CN103788167A - Preparation method for glycyrrhetinic acid monoglucuronide (GAMG) - Google Patents
Preparation method for glycyrrhetinic acid monoglucuronide (GAMG) Download PDFInfo
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Abstract
The invention discloses a preparation method for high-purity GAMG. The preparation method comprises the following steps: with fresh licorice as a raw material, carrying out ultrasonic extraction, taking a supernatant, adjusting the pH value of the supernatant with hydrochloric acid, carrying out centrifugation, taking a precipitate, washing the precipitate with deionized water twice to three times and then drying the washed precipitate in a vacuum drying box for drying so as to obtain a crude GAMG product; and weighing a certain amount of the crude GAMG product, adding water, adjusting a pH value to 8 to 9 by using concentrated ammonia liquor, adding an organic solvent for extraction after complete dissolution, taking a water layer after clear layering of two phases and carrying out further purification by using a semi-preparative high performance liquid chromatograph so as to obtain high-purity GAMG. The preparation method for GAMG has the advantages of simple operation, few experimental steps, small pollution, low cost, a short period and capacity of preparing GAMG with purity of more than 99.5%.
Description
Technical field
The invention belongs to biological medicine technology field, be specifically related to the preparation method of a kind of high purity Glycyrrhetic acid 3-O-mono-BETA-D-glucuronide (GAMG).
Background technology
Radix Glycyrrhizae is a kind of traditional herbal medicine of China, and its major ingredient Potenlini has the effects such as anti-inflammatory, antiviral, antitumor, antianaphylaxis, and it is a kind of sweeting agent, and sugariness is approximately 170 times of sucrose.Glycyrrhetic acid 3-O-mono-BETA-D-glucuronide (glycyrrhetic acid 3-O-mono-β-D-glucuronide, GAMG) as the derivative of Potenlini, both have similar biological activity, and it is equal to or exceedes Potenlini on anti-inflammatory and antiallergic activity, and have significant antitumous effect.GAMG is a kind of novel natural sweeteners, and sugariness is approximately 5 times of GL.Therefore, GAMG has important researching value.Therefore, find the optimum process condition that extracts purifying GAMG, technical process becomes GAMG and utilizes the matter of utmost importance that will solve.
Summary of the invention
The object of the invention is to, at present existing complex process and the not high problem of purity in preparation GAMG process, provides a kind of novel method of preparing high purity GAMG.
For achieving the above object, the technical scheme that the present invention takes is:
A preparation method of GAMG, take Radix Glycyrrhizae as raw material, adopts high performance liquid chromatography to prepare high purity GAMG, and it comprises the following steps:
(1) get fresh Radix Glycyrrhizae, after mixing with containing 70% aqueous ethanolic solution of 0.5% ammoniacal liquor with volume ratio 1:10, after ultrasonic extraction 1 ~ 2h, centrifuging and taking supernatant liquor;
(2) supernatant liquor is adjusted to pH to 3 ~ 4 with hydrochloric acid, acid is heavy spends the night, and centrifuging and taking precipitation is washed after 2-3 time with deionized water, as for dry in vacuum drying oven, obtains GAMG crude product;
(3) get GAMG crude product powder, be dissolved in water and be adjusted to pH8-9 with ammoniacal liquor, until completely dissolved, add after isopyknic organic solvent extraction 2-3 time water intaking layer;
(4) water layer pH is recalled to 5, adopt C18 reverse phase silica gel chromatographic column, first alcohol and water is as moving phase, and degree of grade/gradient elution, carries out after the separation and purification of final step through semi-preparative liquid chromatograph, and the purity of the GAMG collecting is higher than 99.5%.
The invention has the beneficial effects as follows and can select fresh Radix Glycyrrhizae or Radix Glycyrrhizae crude extract to prepare GAMG, process is simple, easy to control, Technology is simplified, production cost is low, this semipreparative chromatographic condition can amplify on year-on-year basis, be applicable to technical grade biological medicine liquid phantom preparing chromatogram device, to realize the suitability for industrialized production of high purity GAMG.In high-performance liquid chromatogram determination, total impurities peak is less than 0.45%, and single contaminant peak is no more than 0.2%.This product meets European Pharmacopoeia standard completely.
Accompanying drawing explanation
Fig. 1 is the color atlas of loading 5mg purifying GAMG
Fig. 2 is the color atlas of loading 10mg purifying GAMG
Fig. 3 is the color atlas of loading 2mg purifying GAMG
Fig. 4 is GAMG sterling liquid chromatography for measuring figure
Embodiment
Embodiment 1
1. after the fresh Radix Glycyrrhizae powder after getting 1kg and having pulverized mixes containing 70% aqueous ethanolic solution of 0.5% ammoniacal liquor with 10 times of volumes, after ultrasonic extraction 1h, centrifuging and taking supernatant liquor;
2. regulate supernatant liquor pH to 3 ~ 4 with concentrated hydrochloric acid, acid is heavy spends the night, and centrifuging and taking precipitation is washed after 2-3 time with deionized water, as for dry in vacuum drying oven, obtains GAMG crude product;
3. take GAMG crude product 0.5g, add in 50ml water, adjust after pH to 8 with ammoniacal liquor, after GAMG crude product dissolves completely, add isopyknic ethyl acetate extraction three times, water intaking layer;
4. water layer pH is recalled to 5, adopt semi-preparative liquid chromatograph to carry out the separation and purification of final step, column size is Φ 10 × 250mm, C18 reverse phase silica gel filler is Hanbon Sci. & Tech. Co., Ltd.'s product, and particle diameter is 10um, applied sample amount 5mg, moving phase is pure methanol-water solution, gradient degree elution requirement is, 0 ~ 25min, methyl alcohol 65% ~ 80%; 25 ~ 50min, methyl alcohol 80% ~ 80%, flow velocity 4ml/min, column temperature is 20 ℃, ultraviolet detection wavelength is 230nm, collects the cut of retention time at 36.5 ~ 38.5min.Through HPLC purity assay >=99.6%.
Embodiment 2
1. after the fresh Radix Glycyrrhizae powder after getting 1kg and having pulverized mixes containing 70% aqueous ethanolic solution of 0.5% ammoniacal liquor with 10 times of volumes, after ultrasonic extraction 1.5h, centrifuging and taking supernatant liquor;
2. regulate supernatant liquor pH to 3 ~ 4 with concentrated hydrochloric acid, acid is heavy spends the night, and centrifuging and taking precipitation is washed after 2-3 time with deionized water, as for dry in vacuum drying oven, obtains GAMG crude product;
3. take GAMG crude product 0.5g, add in 50ml water, adjust after pH to 9 with ammoniacal liquor, after GAMG crude product dissolves completely, add isopyknic acetone extract three times, water intaking layer;
4. water layer pH is recalled to 5, adopt semi-preparative liquid chromatograph to carry out the separation and purification of final step, column size is Φ 10 × 250mm, C18 reverse phase silica gel filler is Hanbon Sci. & Tech. Co., Ltd.'s product, and particle diameter is 5um, applied sample amount 10mg, moving phase is pure methanol-water solution, gradient degree elution requirement is, 0 ~ 20min, methyl alcohol 60% ~ 75%; 20 ~ 35min, methyl alcohol 75% ~ 85%, flow velocity 4ml/min, column temperature is 25 ℃, ultraviolet detection wavelength is 230nm, collects the cut of retention time at 36 ~ 40min, through HPLC purity assay >=99.5%.
Embodiment 3
1. take Radix Glycyrrhizae crude extract 0.5g, add in 50ml water, with after ammoniacal liquor tune pH to 8, if there is not solute, centrifugal, get supernatant liquor;
2. add isopyknic chloroform in supernatant liquor, extract three times, water intaking layer;
4. water layer pH is recalled to 5, adopt semi-preparative liquid chromatograph to carry out the separation and purification of final step, column size is Φ 10 × 250mm, and C18 reverse phase silica gel filler is Hanbon Sci. & Tech. Co., Ltd.'s product, particle diameter is 10um, applied sample amount 2mg, moving phase is pure methanol-water solution, moving phase is 70% methanol-water solution, isocratic elution, flow velocity 4ml/min, 20 ℃ of column temperatures, collect the cut of retention time at 17 ~ 19min.The detection wavelength of the New UV Spectrophotometric detector adopting is 230nm, through HPLC purity assay >=99.7%.
Claims (5)
1. a preparation method for Glycyrrhetic acid 3-O-mono-BETA-D-glucuronide (GAMG), is characterized in that comprising the following steps:
(1) after mixing by Radix Glycyrrhizae with containing 70% aqueous ethanolic solution of 0.5% ammoniacal liquor, ultrasonic extraction 1 ~ 2h, centrifuging and taking supernatant liquor;
(2) supernatant liquor is adjusted to pH to 3 ~ 4 with hydrochloric acid, acid is heavy spends the night, and centrifuging and taking precipitation, washes after 2-3 time with deionized water, is placed in vacuum drying oven dry, obtains GAMG crude product;
(3) by GAMG crude product with water dissolution and with ammoniacal liquor adjust pH to 8-9, after organic solvent extraction 2-3 time, water intaking layer;
(4) water layer pH is recalled to 5, adopt first alcohol and water to do moving phase, use Semipreparative chromatography instrument to prepare high purity GAMG.
2. method according to claim 1, is characterized in that: organic solvent ethyl acetate, acetone or the chloroform described in described step (3).
3. method according to claim 1, it is characterized in that: in described step (4), Semipreparative chromatography instrument adopts the reverse phase silica gel filler that stationary phase is C18, chromatographic column specification: internal diameter 10 ~ 50mm, length 150 ~ 300mm, packing material size is 5 ~ 10 μ m, and moving phase is with the mode wash-out GAMG sample of degree such as grade or gradient elution.
4. isocratic elution mode according to claim 3, is characterized in that: volume ratio (V/V) scope of mobile phase methanol and water is 7:3 ~ 9:1,20 ~ 35 ℃ of column temperatures, flow velocity 4 ~ 30ml/min.
5. gradient elution mode according to claim 3, is characterized in that: moving phase is methanol-water system, and volume ratio is 65:35 ~ 80:20,20 ~ 35 ℃ of column temperatures, flow velocity 4 ~ 30ml/min.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104387439A (en) * | 2014-10-31 | 2015-03-04 | 中国农业大学 | Method for preparing glycyrrhizic acid derivatives by carrying out subcritical hydrolysis reaction |
| CN105669821A (en) * | 2016-01-08 | 2016-06-15 | 中国农业大学 | Preparation method of glycyrrhetinic acid monoglucuronide |
| CN106636290A (en) * | 2017-02-03 | 2017-05-10 | 江南大学 | Method for producing glycyrrhetinic acid monoglucuronide by fermentation, and application |
| CN107286218A (en) * | 2017-06-01 | 2017-10-24 | 亿利耐雀生物科技有限公司 | A kind of preparation method of new oleanane-type triterpene saponin derivative |
| CN112544966A (en) * | 2019-09-25 | 2021-03-26 | 天津科技大学 | Mouth-clearing buccal tablet with anti-inflammatory effect and preparation method thereof |
| CN114605488A (en) * | 2020-12-03 | 2022-06-10 | 中国科学院大连化学物理研究所 | Method for purifying glycyrrhizic acid by using preparative chromatography technology |
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| CN1563073A (en) * | 2004-04-06 | 2005-01-12 | 南开大学 | Method for preparing enoxolone |
| CN102219824A (en) * | 2011-06-15 | 2011-10-19 | 北京联合大学生物化学工程学院 | Method for producing glycyrrhizic acid through enzymolysis |
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Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104387439A (en) * | 2014-10-31 | 2015-03-04 | 中国农业大学 | Method for preparing glycyrrhizic acid derivatives by carrying out subcritical hydrolysis reaction |
| CN104387439B (en) * | 2014-10-31 | 2017-02-01 | 中国农业大学 | Method for preparing glycyrrhizic acid derivatives by carrying out subcritical hydrolysis reaction |
| CN105669821A (en) * | 2016-01-08 | 2016-06-15 | 中国农业大学 | Preparation method of glycyrrhetinic acid monoglucuronide |
| CN105669821B (en) * | 2016-01-08 | 2017-05-10 | 中国农业大学 | Preparation method of glycyrrhetinic acid monoglucuronide |
| CN106636290A (en) * | 2017-02-03 | 2017-05-10 | 江南大学 | Method for producing glycyrrhetinic acid monoglucuronide by fermentation, and application |
| CN106636290B (en) * | 2017-02-03 | 2020-06-09 | 江南大学 | Method for producing glycyrrhetinic acid monoglucuronide through fermentation and application |
| CN107286218A (en) * | 2017-06-01 | 2017-10-24 | 亿利耐雀生物科技有限公司 | A kind of preparation method of new oleanane-type triterpene saponin derivative |
| CN107286218B (en) * | 2017-06-01 | 2019-09-10 | 亿利耐雀生物科技有限公司 | A kind of preparation method of new oleanane-type triterpene saponin derivative |
| CN112544966A (en) * | 2019-09-25 | 2021-03-26 | 天津科技大学 | Mouth-clearing buccal tablet with anti-inflammatory effect and preparation method thereof |
| CN114605488A (en) * | 2020-12-03 | 2022-06-10 | 中国科学院大连化学物理研究所 | Method for purifying glycyrrhizic acid by using preparative chromatography technology |
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