CN104910232A - Preparation method for Zhankuic acid A and application of Zhankuic acid A in anti leukemia drugs - Google Patents
Preparation method for Zhankuic acid A and application of Zhankuic acid A in anti leukemia drugs Download PDFInfo
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- CN104910232A CN104910232A CN201510265019.3A CN201510265019A CN104910232A CN 104910232 A CN104910232 A CN 104910232A CN 201510265019 A CN201510265019 A CN 201510265019A CN 104910232 A CN104910232 A CN 104910232A
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- acid
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
- A61K31/575—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids substituted in position 17 beta by a chain of three or more carbon atoms, e.g. cholane, cholestane, ergosterol, sitosterol
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07J—STEROIDS
- C07J9/00—Normal steroids containing carbon, hydrogen, halogen or oxygen substituted in position 17 beta by a chain of more than two carbon atoms, e.g. cholane, cholestane, coprostane
- C07J9/005—Normal steroids containing carbon, hydrogen, halogen or oxygen substituted in position 17 beta by a chain of more than two carbon atoms, e.g. cholane, cholestane, coprostane containing a carboxylic function directly attached or attached by a chain containing only carbon atoms to the cyclopenta[a]hydrophenanthrene skeleton
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- General Health & Medical Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Epidemiology (AREA)
- Pharmacology & Pharmacy (AREA)
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- Veterinary Medicine (AREA)
- Medicinal Chemistry (AREA)
- Medicines Containing Plant Substances (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)
Abstract
The invention discloses a preparation method and application for a plant extract, and specifically relates to a preparation method for Zhankuic acid A and application of the Zhankuic acid A in anti leukemia drugs. The preparation method for the Zhankuic acid A comprises the following steps: weighing Antrndia cinnamomea sporocarp, then carrying out crushing, carrying out reflux extraction by using low-concentration alcohol, then condensing obtained extract until the extract is free of alcohol, carrying out adsorption through a polyamide resin column, carrying out elution by using acid alcohol, then collecting obtained eluent and recovering a solvent, then carrying out purification by using a high-speed counter current chromatography method, collecting effluent liquid, and carrying out condensation and low-temperature drying so as to obtain high-purity Zhankuic acid A. The preparation method for the Zhankuic acid A provided by the invention has the advantages of simple and convenient process operation, high yield, low pollution and applicability to production of the Zhankuic acid A. The Zhankuic acid A has antitumor effect; meanwhile, pharmacological experiments show that the Zhankuic acid A has good resisting effect to tumor, specifically human lymphocytic leukemia (P388), and can be used to prepare the anti leukemia drugs.
Description
Technical field
The invention discloses preparation method and the application of a Plant Extracts, particularly open up the storehouse acid preparation method of A and the application in anti-leukemia medicine thereof.
Background technology
Exhibition storehouse acid A(Zhankuic acid A), molecular formula is C
29h
40o
5, molecular weight is 468.63, is from polyporaceae (Polyporaceae) beautiful osmanthus Antrodia
antrndia cinnamomeachang & Chou, is separated a kind of activeconstituents obtained in sp.nov. sporophore.Research finds, acid A in exhibition storehouse is cytotoxic activity to _ Lymphoid Leukemic Cells P388, and IC50 is 1.8 μ g/ml.
Leukemia is a class hemopoietic stem cell malignant clone disease.Clonal leukemia cell breeds accumulation in a large number because of mechanism such as proliferation out of control, dysdifferentiation, apoptosis are obstructed in marrow and other hemopoietic tissues, and infiltrates its hetero-organization and organ, and normal hematopoiesis is suppressed simultaneously.Clinical visible anaemia in various degree, hemorrhage, infectious fever and liver, spleen, lymphadenectasis and skeleton pain.It is reported, the leukemic sickness rate in China each department accounts for the 6th in various tumour.
Summary of the invention
For meeting clinical needs, expanding medicine variety, better Therapeutic cancer, providing a kind of and opening up the storehouse acid preparation method of A and the application in anti-leukemia medicine thereof.
To achieve these goals, the present invention is by the following technical solutions:
The preparation method of exhibition storehouse acid A, is characterized in that comprising the following steps:
1) get beautiful osmanthus Antrodia sporophore to pulverize, adopt 4 ~ 10 times amount 20 ~ 40% alcohol reflux 1 ~ 4 time, each 0.5 ~ 1.5h, filter, united extraction liquid, is concentrated into without alcohol, obtains concentrated solution;
2) be added in polyamide resin column by above-mentioned concentrated solution and adsorb, with the acid alcohol wash-out of 4 ~ 8BV, collect elutriant, recycling design, to dry, must open up the sour A crude extract in storehouse;
3) adopt high-speed counter-current chromatograph to be separated above-mentioned exhibition storehouse acid A crude extract, collect elutriant, concentrated, cryodrying must open up storehouse acid A.
It is characterized in that described step 2) in polyamide resin granularity be 80 ~ 120 orders, the envelope-bulk to weight ratio of consumption and medicinal material amount is 1:1-3.
It is characterized in that described step 2) in acid alcohol be 30 ~ 50% ethanolic solns containing 1% acetic acid.
It is characterized in that the parameter setting of the high-speed counter-current chromatograph in described step 3) is: rotating speed 750 ~ 1050r/min, the flow velocity 2 ~ 10ml/min of moving phase.
The exhibition storehouse acid A of described preparation, is preparing the application in anti-leukemia medicine, is is especially preventing and treating the application of lymphoid leukemia and myelocytic leukemia in preparation.
The exhibition storehouse acid A of described preparation, adds intestinal absorption enhancers, comprises one or more in Medium chain fatty hydrochlorate, cholate, CDC, Chitosan-phospholipid complex in its formula of oral.
Adopt this law preparation exhibition storehouse acid A, simple process is easy to operate, and preparation amount is large, and product yield is high, and low stain; Add intestinal absorption enhancers, be conducive to improving bioavailability.
Further illustrate the present invention below in conjunction with embodiment, but the scope of protection of present invention is not limited to following embodiments.
Embodiment
Embodiment 1:
The preparation of exhibition storehouse acid A
Get beautiful osmanthus Antrodia sporophore 1kg, pulverize, add 4L40% alcohol reflux 4 times, each 1.5h, solid-liquid separation obtains extracting solution, be concentrated into without alcohol taste, get 350g80 object polyamide resin, dress post, concentrated solution upper prop, with 30% ethanol elution containing 1% acetic acid of 8BV, elutriant recycling design, storehouse acid A crude extract must be opened up; Get propyl carbinol, acetic acid, water is placed in separating funnel by 3:1:4 volume ratio and mixes, stratification, be separated phase up and down, getting is stationary phase mutually, and lower is moving phase mutually, will open up the lower phased soln of storehouse acid A crude extract, open countercurrent chromatography instrument, enter moving phase with 2ml/min flow pump, and by sampling valve continuous sample introduction, control rotating speed is 750r/min, collect effluent liquid, concentrated, cryodrying must open up storehouse acid A, detection level 99.3%.
Embodiment 2:
The preparation of exhibition storehouse acid A
Get beautiful osmanthus Antrodia sporophore 5kg, pulverize, add 10L20% alcohol reflux 0.5h, solid-liquid separation obtains extracting solution, is concentrated into without alcohol taste, get 5kg120 object polyamide resin, dress post, concentrated solution upper prop, with 50% ethanol elution containing 1% acetic acid of 4BV, elutriant recycling design, must open up storehouse acid A crude extract; Get propyl carbinol, acetic acid, water is placed in separating funnel by 7:3:10 volume ratio and mixes, stratification, be separated phase up and down, getting is stationary phase mutually, and lower is moving phase mutually, will open up the lower phased soln of storehouse acid A crude extract, open countercurrent chromatography instrument, enter moving phase with 3.5ml/min flow pump, and by sampling valve continuous sample introduction, control rotating speed is 1050r/min, collect effluent liquid, concentrated, cryodrying must open up storehouse acid A, detection level 99.1%.
Embodiment 3:
The preparation of exhibition storehouse acid A
Get beautiful osmanthus Antrodia sporophore 10kg, pulverize, add 80L30% alcohol reflux 2 times, each 1h, solid-liquid separation obtains extracting solution, be concentrated into without alcohol taste, get 6kg80 object polyamide resin, dress post, concentrated solution upper prop, with 45% ethanol elution containing 1% acetic acid of 6BV, elutriant recycling design, storehouse acid A crude extract must be opened up; Get propyl carbinol, acetic acid, water is placed in separating funnel by 6:2:7 volume ratio and mixes, stratification, be separated phase up and down, getting is stationary phase mutually, and lower is moving phase mutually, will open up the lower phased soln of storehouse acid A crude extract, open countercurrent chromatography instrument, enter moving phase with 10ml/min flow pump, and by sampling valve continuous sample introduction, control rotating speed is 900r/min, collect effluent liquid, concentrated, cryodrying must open up storehouse acid A, detection level 98.5%.
Embodiment 4:
Its application in anti-leukemia medicine of exhibition storehouse acid A
1, material
Animal: Kunming mouse, male and female half and half property, body weight 20 ± 2g, is provided by Nanjing University of Traditional Chinese Medicine.
Cell strain: P388(leukemia) cell strain, provided by Shanghai Pharmaceutical Inst., Chinese Academy of Sciences.
2, method
Containing in RPMI 1640 substratum of 10% foetal calf serum, be 2 × 10 by tested cell furnishing density
4cell/ml.By above-mentioned cell strain suspension inoculation in 96 well culture plates, every hole 50 μ l, is placed in saturated humidity, 37 DEG C and 5%CO2 incubator is cultivated 24 hours.Add each 50 μ l of dimethyl sulfoxide solution of solvent control 0.1% dimethyl sulfoxide (DMSO), positive control cis-platinum (DDP) and different concns monomeric compound respectively, often group establishes 3 multiple holes.Cultivate 72 hours, first 4 hours are terminated in cultivation, each culture hole adds 5mg/ml tetrazolium father-in-law salt (MTT) 10 μ l, be placed in saturated humidity, 37 DEG C and 5%CO2 incubator are cultivated 4 hours, 150 μ l methyl-sulphoxides are added in every hole, vibration is dissolved and is generated MTT crystal formazan, and survey 570nmO.D value by microplate reader, cell survival rate is by the ratio calculation of sample O.D value for reference substance O.D value.Wherein, storehouse acid A is opened up to the half-inhibition concentration (IC of P388 cell
50) have dose effect curve to obtain.
3, result
Realize result (see table 1) the present invention to open up the storehouse acid growth of A to P388 cell and have obvious restraining effect, it is better than positive control cis-platinum (DDP) to the growth inhibitory activity of P388 cell, illustrate that acid A in exhibition storehouse has splendid antileukemie activity, have good application prospect preparing in anti-leukemia medicine.
Table 1 the present invention open up storehouse acid A to the restraining effect of P388 cell (n=10,
± S)
| Group | IC 50（μM） |
| Blank group | — |
| Positive controls | 6.4±1.8 |
| Exhibition storehouse acid A group | 7.7±2.0 |
Claims (6)
1. open up the preparation method of storehouse acid A, it is characterized in that comprising the following steps:
1) get beautiful osmanthus Antrodia sporophore to pulverize, adopt 4 ~ 10 times amount 20 ~ 40% alcohol reflux 1 ~ 4 time, each 0.5 ~ 1.5h, filter, united extraction liquid, is concentrated into without alcohol, obtains concentrated solution;
2) be added in polyamide resin column by above-mentioned concentrated solution and adsorb, with the acid alcohol wash-out of 4 ~ 8BV, collect elutriant, recycling design, to dry, must open up the sour A crude extract in storehouse;
3) adopt high-speed counter-current chromatograph to be separated above-mentioned exhibition storehouse acid A crude extract, collect elutriant, concentrated, cryodrying must open up storehouse acid A.
2. the preparation method of exhibition storehouse according to claim 1 acid A, is characterized in that described step 2) in polyamide resin granularity be 80 ~ 120 orders, the envelope-bulk to weight ratio of consumption and medicinal material amount is 1:1-3.
3. the preparation method of exhibition storehouse according to claim 1 acid A, is characterized in that described step 2) in acid alcohol be 30 ~ 50% ethanolic solns containing 1% acetic acid.
4. the preparation method of acid A in exhibition storehouse according to claim 1, is characterized in that the parameter setting of the high-speed counter-current chromatograph in described step 3) is: rotating speed 750 ~ 1050r/min, the flow velocity 2 ~ 10ml/min of moving phase.
5., according to acid A in exhibition storehouse prepared by method described in claim 1 ~ 4, preparing the application in anti-leukemia medicine.
6. prepare the application in anti-leukemia medicine according to claim 5, it is characterized in that the application preventing and treating lymphoid leukemia and myelocytic leukemia in preparation.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510265019.3A CN104910232A (en) | 2015-05-22 | 2015-05-22 | Preparation method for Zhankuic acid A and application of Zhankuic acid A in anti leukemia drugs |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510265019.3A CN104910232A (en) | 2015-05-22 | 2015-05-22 | Preparation method for Zhankuic acid A and application of Zhankuic acid A in anti leukemia drugs |
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| Publication Number | Publication Date |
|---|---|
| CN104910232A true CN104910232A (en) | 2015-09-16 |
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Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510265019.3A Pending CN104910232A (en) | 2015-05-22 | 2015-05-22 | Preparation method for Zhankuic acid A and application of Zhankuic acid A in anti leukemia drugs |
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| Country | Link |
|---|---|
| CN (1) | CN104910232A (en) |
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2015
- 2015-05-22 CN CN201510265019.3A patent/CN104910232A/en active Pending
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Application publication date: 20150916 |