CN102649804A - Preparation method for cyasterone - Google Patents
Preparation method for cyasterone Download PDFInfo
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- CN102649804A CN102649804A CN2011100453116A CN201110045311A CN102649804A CN 102649804 A CN102649804 A CN 102649804A CN 2011100453116 A CN2011100453116 A CN 2011100453116A CN 201110045311 A CN201110045311 A CN 201110045311A CN 102649804 A CN102649804 A CN 102649804A
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- cyasterone
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Abstract
The invention discloses a preparation method for cyasterone. The preparation method comprises the steps as follows: A, coarse radix cyathulae powder is taken, extracted with alcohol or methanol and then subjected to slag removal; B, the extract is concentrated and then is subjected to adsorption elution of macroporous resin; C, the prepared liquid is subjected to phase separation; and D, heptane-acetone recrystallization is conducted to obtain cyasterone crystal product with the purity of more than 98.5 percent. The invention has the advantages that the method is simple and has high yield, a great amount of high-purity product can be obtain at a time, and the economic value and the academical value are higher.
Description
Technical field:
The invention belongs to the extracted form natural plant field, be specifically related to a kind of preparation method of cyasterone.
Background technology:
Root of Medicinal Cyathula is a conventional Chinese medicine, is famous birth canal ground, river Chinese medicinal materials.Have by the stasis of blood stimulate the menstrual flow, the effect of easing joint movement, inducing diuresis for treating strangurtia, be used for drenching through closing abdominal mass, placenta retension, injury from falling down, arthralgia due to wind-dampness, sufficient impotence muscle contraction, hematuria blood.The staple of Root of Medicinal Cyathula is vegeto-alkali and sterols material, like cyasterone, capitasterone, ecdysone, red sterone etc., also has FLA in addition.The main pharmacological of Root of Medicinal Cyathula has promoting blood circulation to restore menstrual flow, wind-damp dispelling, easing joint movement, inducing diuresis for treating stranguria syndrome, and to treatment of diseases effects such as hypertensive heart disease and dysmenorrhoeas.
Cyasterone (cyasterone) molecular formula C
29H
44O
8, molecular weight 520.65, is colourless needle crystal by mp.168-173 ℃.Cyasterone all has to separate with Root of Medicinal Cyathula Herba Ajugae and obtains, but the content in Root of Medicinal Cyathula is higher.Reports such as T.Konoshima; From Herba Ajugae, separate the cyasterone that obtains the Epstein-Barr virus EA (EBV-EA) of being induced generation by growth of tumour cell promotor 12-O-te-trade canoylphorbo 1-13-acetate (TPA) is had had strong inhibitory effects, and show the potential anti-tumor activity.Li Wanshou etc. discover that cyasterone has increase trend to the body weight and the uterus coefficient of castration mouse, and the uterus weight in wet base has phenomenal growth, and pointing out it is the effective constituent of the estrogenic activity of Root of Medicinal Cyathula; Cyasterone can increase castrated rats levator ani weight, points out it that protein assimilation is arranged; Experimental result shows that the pharmacologically active of cyasterone conforms to Root of Medicinal Cyathula strengthening the tendons and bones " power also more than merit of its nourishing such as the ox ", cures mainly identical basically with tcm clinical practice medication function.
At present main adopt SX, normal pressure repeatedly silica gel column chromatography or alumina column chromatography separate, have problems such as output is little, yield is low, the production cycle is long, be not suitable for industrial production.
Summary of the invention:
The object of the present invention is to provide a kind of preparation method of cyasterone, this method is simple to operate, yield is high, safety and environmental protection, but the quickly and massively separating high purity cyasterone.
For realizing above-mentioned purpose, the technical scheme that the present invention adopts is following:
(1) extract: get the Root of Medicinal Cyathula pulverizing medicinal materials and become meal, add methyl alcohol or ethanol and be heated to 65 ℃-85 ℃, constant temperature soaked 2-4 hour, leach, filtrate decompression reclaim Root of Medicinal Cyathula medicinal extract;
(2) macroporous resin column enrichment: Root of Medicinal Cyathula medicinal extract is crossed macroporous adsorptive resins, water and ethanol elution, alcohol eluen obtains liquid concentrator after concentrating;
(3) preparation liquid phase separation: get a certain amount of liquid concentrator; With anti-phase C18 chromatographic column on the dissolve with methanol, with ETHYLE ACETATE-methyl alcohol or chloroform-methanol as moving phase, gradient elution; UV-detector detects effluent; Color atlas according to gained is collected the cyasterone component, and the elutriant rotary evaporation is concentrated, and gets the cyasterone coarse-grain;
(4) recrystallization: with heptane-acetone recrystallization, lyophilize obtains high purity cyasterone crystal with the cyasterone coarse-grain.
Nonpolar or low-pole macroporous adsorbent resins such as the optional D1300 of the said macroporous resin of step (2), AB-8, D101, HPD100.
The said macroporous adsorbent resin wash-out of step (2) uses volumes of aqueous ethanol per-cent to be 70-80%, and the wash-out consumption is 6-12 a times of crude drug.
The volume percent of methyl alcohol is 20-80% in the said moving phase ETHYLE ACETATE-methyl alcohol of step (3).
The volume percent of methyl alcohol is 20-80% in the said moving phase chloroform-methanol of step (3).
The said gradient program of step (3) is following:
Chromatographic column filler is C18, and moving phase is ETHYLE ACETATE or chloroform (A) and methyl alcohol (B), and gradient sees the following form, and flow velocity is 2-10ml/min, and column temperature is a room temperature.
The volume ratio of the said heptane-acetone of step (4) is 5-7: 1.
The invention has the advantages that: 1) weak point simple to operate, consuming time is easy to industrialization and amplifies; Solvent load in the steps such as 2) extraction, column chromatography, preparation liquid phase separation is few, and recyclable utilization again; 3) have high economic benefit and learning value, products obtained therefrom purity of the present invention is high, and the further exploitation that can be cyasterone provides pharmacology raw material and practice to support.
Embodiment:
Below in conjunction with specific embodiment the present invention is further specified:
Embodiment 1:
(1) extract:
Take by weighing 5kg Root of Medicinal Cyathula root meal, add 90% ethanolic soln by 1: 5 mass ratio, be heated to 65 ℃ after constant temperature soaked 4 hours, filter cleaner, filtrate decompression reclaim Root of Medicinal Cyathula medicinal extract.
(2) macroporous resin column enrichment:
The medicinal extract of step (1) is passed through the D1300 macroporous adsorptive resins, and it is colourless that first water is washed till elutant, and washing lotion discards, and 70% ethanol with 6 times of amounts of crude drug carries out wash-out again, collects elutriant, obtains liquid concentrator behind the concentrating under reduced pressure.
(3) preparation liquid phase separation:
Adopting filler is the chromatographic column of C18, and moving phase is ETHYLE ACETATE (A) and methyl alcohol (B), and flow velocity is 2ml/min, and the detection wavelength is 246nm, operates under the room temperature, and the gradient elution program is following:
The liquid concentrator of getting step (2) adds methyl alcohol to just dissolving; Solution after the dissolving is got the filtrating sample introduction with 0.45 μ m membrane filtration, every pin sample introduction 2g (in contained dry product weight in the filtrating); The preparation of carrying out cyasterone separates; The UV-detector on-line monitoring, specific aim is collected the preparation cut solution of cyasterone, and reclaim under reduced pressure reagent gets the cyasterone coarse-grain.
(4) recrystallization:
With heptane-acetone (7: 1) recrystallization, lyophilize obtains cyasterone monomer product 1.8g with the coarse-grain of step (3), and measuring its content through the HPLC method is 99.3%.
Embodiment 2:
(1) extract:
Take by weighing 5kg Root of Medicinal Cyathula root meal, add 70% methanol solution by 1: 8 mass ratio, be heated to 85 ℃ after constant temperature soaked 2 hours, filter cleaner, filtrate decompression reclaim Root of Medicinal Cyathula medicinal extract.
(2) macroporous resin column enrichment:
The medicinal extract of step (1) is passed through the HPD100 macroporous adsorptive resins, and it is colourless that first water is washed till elutant, and washing lotion discards, and 80% ethanol with 10 times of amounts of crude drug carries out wash-out again, collects elutriant, obtains liquid concentrator behind the concentrating under reduced pressure.
(3) preparation liquid phase separation:
Adopting filler is the chromatographic column of C18, and moving phase is chloroform (A) and methyl alcohol (B), and flow velocity is 5ml/min, and the detection wavelength is 246nm, operates under the room temperature, and the gradient elution program is following:
The liquid concentrator of getting step (2) adds methyl alcohol to just dissolving; Solution after the dissolving is got the filtrating sample introduction with 0.45 μ m membrane filtration, every pin sample introduction 1g (in contained dry product weight in the filtrating); The preparation of carrying out cyasterone separates; The UV-detector on-line monitoring, specific aim is collected the preparation cut solution of cyasterone, and reclaim under reduced pressure reagent gets the cyasterone coarse-grain.
(4) recrystallization:
With heptane-acetone (5: 1) recrystallization, lyophilize obtains cyasterone monomer product 1.5g with the coarse-grain of step (3), and measuring its content through the HPLC method is 98.8%.
Embodiment 3:
(1) extract:
Take by weighing 5kg Root of Medicinal Cyathula root meal, add 65% methanol solution by 1: 6 mass ratio, be heated to 70 ℃ after constant temperature soaked 3 hours, filter cleaner, filtrate decompression reclaim Root of Medicinal Cyathula medicinal extract.
(2) macroporous resin column enrichment:
The medicinal extract of step (1) is passed through the D101 macroporous adsorptive resins, and it is colourless that first water is washed till elutant, and washing lotion discards, and 75% ethanol with 12 times of amounts of crude drug carries out wash-out again, collects elutriant, obtains liquid concentrator behind the concentrating under reduced pressure.
(3) preparation liquid phase separation:
Adopting filler is the chromatographic column of C18, and moving phase is ETHYLE ACETATE (A) and methyl alcohol (B), and flow velocity is 3.5ml/min, and the detection wavelength is 246nm, operates under the room temperature, and the gradient elution program is following:
The liquid concentrator of getting step (2) adds methyl alcohol to just dissolving; Solution after the dissolving is got the filtrating sample introduction with 0.45 μ m membrane filtration, every pin sample introduction 1g (in contained dry product weight in the filtrating); The preparation of carrying out cyasterone separates; The UV-detector on-line monitoring, specific aim is collected the preparation cut solution of cyasterone, and reclaim under reduced pressure reagent gets the cyasterone coarse-grain.
(4) recrystallization:
With heptane-acetone (6: 1) recrystallization, lyophilize obtains cyasterone monomer product 1.6g with the coarse-grain of step (3), and measuring its content through the HPLC method is 98.6%.
Embodiment 4:
(1) extract:
Take by weighing 5kg Root of Medicinal Cyathula root meal, add 70% ethanolic soln by 1: 8 mass ratio, be heated to 70 ℃ after constant temperature soaked 3 hours, filter cleaner, filtrate decompression reclaim Root of Medicinal Cyathula medicinal extract.
(2) macroporous resin column enrichment:
The medicinal extract of step (1) is passed through the AB-8 macroporous adsorptive resins, and it is colourless that first water is washed till elutant, and washing lotion discards, and 65% ethanol with 8 times of amounts of crude drug carries out wash-out again, collects elutriant, obtains liquid concentrator behind the concentrating under reduced pressure.
(3) preparation liquid phase separation:
Adopting filler is the chromatographic column of C18, and moving phase is chloroform (A) and methyl alcohol (B), and flow velocity is 4.0ml/min, and the detection wavelength is 246nm, operates under the room temperature, and the gradient elution program is following:
The liquid concentrator of getting step (2) adds methyl alcohol to just dissolving; Solution after the dissolving is got the filtrating sample introduction with 0.45 μ m membrane filtration, every pin sample introduction 1g (in contained dry product weight in the filtrating); The preparation of carrying out cyasterone separates; The UV-detector on-line monitoring, specific aim is collected the preparation cut solution of cyasterone, and reclaim under reduced pressure reagent gets the cyasterone coarse-grain.
(4) recrystallization:
With heptane-acetone (5: 1) recrystallization, lyophilize obtains cyasterone monomer product 1.4g with the coarse-grain of step (3), and measuring its content through the HPLC method is 99.1%.
Claims (6)
1. the preparation method of a cyasterone is characterized in that it may further comprise the steps:
(1) extract: get the Root of Medicinal Cyathula pulverizing medicinal materials and become meal, add methyl alcohol or ethanol and be heated to 65 ℃-85 ℃, constant temperature soaked 2-4 hour, leach, filtrate decompression reclaim Root of Medicinal Cyathula medicinal extract;
(2) macroporous adsorptive resins enrichment: Root of Medicinal Cyathula medicinal extract is crossed macroporous adsorptive resins, water and ethanol elution, alcohol eluen obtains liquid concentrator after concentrating;
(3) preparation liquid phase separation: get a certain amount of liquid concentrator, with anti-phase C18 chromatographic column on the dissolve with methanol, with ETHYLE ACETATE-methyl alcohol or chloroform-methanol as moving phase; Gradient elution; Collect the cyasterone component, the elutriant rotary evaporation is concentrated, get the cyasterone coarse-grain;
(4) recrystallization: with heptane-acetone recrystallization, lyophilize obtains high purity cyasterone crystal with the cyasterone coarse-grain.
2. the preparation method of cyasterone according to claim 1 is characterized in that nonpolar or low-pole macroporous adsorbent resins such as the optional D1300 of the said macroporous resin of step (2), AB-8, D101, HPD100.
3. the preparation method of cyasterone according to claim 1 is characterized in that the said macroporous adsorbent resin wash-out of step (2) uses volumes of aqueous ethanol per-cent to be 70-80%, and the wash-out consumption is 6-12 a times of crude drug.
4. the preparation method of cyasterone according to claim 1 is characterized in that the volume percent of methyl alcohol in the said moving phase ETHYLE ACETATE-methyl alcohol of step (3) is 20-80%.
5. the preparation method of cyasterone according to claim 1 is characterized in that the volume percent of methyl alcohol in the said moving phase chloroform-methanol of step (3) is 20-80%.
6. the preparation method of cyasterone according to claim 1 is characterized in that the volume ratio of the said heptane-acetone of step (4) is 5-7: 1.
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| CN2011100453116A CN102649804A (en) | 2011-02-25 | 2011-02-25 | Preparation method for cyasterone |
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| CN2011100453116A CN102649804A (en) | 2011-02-25 | 2011-02-25 | Preparation method for cyasterone |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108218944A (en) * | 2018-03-05 | 2018-06-29 | 上海诗丹德标准技术服务有限公司 | A kind of preparation method of cyasterone |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101612177A (en) * | 2009-07-20 | 2009-12-30 | 雅安三九中药材科技产业化有限公司 | The construction method of Radix Cyathulae medicinal materials fingerprint and standard finger-print thereof |
-
2011
- 2011-02-25 CN CN2011100453116A patent/CN102649804A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101612177A (en) * | 2009-07-20 | 2009-12-30 | 雅安三九中药材科技产业化有限公司 | The construction method of Radix Cyathulae medicinal materials fingerprint and standard finger-print thereof |
Non-Patent Citations (1)
| Title |
|---|
| 刘晶晶 等: "大孔吸附树脂富集纯化川牛膝中杯苋甾酮的研究", 《时珍国医国药》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108218944A (en) * | 2018-03-05 | 2018-06-29 | 上海诗丹德标准技术服务有限公司 | A kind of preparation method of cyasterone |
| CN108218944B (en) * | 2018-03-05 | 2020-10-02 | 上海诗丹德标准技术服务有限公司 | Preparation method of cyasterone |
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Application publication date: 20120829 |