CN102010457A - Method for preparing raddeanin A - Google Patents
Method for preparing raddeanin A Download PDFInfo
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- CN102010457A CN102010457A CN2010105201920A CN201010520192A CN102010457A CN 102010457 A CN102010457 A CN 102010457A CN 2010105201920 A CN2010105201920 A CN 2010105201920A CN 201010520192 A CN201010520192 A CN 201010520192A CN 102010457 A CN102010457 A CN 102010457A
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- anemone rhizome
- radde anemone
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Abstract
The invention belongs to the technical field of medicaments, and discloses a method for extracting raddeanin A from rhizoma anemones raddeanae. The method is characterized by comprising the following steps of: performing alcohol extraction and water extraction on the rhizoma anemones raddeanae serving as a raw material; concentrating until alcohol taste is eliminated; extracting with an organic solvent; enriching and separating a water layer with macroporous resin and recrystallizing; and purifying by a semi-prepared high-efficiency liquid phase chromatography to finally obtain the raddeanin A. The method has the advantages of less sample loss, good separation effect, high product purity and high controllability and is suitable for automatic production; and the obtained raddeanin A has various pharmacological activities and the application prospect of being prepared into medicaments.
Description
Technical field:
The invention belongs to medical technical field, disclose the preparation method of the plain A of a kind of Radde Anemone Rhizome.
Background technology:
The plain A of Radde Anemone Rhizome is the triterpene glycosides compound.Have another name called the plain A of anemone raddeana Regel, the plain A of rhizoma anemones raddeanae; English name: Anemodeanin A.
The plain A of Radde Anemone Rhizome is the white powder crystallization, mp.242-244 ℃.
(c=0.8, methyl alcohol).Molecular formula is C
47H
76O
16, molecular weight is 897.10.Soluble in water, methyl alcohol, ethanol are insoluble to low polar solvents such as acetone, ethyl acetate.Molecular structural formula is:
The plain A of Radde Anemone Rhizome derives from Ranunculaceae (Ranunculaceae) anemone raddeana Regel (Radde Anemone Rhizome) Anemone raddeanaRegel rhizome.Originate in ground such as the Northeast and Shandong, with Jilin Province's output maximum.This medicine head is stated from the Liu Wen of Ming Dynasty Thailand and shows in " Bencao Pinhui Jingyao ", and the effect of " dispelling rheumatism subduing inflammation " is arranged clinically, is used for diseases such as " arthralgia due to wind-cold-dampness pathogen BI syndrome, spasm of the limbs, arthralgia, sore carbuncle are festered ".
The plain A of Radde Anemone Rhizome is one of biapiculate main effective constituent, has stronger physiologically active.Woods is little to be waited by the emperor himself with plain A of the Radde Anemone Rhizome of different mass concentration and HepG2 cell co-cultivation, adopts MTT colorimetry and cell colony forming method to detect the influence of Radde Anemone Rhizome element A to the growth of HepG2 cell, propagation.The effect of plain each dosage of A of Radde Anemone Rhizome is as a result compared with untreated fish group, all there were significant differences at each time point, thereby obtain conclusion: cell proliferation has obvious restraining effect to the plain A of Radde Anemone Rhizome to HepG2, strengthens along with the rising of reagent mass concentration and the prolongation of action time within the specific limits.Usefulness hydrolysis such as Wang Mingkui and solvent-extracted method are extracted from Chinese medicine is pointed at both ends and have been prepared total saponins to be measured (anemonin A account for total glycosides 20%), and studied the external and intravital antitumor activity of total saponins, pointed at both ends total glycosides of finding preparation has the activity that suppresses tumor growth preferably, is a potential cancer therapy drug.
Liu Dayou etc. identify separation and the structure of having carried out pointed at both ends, employing be the normal pressure silica gel column method, but it adopts n-butanol extraction to go up macroporous resin again, the n-butanol extraction rate is lower, the reservation composition loses bigger.The report that does not have at present report the plain A of Radde Anemone Rhizome to be carried out separation and purification as yet with half preparative chromatography.Therefore for pointed at both ends and preparation are carried out quality control, the present invention adopts the method that conventional macroporous resin column is separated and high performance liquid chromatography combines to prepare the plain A of Radde Anemone Rhizome.This method is simple to operate, and the product purity height can reach more than 98%.
Summary of the invention:
The preparation method who the purpose of this invention is to provide the plain A of a kind of Radde Anemone Rhizome specifically, is to adopt macroporous resin and liquid phase bonded method to prepare the Radde Anemone Rhizome element.
The preparation method of the plain A of a kind of Radde Anemone Rhizome of the present invention is characterized in that comprising following steps:
(1) extract: with pulverizing pointed at both ends, behind extraction using alcohol, water extraction, filtered while hot, united extraction liquid, being evaporated to does not have the alcohol flavor;
(2) extraction:, discard extraction liquid with the extract ethyl acetate extraction;
(3) column chromatography: with macroporous resin column absorption on the water layer, acetone-methanol-water wash-out, substep is collected, and every part of about 500ml merges the plain A flow point of Radde Anemone Rhizome, and cryoconcentration is to there being precipitation to separate out;
(4) purifying: with wash-out concentrated solution standing over night, leach precipitation, throw out is dissolved in the ethanolic soln, ultrasonic dissolution with half preparative liquid chromatography purifying, is collected the reservation flow point, and concentrating under reduced pressure, crystallization and drying obtain colourless crystallization.
The alcohol concn volume percent is 70-90% described in the step (1), extracts twice, and water is carried once, and the liquid material is than being 3-10: 1 (V/W).
Macroporous resin described in the step (3) is HPD300, HPD450, NKA-9 type macroporous resin, and described elution requirement shoals for washing with water earlier to color, uses acetone-methanol-water (55: 35: 10) wash-out then instead.
Low temperature is 0-10 ℃ described in the step (3).
Half preparative high performance liquid chromatography condition is described in the step (4): ODS C18, and 5 μ m, 250*20.0mm (I.D.), moving phase is formed volume ratio: methanol-water (68: 32), flow velocity 5.0ml/min, column temperature: room temperature; Detect wavelength: UV 207nm; Sample size: 50 microlitres.
Positively effect of the present invention is: the plain A purity of the Radde Anemone Rhizome of the present invention preparation height, have stronger antitumour activity, abundant raw material is easy to get, and has a extensive future.
The analysis mode high-efficient liquid phase chromatogram condition: ODS C18,5 μ m, 250*4.5mm (I.D.), moving phase is formed volume ratio: methyl alcohol: water (75: 25), flow velocity 1.0ml/min, column temperature: room temperature; Detect wavelength: UV 207nm; Sample size: 10 microlitres.
Further specify the present invention below in conjunction with embodiment, but the scope of protection of present invention is not limited to following embodiment.
Embodiment:
Embodiment 1:
300g medicinal material pointed at both ends is 85% alcohol reflux 2 times according to 1: 5 mass ratio concentration of volume percent of solid-liquid ratio after crushed, filtered while hot, filter residue extracts 1 time with 4 times of water heating again, united extraction liquid, be evaporated to and do not have the alcohol flavor, with the extract ethyl acetate extraction, discard extraction liquid, HPD300 macroporous resin column absorption on the water layer washes with water earlier to color and shoals, use acetone-methanol-water (55: 35: 10) wash-out again instead, substep is collected, and every part of about 500ml merges the plain A flow point of Radde Anemone Rhizome, 5 ℃ of cryoconcentration are to there being precipitation to separate out, throw out is dissolved in the ethanolic soln, and ultrasonic dissolution is made with extra care with half preparative high performance liquid chromatography instrument, used column packing is C18,5 μ m, 250*20.0mm (I.D.), moving phase is formed volume ratio: methanol-water (68: 32), flow velocity 5.0ml/min, detecting wavelength is UV 207nm; Sample size: 50 μ l, column temperature: room temperature.Cut collection according to chromatographic peak and merge, concentrating under reduced pressure leaves standstill crystallization, and drying obtains the plain A colourless crystallization of Radde Anemone Rhizome, content 99.2%.
Embodiment 2:
500g medicinal material pointed at both ends is 70% alcohol reflux 2 times according to 1: 8 mass ratio concentration of volume percent of solid-liquid ratio after crushed, filtered while hot, filter residue extracts 1 time with 5 times of water heating again, united extraction liquid, be evaporated to and do not have the alcohol flavor, with the extract ethyl acetate extraction, discard extraction liquid, HPD450 macroporous resin column absorption on the water layer washes with water earlier to color and shoals, use acetone-methanol-water (55: 35: 10) wash-out again instead, substep is collected, and every part of about 500ml merges the plain A flow point of Radde Anemone Rhizome, 10 ℃ of cryoconcentration are to there being precipitation to separate out, throw out is dissolved in the ethanolic soln, and ultrasonic dissolution is made with extra care with half preparative high performance liquid chromatography instrument, used column packing is C18,5 μ m, 250*20.0mm (I.D.), moving phase is formed volume ratio: methanol-water (68: 32), flow velocity 5.0ml/min, detecting wavelength is UV 207nm; Sample size: 50 μ l, column temperature: room temperature.Cut collection according to chromatographic peak and merge, concentrating under reduced pressure leaves standstill crystallization, and drying obtains the plain A colourless crystallization of Radde Anemone Rhizome, content 99.4%.
Embodiment 3:
500g medicinal material pointed at both ends is 85% alcohol reflux 2 times according to 1: 6 mass ratio concentration of volume percent of solid-liquid ratio after crushed, filtered while hot, filter residue extracts 1 time with 3 times of water heating again, united extraction liquid, be evaporated to and do not have the alcohol flavor, with the extract ethyl acetate extraction, discard extraction liquid, HPD300 macroporous resin column absorption on the water layer washes with water earlier to color and shoals, use acetone-methanol-water (55: 35: 10) wash-out again instead, substep is collected, and every part of about 500ml merges the plain A flow point of Radde Anemone Rhizome, 8 ℃ of cryoconcentration are to there being precipitation to separate out, throw out is dissolved in the ethanolic soln, and ultrasonic dissolution is made with extra care with half preparative high performance liquid chromatography instrument, used column packing is C18,5 μ m, 250*20.0mm (I.D.), moving phase is formed volume ratio: methanol-water (68: 32), flow velocity 5.0ml/min, detecting wavelength is UV 207nm; Sample size: 50 μ l, column temperature: room temperature.Cut collection according to chromatographic peak and merge, concentrating under reduced pressure leaves standstill crystallization, and drying obtains the plain A colourless crystallization of Radde Anemone Rhizome, content 98.5%.
Embodiment 4:
1kg medicinal material pointed at both ends is 90% alcohol reflux 2 times according to 1: 10 mass ratio concentration of volume percent of solid-liquid ratio after crushed, filtered while hot, filter residue extracts 1 time with 5 times of water heating again, united extraction liquid, and being evaporated to does not have the alcohol flavor, with the extract ethyl acetate extraction, discard extraction liquid, NKA-9 macroporous resin column absorption on the water layer washes with water earlier to color and shoals, use acetone-methanol-water (55: 35: 10) wash-out again instead, substep is collected, and every part of about 500ml merges the plain A flow point of Radde Anemone Rhizome, 6 ℃ of cryoconcentration are to there being precipitation to separate out, throw out is dissolved in the ethanolic soln, and ultrasonic dissolution is made with extra care with half preparative high performance liquid chromatography instrument, used column packing is C18,5 μ m, 250*20.0mm (I.D.), moving phase is formed volume ratio: methanol-water (68: 32), flow velocity 5.0ml/min, detecting wavelength is UV 207nm; Sample size: 50 μ l, column temperature: room temperature.Cut collection according to chromatographic peak and merge, concentrating under reduced pressure leaves standstill crystallization, and drying obtains the plain A colourless crystallization of Radde Anemone Rhizome, content 98.7%.
Claims (5)
1. the preparation method of the plain A of Radde Anemone Rhizome is characterized in that comprising the following steps:
(1) extract: with pulverizing pointed at both ends, behind extraction using alcohol, water extraction, filtered while hot, united extraction liquid, being evaporated to does not have the alcohol flavor;
(2) extraction:, discard extraction liquid with the extract ethyl acetate extraction;
(3) column chromatography: with macroporous resin column absorption on the water layer, acetone-methanol-water wash-out, substep is collected, and every part of about 500ml merges the plain A flow point of Radde Anemone Rhizome, and cryoconcentration is to there being precipitation to separate out;
(4) purifying: with wash-out concentrated solution standing over night, leach precipitation, throw out is dissolved in the ethanolic soln, ultrasonic dissolution with half preparative liquid chromatography purifying, is collected the reservation flow point, and concentrating under reduced pressure, crystallization and drying obtain colourless crystallization.
2. the preparation method of the plain A of a kind of Radde Anemone Rhizome according to claim 1 is characterized in that the alcohol concn volume percent is 70-90% described in the step (1), extracts twice, and water is carried once, and the liquid material is than being 3-10: 1 (V/W).
3. the preparation method of the plain A of a kind of Radde Anemone Rhizome according to claim 1, it is characterized in that macroporous resin described in the step (3) is HPD300, HPD450, NKA-9 type macroporous resin, described elution requirement shoals for washing with water earlier to color, uses acetone-methanol-water (55: 35: 10) wash-out then instead.
4. the preparation method of the plain A of a kind of Radde Anemone Rhizome according to claim 1 is characterized in that low temperature is 0-10 ℃ described in the step (3).
5. the preparation method of the plain A of a kind of Radde Anemone Rhizome according to claim 1, it is characterized in that half preparative high performance liquid chromatography condition is described in the step (4): ODS C18,5 μ m, 250*20.0mm (I.D.), moving phase is formed volume ratio: methanol-water (68: 32), flow velocity 5.0ml/min, column temperature: room temperature; Detect wavelength: UV 207nm; Sample size: 50 microlitres.
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|---|---|---|---|
| CN2010105201920A CN102010457A (en) | 2010-10-26 | 2010-10-26 | Method for preparing raddeanin A |
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| CN2010105201920A CN102010457A (en) | 2010-10-26 | 2010-10-26 | Method for preparing raddeanin A |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103588849A (en) * | 2012-08-14 | 2014-02-19 | 江苏汉邦科技有限公司 | Preparation method for glycyrrhetinic acid |
| CN105616552A (en) * | 2014-11-28 | 2016-06-01 | 天津耀宇生物技术有限公司 | Preparation method and application of Rhizoma Anemones Raddeanae extract |
| CN105641078A (en) * | 2014-11-28 | 2016-06-08 | 天津耀宇生物技术有限公司 | Method for extracting active component from rhizoma anemones raddeanae and application of active component |
-
2010
- 2010-10-26 CN CN2010105201920A patent/CN102010457A/en active Pending
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103588849A (en) * | 2012-08-14 | 2014-02-19 | 江苏汉邦科技有限公司 | Preparation method for glycyrrhetinic acid |
| CN105616552A (en) * | 2014-11-28 | 2016-06-01 | 天津耀宇生物技术有限公司 | Preparation method and application of Rhizoma Anemones Raddeanae extract |
| CN105641078A (en) * | 2014-11-28 | 2016-06-08 | 天津耀宇生物技术有限公司 | Method for extracting active component from rhizoma anemones raddeanae and application of active component |
| CN105641078B (en) * | 2014-11-28 | 2019-09-17 | 天津耀宇生物技术有限公司 | From the middle method and its application for extracting active component pointed at both ends |
| CN105616552B (en) * | 2014-11-28 | 2019-10-15 | 天津耀宇生物技术有限公司 | The preparation method and applications of Radde anemone rhizome extract |
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Application publication date: 20110413 |