25% (containing 0.02%EDTA) digests, and with 1 * 104 every hole of cell, is inoculated in 96 orifice plates, reserved three zeroing holes, zeroing hole only adds not containing 1640 culture medium of hyclone, does not add cell.
3, dosing
Cell in 96 orifice plates covers with after monolayer, discard culture medium, zeroing group, blank group add 100 μ L and do not contain 1640 culture medium of hyclone, it is 10 μ g/ml cisplatin solution 100 μ l that positive controls adds concentration, the every hole of other test group adds respectively the 1640 culture medium 100 μ l containing variable concentrations screening of medicaments, and each concentration medicine adds 6 holes.Then cell continues to cultivate 24h in 37 ℃, 5%CO2 incubator.
4, measure:
Mtt assay is measured cell survival rate: in every hole, add 5mg/mlMTT solution 20 μ l lucifuge reaction 4h in incubator.Then add 100 μ L DMSO in 492nm, to measure OD value by microplate reader, calculate cell survival rate: computing formula: cell survival rate (%)=(medication group or model group A490/ control group A 490) * 100%.
5. statistical method
Adopt GraphPad Prism5 software analysis deal with data, utilize each group of t survey and between blank group, whether have significant difference, all data represent with X ± SD, the significance of t check comparison group difference, and P<0.05 is significant difference.
6MTT method is measured stomach cancer cell suppression ratio result
Table 2 total alkaloids to the suppression ratio of stomach cancer cell (n=6,
| Medicine | Concentration (μ g/ml) | Suppression ratio (%) |
| Cisplatin | 10 | 61.86±1.99* |
| Total alkaloids extract | 25 | 9.26±4.76 |
| Total alkaloids extract | 50 | 21.73±7.97 |
| Total alkaloids extract | 100 | 35.65±6.27* |
| Total alkaloids extract | 200 | 44.55±11.23* |
* with the comparison of blank group, P<0.05
Table 2 result shows, cisplatin group and total alkaloids 100 and 200 μ g/ml have significant difference with blank group, and it is not obvious that other each groups and blank are organized difference, illustrates that total alkaloids can significantly reduce stomach cancer cell activity more than 100 μ g/ml.
Table 3 total alkaloids to the suppression ratio of breast cancer cell (n=6,
| Medicine | Concentration (μ g/ml) | Suppression ratio (%) |
| Cisplatin | 10 | 55.88±3.79* |
| Total alkaloids extract | 25 | 6.30±4.12 |
| Total alkaloids extract | 50 | 5.74±7.96 |
| Total alkaloids extract | 100 | 28.76±9.68* |
| Total alkaloids extract | 200 | 37.48±11.96* |
* with the comparison of blank group, P<0.05
Table 3 result shows, cisplatin group and total alkaloids 100 and 200 μ g/ml have significant difference with blank group, and it is not obvious that other each groups and blank are organized difference, illustrates that total alkaloids can significantly reduce breast carcinoma tumor cell viability more than 100 μ g/ml.
Table 4 total alkaloids to the suppression ratio of lung carcinoma cell (n=6,
| Medicine | Concentration (μ g/ml) | Suppression ratio (%) |
| Cisplatin | 10 | 37.33±3.79* |
| Total alkaloids extract | 31.25 | 7.78±9.68 |
| Total alkaloids extract | 62.5 | 2.22±5.84 |
| Total alkaloids extract | 125 | 22.22±7.19* |
| Total alkaloids extract | 250 | 27.59±8.41* |
* with the comparison of blank group, P<0.05
Table 4 result shows, cisplatin group and total alkaloids 125 and 250 μ g/ml have significant difference with blank group, and it is not obvious that other each groups and blank are organized difference, illustrates that total alkaloids can significantly reduce lung carcinoma cell activity more than 125 μ g/ml.
Table 5 total alkaloids to the suppression ratio of ovarian cancer cell (n=6,
| Medicine | Concentration (μ g/ml) | Suppression ratio (%) |
| Cisplatin | 10 | 25.55±4.09* |
| Total alkaloids extract | 31.25 | 15.33±5.96 |
| Total alkaloids extract | 62.5 | 11.68±8.04 |
| Total alkaloids extract | 125 | 17.52±4.28* |
| Total alkaloids extract | 250 | 19.42±5.89* |
* with the comparison of blank group, P<0.05
Table 5 result shows, cisplatin group and total alkaloids 125 and 250 μ g/ml have significant difference with blank group, and it is not obvious that other each groups and blank are organized difference, illustrates that total alkaloids can significantly reduce ovarian cancer cell activity more than 125 μ g/ml.
The specific embodiment
The present invention's Corydalis impatiens (Pall.) Fisch. used is taken from Qinghai Province, through the fame president of Qinghai Province Tibetan medicine hospital, Qinghai Province's Tibetan medicine principal expert's Nima, be accredited as small-mouthed jar section Genus Corydalis Corydalis impatiens (Pall.) Fisch. (Corydalis impatiens (Pall.) Fisch) medical material, the position that all embodiment of the present invention adopt is the position, ground that Corydalis impatiens (Pall.) Fisch. is removed root.
Getting Corydalis impatiens (Pall.) Fisch. pulverizing medicinal materials becomes coarse powder to cross 10 mesh sieves, adding volumetric concentration is 50% saturated alkaline ethanol, and at 50 ℃, reflux, extract, is 3 times, extracts 3 hours at every turn, obtain Corydalis impatiens (Pall.) Fisch. extracting solution, wherein in this step, the consumption of saturated alkaline ethanol is 10 times of Corydalis impatiens (Pall.) Fisch. quality of medicinal material; By above-mentioned Corydalis impatiens (Pall.) Fisch. extracting solution reclaim under reduced pressure alkaline ethanol at 60 ℃, reconcentration to relative density is 1.1(50 ℃ of survey) clear paste, spraying is dry, obtains Corydalis impatiens (Pall.) Fisch. alkaline ethanol extract; To adding volumetric concentration in Corydalis impatiens (Pall.) Fisch. alkaline ethanol extract, be that 0.5% dissolving with hydrochloric acid filters and repeats 3 times, wherein in each course of dissolution, add the acid liquid of 6 times of amounts of amount of solid quality, the filtrate merging after filtering for 3 times forms acid liquid, in described acid liquid, add volumetric concentration 4% sodium hydrate aqueous solution to regulate pH value to 10, obtain acid-alkali treatment liquid; In described acid-alkali treatment liquid, add dichloromethane to extract 5 times, the addition that wherein each dichloro adds alkane is 3 times of volume total alkali liquid measures, and combined dichloromethane liquid reclaims, and the dry Corydalis impatiens (Pall.) Fisch. total alkaloids crude extract that obtains.Get silica gel ethyl acetate wet method dress post, in Corydalis impatiens (Pall.) Fisch. total alkaloids crude extract, add 2 times of loadings after the acetic acid ethyl dissolution of its quality, adopt ethyl acetate eluting, dry, obtain required Corydalis impatiens (Pall.) Fisch. total alkaloids extract, wherein silica gel consumption is 5 times of described Corydalis impatiens (Pall.) Fisch. total alkaloids crude extract quality, and the consumption of eluent ethyl acetate agent is 50 times of described Corydalis impatiens (Pall.) Fisch. total alkaloids crude extract quality.Total alkaloids yield is 0.33wt% after measured, extracting species total alkaloid content is 52wt%, its Protopine 10wt%, α-Bicuculline 8wt%, the bright alkali 10wt% of Herba Corydalis ochotensis, contains the peak of β-Bicuculline, β-hydrystine in its high-efficient liquid phase chromatogram.To measure the activity of tumor cells inhibition percentage of In vitro culture, its anticancer onset dosage is 100 μ g/ml.
Getting Corydalis impatiens (Pall.) Fisch. pulverizing medicinal materials becomes coarse powder to cross 50 mesh sieves, adding volumetric concentration is 95% saturated alkaline ethanol, and at 70 ℃, reflux, extract, is 3 times, extracts 2 hours at every turn, obtain Corydalis impatiens (Pall.) Fisch. extracting solution, wherein in this step, the consumption of saturated alkaline ethanol is 8 times of Corydalis impatiens (Pall.) Fisch. quality of medicinal material, by above-mentioned Corydalis impatiens (Pall.) Fisch. extracting solution reclaim under reduced pressure alkaline ethanol at 60 ℃, reconcentration to relative density is 1.05(50 ℃ of survey) clear paste, spraying is dry, obtains Corydalis impatiens (Pall.) Fisch. alkaline ethanol extract, to adding volumetric concentration in Corydalis impatiens (Pall.) Fisch. alkaline ethanol extract, be that 4% dissolving with hydrochloric acid filters and repeats 4 times, wherein in each course of dissolution, add the acid liquid of 1 times of amount of solid masses, the filtrate merging after filtering forms acid liquid, in described acid liquid, add volumetric concentration 10% sodium hydrate aqueous solution to regulate pH value to 10, obtain acid-alkali treatment liquid, in described acid-alkali treatment liquid, add dichloromethane to extract 4 times, wherein the addition of each dichloromethane is the volume of 5 times of total alkali liquor, combined dichloromethane liquid, reclaim, and the dry Corydalis impatiens (Pall.) Fisch. total alkaloids crude extract that obtains, get silica gel ethyl acetate wet method dress post, in Corydalis impatiens (Pall.) Fisch. total alkaloids crude extract, add 2 times of loadings after the acetic acid ethyl dissolution of its quality, adopt ethyl acetate eluting, dry, obtain required Corydalis impatiens (Pall.) Fisch. total alkaloids extract, wherein silica gel consumption is 10 times of described Corydalis impatiens (Pall.) Fisch. total alkaloids crude extract quality, the consumption of eluent ethyl acetate agent is 100 times of described Corydalis impatiens (Pall.) Fisch. total alkaloids crude extract quality.Total alkaloids yield is 0.3wt% after measured, total alkaloid content is 55wt%, its Protopine 11wt%, α-Bicuculline 9wt%, the bright alkali 13wt% of Herba Corydalis ochotensis, the peak that contains β-Bicuculline, β-hydrystine in its high-efficient liquid phase chromatogram, to measure the activity of tumor cells inhibition percentage of In vitro culture, its anticancer onset dosage is 100 μ g/ml.
Embodiment 3
Getting Corydalis impatiens (Pall.) Fisch. pulverizing medicinal materials becomes coarse powder to cross 50 mesh sieves, adding volumetric concentration is 90% saturated alkaline ethanol, and at 80 ℃, reflux, extract, is 3 times, extracts 2.5 hours at every turn, obtain Corydalis impatiens (Pall.) Fisch. extracting solution, wherein in this step, the consumption of saturated alkaline ethanol is 7 times of Corydalis impatiens (Pall.) Fisch. quality of medicinal material; By above-mentioned Corydalis impatiens (Pall.) Fisch. extracting solution reclaim under reduced pressure alkaline ethanol at 60 ℃, reconcentration to relative density is 1.1(50 ℃ of survey) clear paste, spraying is dry, obtains Corydalis impatiens (Pall.) Fisch. alkaline ethanol extract; To adding volumetric concentration in Corydalis impatiens (Pall.) Fisch. alkaline ethanol extract, be that 1% sulfuric acid dissolution filters and repeats 3 times, wherein in each course of dissolution, add the sulfuric acid solution of 3 times of amounts of amount of solid quality, the filtrate merging after filtering for 3 times forms acid liquid, in described acid liquid, add volumetric concentration 10% aqueous sodium carbonate to regulate pH value to 10, obtain acid-alkali treatment liquid; In described acid-alkali treatment liquid, add dichloromethane to extract 6 times, the addition that wherein each dichloro adds alkane is the volume of 3 times of total alkali liquor, and combined dichloromethane liquid reclaims, and the dry Corydalis impatiens (Pall.) Fisch. total alkaloids crude extract that obtains.Get silica gel ethyl acetate wet method dress post, in Corydalis impatiens (Pall.) Fisch. total alkaloids crude extract, add 2 times of loadings after the acetic acid ethyl dissolution of its quality, adopt ethyl acetate eluting, dry, obtain required Corydalis impatiens (Pall.) Fisch. total alkaloids extract, wherein silica gel consumption is 7 times of described Corydalis impatiens (Pall.) Fisch. total alkaloids crude extract quality, and the consumption of eluent ethyl acetate agent is 80 times of described Corydalis impatiens (Pall.) Fisch. total alkaloids crude extract quality.Total alkaloids yield is 0.31wt% after measured, total alkaloid content is 60wt%, its Protopine 12wt%, α-Bicuculline 10wt%, the bright alkali 15wt% of Herba Corydalis ochotensis, contains the peak of β-Bicuculline, β-hydrystine in its high-efficient liquid phase chromatogram, to measure the activity of tumor cells inhibition percentage of In vitro culture, its anticancer onset dosage is 100 μ g/ml.
Embodiment 4
Getting Corydalis impatiens (Pall.) Fisch. pulverizing medicinal materials becomes coarse powder to cross 50 mesh sieves, adding volumetric concentration is 80% saturated alkaline ethanol, and at 70 ℃, reflux, extract, is 3 times, extracts 2.5 hours at every turn, obtain Corydalis impatiens (Pall.) Fisch. extracting solution, wherein in this step, the consumption of saturated alkaline ethanol is 7 times of Corydalis impatiens (Pall.) Fisch. quality of medicinal material; By above-mentioned Corydalis impatiens (Pall.) Fisch. extracting solution reclaim under reduced pressure alkaline ethanol at 60 ℃, reconcentration to relative density is 1.1(50 ℃ of survey) clear paste, spraying is dry, obtains Corydalis impatiens (Pall.) Fisch. alkaline ethanol extract; To add volumetric concentration in Corydalis impatiens (Pall.) Fisch. alkaline ethanol extract be 2% phosphoric acid dissolution filter and repeat 3 times, wherein in each course of dissolution, add the phosphoric acid solution of 3 times of amounts of amount of solid quality, the filtrate merging after filtering for 3 times forms acid liquid, in described acid liquid, add volumetric concentration 15% sodium bicarbonate aqueous solution to regulate pH value to 10, obtain acid-alkali treatment liquid; In described acid-alkali treatment liquid, add ethyl acetate to extract 4 times, the addition that wherein each dichloro adds alkane is 5 times of total alkali liquid accumulated amounts, and combined ethyl acetate liquid reclaims, and the dry Corydalis impatiens (Pall.) Fisch. total alkaloids crude extract that obtains.Get silica gel ethyl acetate wet method dress post, in Corydalis impatiens (Pall.) Fisch. total alkaloids crude extract, add 2 times of loadings after the acetic acid ethyl dissolution of its quality, adopt ethyl acetate eluting, dry, obtain required Corydalis impatiens (Pall.) Fisch. total alkaloids extract, wherein silica gel consumption is 8 times of described Corydalis impatiens (Pall.) Fisch. total alkaloids crude extract quality, and the consumption of eluent ethyl acetate agent is 50 times of described Corydalis impatiens (Pall.) Fisch. total alkaloids crude extract quality.Total alkaloids yield is 0.33wt% after measured, total alkaloid content is 53wt%, its Protopine 10wt%, α-Bicuculline 9wt%, the bright alkali 12wt% of Herba Corydalis ochotensis, the peak that contains β-Bicuculline, β-hydrystine in its high-efficient liquid phase chromatogram, to measure the activity of tumor cells inhibition percentage of In vitro culture, its anticancer onset dosage is 100 μ g/ml.
Embodiment 5
Getting Corydalis impatiens (Pall.) Fisch. pulverizing medicinal materials becomes coarse powder to cross 50 mesh sieves, adding volumetric concentration is 60% saturated alkaline ethanol, and at 80 ℃, reflux, extract, is 3 times, extracts 2.5 hours at every turn, obtain Corydalis impatiens (Pall.) Fisch. extracting solution, wherein in this step, the consumption of saturated alkaline ethanol is 7 times of Corydalis impatiens (Pall.) Fisch. quality of medicinal material; By above-mentioned Corydalis impatiens (Pall.) Fisch. extracting solution reclaim under reduced pressure alkaline ethanol at 60 ℃, reconcentration to relative density is 1.1(50 ℃ of survey) clear paste, spraying is dry, obtains Corydalis impatiens (Pall.) Fisch. alkaline ethanol extract; To adding volumetric concentration in Corydalis impatiens (Pall.) Fisch. alkaline ethanol extract, be that 3% acetate dissolution filters and repeats 3 times, wherein in each course of dissolution, add the acetum of 3 times of amounts of amount of solid quality, the filtrate merging after filtering for 3 times forms acid liquid, in described acid liquid, add volumetric concentration 10% aqueous sodium carbonate to regulate pH value to 10, obtain acid-alkali treatment liquid; In described acid-alkali treatment liquid, add dichloromethane to extract 6 times, the volume that adds that wherein each dichloro adds alkane is the volume of 4 times of total alkali liquor, and combined dichloromethane liquid reclaims, and the dry Corydalis impatiens (Pall.) Fisch. total alkaloids crude extract that obtains.Get silica gel ethyl acetate wet method dress post, in Corydalis impatiens (Pall.) Fisch. total alkaloids crude extract, add 2 times of loadings after the acetic acid ethyl dissolution of its quality, adopt ethyl acetate eluting, dry, obtain required Corydalis impatiens (Pall.) Fisch. total alkaloids extract, wherein silica gel consumption is 8 times of described Corydalis impatiens (Pall.) Fisch. total alkaloids crude extract quality, and the consumption of eluent ethyl acetate agent is 50 times of described Corydalis impatiens (Pall.) Fisch. total alkaloids crude extract quality.Total alkaloids yield is 0.34% after measured, and total alkaloid content is 55wt%, its Protopine 11wt%, and α-Bicuculline 8wt%, the bright alkali 14wt% of Herba Corydalis ochotensis, contains the peak of β-Bicuculline, β-hydrystine in its high-efficient liquid phase chromatogram.To measure the activity of tumor cells inhibition percentage of In vitro culture, its anticancer onset dosage is 100 μ g/ml.
Embodiment 6
Getting Corydalis impatiens (Pall.) Fisch. pulverizing medicinal materials becomes coarse powder to cross 30 mesh sieves, adding volumetric concentration is 80% saturated alkaline ethanol, and at 80 ℃, reflux, extract, is 4 times, extracts 3 hours at every turn, obtain Corydalis impatiens (Pall.) Fisch. extracting solution, wherein in this step, the consumption of saturated alkaline ethanol is 10 times of Corydalis impatiens (Pall.) Fisch. quality of medicinal material; By above-mentioned Corydalis impatiens (Pall.) Fisch. extracting solution reclaim under reduced pressure alkaline ethanol at 60 ℃, reconcentration to relative density is 1.1(50 ℃ of survey) clear paste, spraying is dry, obtains Corydalis impatiens (Pall.) Fisch. alkaline ethanol extract; To adding volumetric concentration in Corydalis impatiens (Pall.) Fisch. alkaline ethanol extract, be that 0.5% dissolving with hydrochloric acid filters and repeats 3 times, wherein in each course of dissolution, add the hydrochloric acid solution of 5 times of amounts of amount of solid quality, the filtrate merging after filtering for 3 times forms acid liquid, in described acid liquid, add volumetric concentration 10% sodium hydrate aqueous solution to regulate pH value to 10, obtain acid-alkali treatment liquid; In described acid-alkali treatment liquid, add dichloromethane to extract 3 times, the volume that adds that wherein each dichloro adds alkane is the volume of 3 times of total alkali liquor, and combined dichloromethane liquid reclaims, and the dry Corydalis impatiens (Pall.) Fisch. total alkaloids crude extract that obtains.Get silica gel ethyl acetate wet method dress post, in Corydalis impatiens (Pall.) Fisch. total alkaloids crude extract, add 2 times of loadings after the acetic acid ethyl dissolution of its quality, adopt ethyl acetate eluting, dry, obtain required Corydalis impatiens (Pall.) Fisch. total alkaloids extract, wherein silica gel consumption is 10 times of described Corydalis impatiens (Pall.) Fisch. total alkaloids crude extract quality, and the consumption of eluent ethyl acetate agent is 80 times of described Corydalis impatiens (Pall.) Fisch. total alkaloids crude extract quality.Total alkaloids yield is 0.34wt% after measured, and total alkaloid content is 55wt%, its Protopine 11wt%, and α-Bicuculline 9wt%, the bright alkali 13% of Herba Corydalis ochotensis, contains the peak of β-Bicuculline, β-hydrystine in its high-efficient liquid phase chromatogram.To measure the activity of tumor cells inhibition percentage of In vitro culture, its anticancer onset dosage is 100 μ g/ml.
Embodiment 7
Getting Corydalis impatiens (Pall.) Fisch. pulverizing medicinal materials becomes coarse powder to cross 5-30 mesh sieve, adding volumetric concentration is 70% saturated alkaline ethanol, and at 40 ℃, reflux, extract, is 5 times, extracts 3 hours at every turn, obtain Corydalis impatiens (Pall.) Fisch. extracting solution, wherein in this step, the consumption of saturated alkaline ethanol is 7 times of Corydalis impatiens (Pall.) Fisch. quality of medicinal material; By above-mentioned Corydalis impatiens (Pall.) Fisch. extracting solution reclaim under reduced pressure alkaline ethanol at 60 ℃, reconcentration to relative density is 1.04(50 ℃ of survey) clear paste, spraying is dry, obtains Corydalis impatiens (Pall.) Fisch. alkaline ethanol extract; To adding volumetric concentration in Corydalis impatiens (Pall.) Fisch. alkaline ethanol extract, be that 2% acetate dissolution filters and repeats 3 times, wherein in each course of dissolution, add the acetum of 2 times of amounts of amount of solid quality, the filtrate merging after filtering for 3 times forms acid liquid, in described acid liquid, add volumetric concentration 12% aqueous sodium carbonate to regulate pH value to 9, obtain acid-alkali treatment liquid; In described acid-alkali treatment liquid, add dichloromethane to extract 3 times, the volume that adds that wherein each dichloro adds alkane is the volume of 2 times of total alkali liquor, and combined dichloromethane liquid reclaims, and the dry Corydalis impatiens (Pall.) Fisch. total alkaloids crude extract that obtains.Get silica gel ethyl acetate wet method dress post, in Corydalis impatiens (Pall.) Fisch. total alkaloids crude extract, add 1 times of loading after the acetic acid ethyl dissolution of its quality, adopt ethyl acetate eluting, elution flow rate is 1 volume post per hour, wherein silica gel consumption is 5 times of described Corydalis impatiens (Pall.) Fisch. total alkaloids crude extract quality, the consumption of eluent ethyl acetate agent is 60 times of described Corydalis impatiens (Pall.) Fisch. total alkaloids crude extract quality, after elution process completes, by the first reclaim under reduced pressure ethyl acetate of eluent, then drying under reduced pressure at 60 ℃, obtains required Corydalis impatiens (Pall.) Fisch. total alkaloids extract.Total alkaloids yield is 0.32% after measured, and total alkaloid content is 52wt%, its Protopine 11wt%, and α-Bicuculline 10wt%, the bright alkali 14wt% of Herba Corydalis ochotensis, contains the peak of β-Bicuculline, β-hydrystine in its high-efficient liquid phase chromatogram.To measure the activity of tumor cells inhibition percentage of In vitro culture, its anticancer onset dosage is 100 μ g/ml.
Embodiment 8
Getting Corydalis impatiens (Pall.) Fisch. pulverizing medicinal materials becomes coarse powder to cross 20-50 mesh sieve, adding volumetric concentration is 75% saturated alkaline ethanol, and at 65 ℃, reflux, extract, is 3 times, extracts 3 hours at every turn, obtain Corydalis impatiens (Pall.) Fisch. extracting solution, wherein in this step, the consumption of saturated alkaline ethanol is 7 times of Corydalis impatiens (Pall.) Fisch. quality of medicinal material; By above-mentioned Corydalis impatiens (Pall.) Fisch. extracting solution reclaim under reduced pressure alkaline ethanol at 60 ℃, reconcentration to relative density is 1.06(50 ℃ of survey) clear paste, spraying is dry, obtains Corydalis impatiens (Pall.) Fisch. alkaline ethanol extract; To add volumetric concentration in Corydalis impatiens (Pall.) Fisch. alkaline ethanol extract be 0.5% phosphoric acid dissolution filter and repeat 3 times, wherein in each course of dissolution, add the phosphoric acid solution of 2 times of amounts of amount of solid quality, the filtrate merging after filtering for 3 times forms acid liquid, in described acid liquid, add volumetric concentration 10% aqueous sodium carbonate to regulate pH value to 10, obtain acid-alkali treatment liquid; In described acid-alkali treatment liquid, add dichloromethane to extract 6 times, the volume that adds that wherein each dichloro adds alkane is the volume of 4 times of total alkali liquor, and combined dichloromethane liquid reclaims, and the dry Corydalis impatiens (Pall.) Fisch. total alkaloids crude extract that obtains.Get silica gel ethyl acetate wet method dress post, in Corydalis impatiens (Pall.) Fisch. total alkaloids crude extract, add 2 times of loadings after the acetic acid ethyl dissolution of its quality, adopt ethyl acetate eluting, elution flow rate is 2 volume posts per hour, wherein silica gel consumption is 5 times of described Corydalis impatiens (Pall.) Fisch. total alkaloids crude extract quality, the consumption of eluent ethyl acetate agent is 50 times of described Corydalis impatiens (Pall.) Fisch. total alkaloids crude extract quality, after elution process completes, by the first reclaim under reduced pressure ethyl acetate of eluent, then drying under reduced pressure at 60 ℃, obtains required Corydalis impatiens (Pall.) Fisch. total alkaloids extract.Total alkaloids yield is 0.31wt% after measured, total alkaloid content is 54wt%, its Protopine 11wt%, α-Bicuculline 9wt%, the bright alkali 12wt% of Herba Corydalis ochotensis, the peak that contains β-Bicuculline, β-hydrystine in its high-efficient liquid phase chromatogram, to measure the activity of tumor cells inhibition percentage of In vitro culture, its anticancer onset dosage is 100 μ g/ml.
Embodiment 9
Getting Corydalis impatiens (Pall.) Fisch. pulverizing medicinal materials becomes coarse powder to cross 30 mesh sieves, adding volumetric concentration is 50% saturated alkaline ethanol, and at 60 ℃, reflux, extract, is 3 times, extracts 3 hours at every turn, obtain Corydalis impatiens (Pall.) Fisch. extracting solution, wherein in this step, the consumption of saturated alkaline ethanol is 5 times of Corydalis impatiens (Pall.) Fisch. quality of medicinal material; By above-mentioned Corydalis impatiens (Pall.) Fisch. extracting solution reclaim under reduced pressure alkaline ethanol at 60 ℃, reconcentration to relative density is 1.1(50 ℃ of survey) clear paste, spraying is dry, obtains Corydalis impatiens (Pall.) Fisch. alkaline ethanol extract; To adding volumetric concentration in Corydalis impatiens (Pall.) Fisch. alkaline ethanol extract, be that 3% acetate dissolution filters and repeats 3 times, wherein in each course of dissolution, add the acetum of 3 times of amounts of amount of solid quality, the filtrate merging after filtering for 3 times forms acid liquid, in described acid liquid, add volumetric concentration 10% aqueous sodium carbonate to regulate pH value to 9, obtain acid-alkali treatment liquid; In described acid-alkali treatment liquid, add dichloromethane to extract 3 times, the volume that adds that wherein each dichloro adds alkane is the volume of 4 times of total alkali liquor, and combined dichloromethane liquid reclaims, and the dry Corydalis impatiens (Pall.) Fisch. total alkaloids crude extract that obtains.Get silica gel ethyl acetate wet method dress post, in Corydalis impatiens (Pall.) Fisch. total alkaloids crude extract, add 1 times of loading after the acetic acid ethyl dissolution of its quality, adopt ethyl acetate eluting, elution flow rate is 1 volume post per hour, wherein silica gel consumption is 10 times of described Corydalis impatiens (Pall.) Fisch. total alkaloids crude extract quality, the consumption of eluent ethyl acetate agent is 80 times of described Corydalis impatiens (Pall.) Fisch. total alkaloids crude extract quality, after elution process completes, by the first reclaim under reduced pressure ethyl acetate of eluent, then drying under reduced pressure at 60 ℃, obtains required Corydalis impatiens (Pall.) Fisch. total alkaloids extract.Total alkaloids yield is 0.34% after measured, and total alkaloid content is 54wt%, its Protopine 10wt%, α-Bicuculline 9wt%, the bright alkali 12wt% of Herba Corydalis ochotensis, and the peak that contains β-Bicuculline, β-hydrystine in its high-efficient liquid phase chromatogram.To measure the activity of tumor cells inhibition percentage of In vitro culture, its anticancer onset dosage is 100 μ g/ml.
Comparative example 1
Getting Corydalis impatiens (Pall.) Fisch. pulverizing medicinal materials becomes coarse powder to cross 10 mesh sieves, 50% ethanol that adds 10 times of Herba corydalis edulis quality of medicinal materials, at 50 ℃, reflux, extract, is 3 times, each extraction 3 hours, obtain Corydalis impatiens (Pall.) Fisch. extracting solution, by above-mentioned Corydalis impatiens (Pall.) Fisch. extracting solution decompression recycling ethanol at 60 ℃, reconcentration to relative density is 1.10(50 ℃ of survey) clear paste is Corydalis impatiens (Pall.) Fisch. ethanol extraction, to adding volumetric concentration in Corydalis impatiens (Pall.) Fisch. ethanol extraction, it is 0.5% dissolving with hydrochloric acid to regulate pH be 3, after standing over night, filter and obtain filtrate, in described filtrate, add volumetric concentration 10% aqueous sodium carbonate to regulate pH value to 10, obtain acid-alkali treatment liquid, to after described acid-alkali treatment liquid standing over night, add dichloromethane to extract 5 times, the addition that wherein each dichloro adds alkane be 3 times of total alkali liquid accumulated amounts, and combined dichloromethane liquid reclaims, and the dry Corydalis impatiens (Pall.) Fisch. total alkaloids crude extract that obtains.Get silica gel ethyl acetate wet method dress post, in Corydalis impatiens (Pall.) Fisch. total alkaloids crude extract, add 2 times of loadings after the acetic acid ethyl dissolution of its quality, adopt ethyl acetate eluting, dry, obtain required Corydalis impatiens (Pall.) Fisch. total alkaloids, wherein silica gel consumption is 5 times of described Corydalis impatiens (Pall.) Fisch. total alkaloids crude extract quality, and the consumption of eluent ethyl acetate agent is 50 times of described Corydalis impatiens (Pall.) Fisch. total alkaloids crude extract quality.Total alkaloids yield is 0.24wt% after measured, and total alkaloid content is 42wt%, its Protopine 8wt%, and α-Bicuculline 2wt%, the bright alkali 5wt% of Herba Corydalis ochotensis, does not contain the peak of β-Bicuculline and β-hydrystine in its high-efficient liquid phase chromatogram.
Comparative example 2
Getting Corydalis impatiens (Pall.) Fisch. pulverizing medicinal materials becomes coarse powder to cross 50 mesh sieves, the dilute hydrochloric acid that adds 7 times of Herba corydalis edulis quality of medicinal materials, at 80 ℃, reflux, extract, is 2 times, each extraction 2 hours, obtain Corydalis impatiens (Pall.) Fisch. extracting solution, by above-mentioned Corydalis impatiens (Pall.) Fisch. extracting solution concentrating under reduced pressure at 70 ℃, to relative density be 1.12(50 ℃ of survey) clear paste, in described clear paste, add volumetric concentration 10% sodium hydrate aqueous solution to regulate pH value to 10, obtain alkali treatment liquid; In described alkali treatment liquid, add dichloromethane to extract 6 times, the addition that wherein each dichloro adds alkane is 4 times of total alkali liquid accumulated amounts, and combined dichloromethane liquid reclaims, and the dry Corydalis impatiens (Pall.) Fisch. total alkaloids crude extract that obtains.Get silica gel ethyl acetate wet method dress post, in Corydalis impatiens (Pall.) Fisch. total alkaloids crude extract, add 2 times of loadings after the acetic acid ethyl dissolution of its quality, adopt ethyl acetate eluting, dry, obtain required Corydalis impatiens (Pall.) Fisch. total alkaloids, wherein silica gel consumption is 10 times of described Corydalis impatiens (Pall.) Fisch. total alkaloids crude extract quality, and the consumption of eluent ethyl acetate agent is 50 times of described Corydalis impatiens (Pall.) Fisch. total alkaloids crude extract quality.Total alkaloids yield is 0.38wt% after measured, and total alkaloid content is 50wt%, its Protopine 8wt%, and α-Bicuculline 6wt%, the bright alkali 10wt% of Herba Corydalis ochotensis, does not contain the peak of β-hydrystine in its high-efficient liquid phase chromatogram.
Comparative example 3
Getting Corydalis impatiens (Pall.) Fisch. pulverizing medicinal materials becomes coarse powder to cross 10 mesh sieves, adding volumetric concentration is 30% saturated alkaline ethanol, and at 30 ℃, reflux, extract, is 6 times, extracts 3 hours at every turn, obtain Corydalis impatiens (Pall.) Fisch. extracting solution, wherein in this step, the consumption of saturated alkaline ethanol is 10 times of Corydalis impatiens (Pall.) Fisch. quality of medicinal material; By above-mentioned Corydalis impatiens (Pall.) Fisch. extracting solution reclaim under reduced pressure alkaline ethanol at 60 ℃, reconcentration to relative density is 1.1(50 ℃ of survey) clear paste, spraying is dry, obtains Corydalis impatiens (Pall.) Fisch. alkaline ethanol extract; To adding volumetric concentration in Corydalis impatiens (Pall.) Fisch. alkaline ethanol extract, be that 0.1% dissolving with hydrochloric acid filters and repeats 3 times, wherein in each course of dissolution, add the acid liquid of 5 times of amounts of amount of solid quality, the filtrate merging after filtering for 3 times forms acid liquid, in described acid liquid, add volumetric concentration 10% sodium hydrate aqueous solution to regulate pH value to 8, obtain acid-alkali treatment liquid; In described acid-alkali treatment liquid, add dichloromethane to extract 5 times, the addition that wherein each dichloro adds alkane is 3 times of volume total alkali liquid measures, and combined dichloromethane liquid reclaims, and the dry Corydalis impatiens (Pall.) Fisch. total alkaloids crude extract that obtains.Get silica gel ethyl acetate wet method dress post, in Corydalis impatiens (Pall.) Fisch. total alkaloids crude extract, add 2 times of loadings after the acetic acid ethyl dissolution of its quality, adopt ethyl acetate eluting, dry, obtain required Corydalis impatiens (Pall.) Fisch. total alkaloids, wherein silica gel consumption is 5 times of described Corydalis impatiens (Pall.) Fisch. total alkaloids crude extract quality, and the consumption of eluent ethyl acetate agent is 80 times of described Corydalis impatiens (Pall.) Fisch. total alkaloids crude extract quality.Total alkaloids yield is 0.28wt% after measured, and extracting species total alkaloid content is 44wt%, its Protopine 12wt%, and α-Bicuculline 6wt%, the bright alkali 8wt% of Herba Corydalis ochotensis, contains the peak of β-Bicuculline, β-hydrystine in its high-efficient liquid phase chromatogram.
Obviously, above-described embodiment is only for example is clearly described, and the not restriction to embodiment.For those of ordinary skill in the field, can also make other changes in different forms on the basis of the above description.Here exhaustive without also giving all embodiments.And the apparent variation of being extended out thus or change are still among the protection domain in the invention.
Claims (8)
1. the Corydalis impatiens (Pall.) Fisch. total alkaloids extract for the preparation of cancer therapy drug, it is characterized in that, in described Corydalis impatiens (Pall.) Fisch. total alkaloids extract, the content of total alkaloids is not less than 50wt%, the protopine that comprises β-Bicuculline, β-hydrystine, 10-12wt%, α-Bicuculline of 8-10wt% and the bright alkali of the Herba Corydalis ochotensis of 10-15wt%.
2. the Corydalis impatiens (Pall.) Fisch. total alkaloids extract for the preparation of cancer therapy drug according to claim 1, it is characterized in that, in described Corydalis impatiens (Pall.) Fisch. total alkaloids extract, the content of total alkaloids is not less than 50wt%, comprise β-Bicuculline, β-hydrystine, 11wt% protopine, 9wt% α-Bicuculline and the bright alkali of 13wt% Herba Corydalis ochotensis.
3. the Corydalis impatiens (Pall.) Fisch. total alkaloids extract for the preparation of cancer therapy drug according to claim 1 and 2, is characterized in that, described cancer therapy drug is the medicine of anti-breast cancer, ovarian cancer, gastric cancer or pulmonary carcinoma.
4. the extract of Corydalis impatiens (Pall.) Fisch. total alkaloids, for the preparation of an application for cancer therapy drug, is characterized in that, described extract is extracted by following steps,
(1) by Corydalis impatiens (Pall.) Fisch. pulverizing medicinal materials, and utilize the saturated alkaline ethanol of 50-95% volumetric concentration or saturated alkaline methanol, reflux, extract, at 40-80 ℃, obtain Corydalis impatiens (Pall.) Fisch. extracting solution, the 7-10 of Corydalis impatiens (Pall.) Fisch. quality of medicinal material doubly described in the consumption of wherein said saturated alkaline ethanol or described saturated alkaline methanol;
(2) by concentrated, the dry Corydalis impatiens (Pall.) Fisch. Extract that obtains of described Corydalis impatiens (Pall.) Fisch. extracting solution;
(3) to adding volumetric concentration in described Corydalis impatiens (Pall.) Fisch. Extract, be that the acid solution of 0.5-4% dissolves, wherein, the addition of acid solution and the mass ratio of described Corydalis impatiens (Pall.) Fisch. Extract are (1-6): 1, filtration obtains acid liquid, to adding volumetric concentration in described acid liquid, it is the aqueous slkali of 4-15%, regulator solution pH value, to 9-10, obtains acid-alkali treatment liquid;
(4) in described acid-alkali treatment liquid, add organic solvent extraction, reclaim described organic solvent and obtain Corydalis impatiens (Pall.) Fisch. total alkaloids crude extract, wherein the each addition of organic solvent is 2-4 times of long-pending volume of described acid-alkali treatment liquid.
(5) get silica gel ethyl acetate wet method dress post, and the described Corydalis impatiens (Pall.) Fisch. total alkaloids crude extract obtaining is added to loading after acetic acid ethyl dissolution, and adopt ethyl acetate eluting, dry, obtain required Corydalis impatiens (Pall.) Fisch. total alkaloids, wherein for dissolving the 1-3 that the consumption of the ethyl acetate of total alkaloids crude extract is its quality, doubly measure, silica gel consumption be described Corydalis impatiens (Pall.) Fisch. total alkaloids crude extract quality 5-10 doubly, for the consumption of the ethyl acetate of eluting be described Corydalis impatiens (Pall.) Fisch. total alkaloids crude extract quality 50-100 doubly.
5. Corydalis impatiens (Pall.) Fisch. total alkaloids extract according to claim 4, for the preparation of the application of cancer therapy drug, is characterized in that, its extraction step is as follows,
(1) by Corydalis impatiens (Pall.) Fisch. pulverizing medicinal materials to 5-50 order, and utilize the saturated alkaline ethanol of 50-95% volumetric concentration or saturated alkaline methanol, at 40-80 ℃, reflux, extract, is 3-8 time, obtain Corydalis impatiens (Pall.) Fisch. extracting solution, the 7-10 of Corydalis impatiens (Pall.) Fisch. quality of medicinal material doubly described in the consumption of wherein said saturated alkaline ethanol or described saturated alkaline methanol;
(2) described Corydalis impatiens (Pall.) Fisch. extracting solution is concentrated into clear paste, the dry Corydalis impatiens (Pall.) Fisch. Extract that obtains;
(3) to adding volumetric concentration in described Corydalis impatiens (Pall.) Fisch. Extract, be the acid solution dissolution filter several times of 0.5-4%, in each dissolution filter step, the addition of acid solution and the mass ratio of described Corydalis impatiens (Pall.) Fisch. Extract are (1-6): 1, collect to filter and obtain all filtrate formation acid liquids, to adding volumetric concentration in described acid liquid, it is the aqueous slkali of 4-15%, regulator solution pH value, to 9-10, obtains acid-alkali treatment liquid;
(4) in described acid-alkali treatment liquid, add organic solvent extraction 3-6 time, reclaim described organic solvent and obtain Corydalis impatiens (Pall.) Fisch. total alkaloids crude extract, wherein the each addition of organic solvent is 2-4 times of long-pending volume of described acid-alkali treatment liquid.
(5) get silica gel ethyl acetate wet method dress post, and the described Corydalis impatiens (Pall.) Fisch. total alkaloids crude extract obtaining is added to loading after acetic acid ethyl dissolution, and adopt ethyl acetate to carry out eluting with the elution flow rate of a 1-2 per hour column volume, dry, obtain required Corydalis impatiens (Pall.) Fisch. total alkaloids, wherein for dissolving the 1-3 that the consumption of the ethyl acetate of total alkaloids crude extract is its quality, doubly measure, silica gel consumption is 5-10 times of described Corydalis impatiens (Pall.) Fisch. total alkaloids crude extract quality, the consumption that is used for the ethyl acetate of eluting is 50-100 times of described Corydalis impatiens (Pall.) Fisch. total alkaloids crude extract quality, described drying steps is the first reclaim under reduced pressure ethyl acetate of eluent, then at 60-70 ℃ of drying under reduced pressure.
6. the application for the preparation of cancer therapy drug according to the Corydalis impatiens (Pall.) Fisch. total alkaloids extract described in claim 4 or 5, is characterized in that,
Described acid solution is hydrochloric acid, sulphuric acid, phosphoric acid or vinegar aqueous acid;
Described aqueous slkali is the aqueous solution of sodium hydroxide, sodium carbonate, sodium bicarbonate;
Described organic solvent is dichloromethane, chloroform or ethyl acetate.
7. Corydalis impatiens (Pall.) Fisch. total alkaloids extract according to claim 5, for the preparation of the application of cancer therapy drug, is characterized in that,
(1) by Corydalis impatiens (Pall.) Fisch. pulverizing medicinal materials to 5-50 order, and the saturated alkaline ethanol that utilizes 80% volumetric concentration reflux, extract, 4 times at 40-80 ℃, mention 3 hours at every turn, obtain Corydalis impatiens (Pall.) Fisch. extracting solution, wherein the addition of saturated alkaline ethanol is 10 times of Corydalis impatiens (Pall.) Fisch. quality of medicinal material;
(2) by described Corydalis impatiens (Pall.) Fisch. extracting solution at 60 ℃ of decompression recycling ethanols, reconcentration is to clear paste shape, the dry Corydalis impatiens (Pall.) Fisch. Extract that obtains of spraying;
(3) to adding volumetric concentration in described Corydalis impatiens (Pall.) Fisch. Extract, be 0.5% dissolve with hydrochloric acid solution filter 23, wherein, each addition of acid solution and the mass ratio of solid are 5:1, merge the filtrate of filtering for 3 times and obtain acid liquid, to adding volumetric concentration in described acid liquid, it is 10% sodium hydrate aqueous solution, regulator solution pH value to 10, obtains acid-alkali treatment liquid;
(4) in described acid-alkali treatment liquid, add organic solvent extraction 3-6 time, reclaim described organic solvent and obtain Corydalis impatiens (Pall.) Fisch. total alkaloids crude extract, wherein the each addition of organic solvent is 3 times of long-pending volumes of acid-alkali treatment liquid.
(5) get silica gel ethyl acetate wet method dress post, and the described Corydalis impatiens (Pall.) Fisch. total alkaloids crude extract obtaining is added to loading after acetic acid ethyl dissolution, and adopt ethyl acetate to carry out eluting with the elution flow rate of a 1-2 per hour column volume, dry, obtain required Corydalis impatiens (Pall.) Fisch. total alkaloids, wherein for dissolving the 1-3 that the consumption of the ethyl acetate of total alkaloids crude extract is its quality, doubly measure, silica gel consumption is 10 times of described Corydalis impatiens (Pall.) Fisch. total alkaloids crude extract quality, the consumption of eluent ethyl acetate agent is 80 times of described Corydalis impatiens (Pall.) Fisch. total alkaloids crude extract quality, described drying steps is the first reclaim under reduced pressure ethyl acetate of eluent, then at 60-70 ℃ of drying under reduced pressure.
8. the application for the preparation of cancer therapy drug according to the arbitrary described Corydalis impatiens (Pall.) Fisch. total alkaloids extract of claim 4-7, is characterized in that, described cancer therapy drug is the medicine of anti-breast cancer, ovarian cancer, gastric cancer or pulmonary carcinoma.
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