CN101182346A - Method for preparing asiaticoside A and B simultaneously from herba centellae total saponins - Google Patents
Method for preparing asiaticoside A and B simultaneously from herba centellae total saponins Download PDFInfo
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- CN101182346A CN101182346A CNA200710164474XA CN200710164474A CN101182346A CN 101182346 A CN101182346 A CN 101182346A CN A200710164474X A CNA200710164474X A CN A200710164474XA CN 200710164474 A CN200710164474 A CN 200710164474A CN 101182346 A CN101182346 A CN 101182346A
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Abstract
The invention discloses a method of synchronously preparing for asiaticoside A and asiaticoside B from Asiatic pennywort total saponin. The method is that the Asiatic pennywort total saponin is dissolved by methanol and stirred with silica gel to be evaporated to dryness to obtain stirred silica gel; the silica gel, stirred silica gel are added into a chromatography column by the dry method for filling into the column; a three-element mixed solvent of organic solvent I-organic solvent II-water is used for liquid-phase elution; eluted liquid is analyzed by TLC to be combined into three fractions, wherein, the fraction contains the asiaticoside A and the asiaticoside B abundantly is taken for vacuum concentration and vacuum drying to be dissolved and filtered to obtain filtrate; C18 silica gel is filled into the column by the wet method, the upper of the filtrate is eluted by the mixed solvent of acetonitrile-water; the eluted liquid is analyzed by TLC to be combined into three fractions, wherein, the fractions which contain the asiaticoside B and the asiaticoside A abundantly are taken respectively for the vacuum concentration, crystallization and vacuum drying. The method has the advantages of simple technical process, low operation expense, the high purity of the asiaticoside A and the asiaticoside B etc. and can be applied to the development of the new drug.
Description
Technical field
The present invention relates to a kind of method for preparing centella asiatica glucoside A and centella asiatica glucoside B from asiatic centella total saponins simultaneously.
Background technology
Herba Centellae [Centella asiatica (L.) UrBan] is a samphire, and how (Linnaeus Carolus) was included into Lawn Pennywort Herb in 1764 with Herba Centellae and belongs to (Hydrocotyle), and names the L. into Hydrocotyleasiatica the botanist woods.In 1879, with its independent from Lawn Pennywort Herb belongs to and set up Centella (Centella), be included into this genus and rename Centella asiatica (L.) Urb. as by Herba Centellae according to the rib number of the arrangement of petal and fruit for fritz UrBan.Herba Centellae mainly is distributed in the southern hemisphere and northern hemisphere torrid zone and subtropical zone, and main product ground is South Africa, India, Sri Lanka, Malaysia, Indonesia, Australia, Japan etc., and China also produces Herba Centellae.Herba Centellae for two thousand years, among the people is usually used in treating flu, tonsillitis, infectious hepatitis, dysentery, wound, furuncle swelling toxin, traumatic hemorrhage etc. at the medicinal history of China.Research of Herba Centellae modern pharmacology and application have had very great development, show that Herba Centellae can be used for treating dysthymia disorders, skin wound, stomach ulcer, infectious hepatitis, tetter and meningococcal meningitis etc.
The complex chemical composition of Herba Centellae mainly contains triterpenes, flavonoid, volatile oil, polyyne alkene class etc.Wherein triterpene compound is main effective component, comprises centella asiatica glucoside, centella asiatica glucoside A, ripple hot-die glycosides, Bo Remi glycosides, thankuniside, isothankuniside, asiatic acid, hydroxyl Herba Centellae Bo Remi acid, madasiatic acid etc.Asiatic centella total saponins is a general name of extracting the saponins material that obtains from Herba Centellae, mainly form by triterpene glycoside material, wherein representative composition mainly contains centella asiatica glucoside (Aisaticoside, CAS No.:16830-15-2), centella asiatica glucoside A (Asaiticoside A; Madecassoside, CAS No.:34540-22-2) and centella asiatica glucoside B (Asaiticoside B, CAS No.:125265-68-1).Centella asiatica glucoside A also is asiaticoside, and centella asiatica glucoside B and centella asiatica glucoside A are a pair of isomerss.How centella asiatica glucoside A has compared a hydroxyl with centella asiatica glucoside B with centella asiatica glucoside, polarity difference is bigger, easily realize separating, but the difference of centella asiatica glucoside A and centella asiatica glucoside B only is that one of them methyl ownership is different, and two different ownership are in the ortho position relation, this species diversity seems more small down in R base (Glu-Glu-Rha sugar key) influence, thus centella asiatica glucoside A and centella asiatica glucoside B separate very difficulty, still do not have effective means report at present.
The structural formula of centella asiatica glucoside (a), centella asiatica glucoside A (b) and centella asiatica glucoside B (c) is as follows respectively:
Studies show that the mixture of centella asiatica glucoside A and centella asiatica glucoside B has pharmacological actions such as promoting wound healing, crease-resistant, anti-inflammatory, antidepressant, promotion intelligence.Relatively centella asiatica glucoside promotes wound healing, and the pharmacological action of treatment scar can find that pharmacological actions such as the anti-inflammatory of asiatic centella total saponins, antidepressant, promotion intelligence may be that centella asiatica glucoside A and centella asiatica glucoside B have played main effect.Also there are some researches show simultaneously, the centella asiatica glucoside A that different concns and different mass are formed and the anti-inflammatory pharmacology of centella asiatica glucoside B mixture have evident difference, so be necessary very much to separate centella asiatica glucoside A and the pure product of centella asiatica glucoside B of obtaining, with the pharmacological action of further research centella asiatica glucoside A and centella asiatica glucoside B, for the exploitation of new drug provides the basis.
Summary of the invention
The purpose of this invention is to provide a kind of method for preparing centella asiatica glucoside A and centella asiatica glucoside B from asiatic centella total saponins simultaneously.
The step of method is as follows:
1) asiatic centella total saponins adds the silica gel mixing and stirring after with dissolve with methanol, must mix sample silica gel behind the evaporate to dryness, and the mass ratio of asiatic centella total saponins and silica gel is 0.05～0.2: 1;
2) add silica gel in the chromatography column in regular turn and mix sample silica gel, adopt dry column-packing, the aspect ratio of dry method chromatography column is 3～9, and silica gel is 3～8 with the aspect ratio of mixing sample silica gel;
3) be moving phase with organic solvent I-organic solvent II-water ternary mixed solvent, with the flow velocity wash-out of 0.5～3 bed volume/h;
4) elutriant is merged into three cuts after TLC analyzes, and cut 1 is a centella asiatica glucoside solution, and cut 2 is the mixing solutions of centella asiatica glucoside, centella asiatica glucoside A, centella asiatica glucoside B, and cut 3 is the mixing solutions of centella asiatica glucoside A and centella asiatica glucoside B;
5) after using the dissolving of acetonitrile-water mixed solvent, filtration after cut 3 vacuum concentration, the vacuum-drying, get filtrate;
6) C18 silica gel adopts wet method dress post, and the aspect ratio of pillar is 4～12, washes pillar mutually with the acetonitrile-water mixed solvent;
7) filtrate is in sample on the ratio of mixture/1g C18 silica gel of 0.01～0.1g centella asiatica glucoside A and centella asiatica glucoside B, and the acetonitrile-water mixed solvent is with the flow velocity wash-out of 0.5～3 bed volume/h;
8) elutriant is merged into three cuts after HPLC analyzes, cut 4 is a centella asiatica glucoside B solution, cut 5 is the mixing solutions of centella asiatica glucoside B and centella asiatica glucoside A, cut 6 is a centella asiatica glucoside A solution, and cut 4 gets centella asiatica glucoside B and centella asiatica glucoside A product respectively with cut 6 after vacuum concentration, crystallization, vacuum-drying;
9) the C18 post can be gone up sample once more after acetonitrile-water mixed solvent flushing regeneration.
Ratio adding silica gel with 0.08～0.15g asiatic centella total saponins/1g silica gel in the described step 1) stirs.
Organic solvent I is chloroform or methylene dichloride or ethyl acetate in the step 3).
Organic solvent II is methyl alcohol or ethanol in the step 3).
The volumetric concentration of organic solvent I is preferably 60～80% in step 3) organic solvent I-organic solvent II-water ternary mixed solvent.
The volumetric concentration of organic solvent II is preferably 18～38% in step 3) organic solvent I-organic solvent II-water ternary mixed solvent.
The volumetric concentration of water is preferably 2～10% in step 3) organic solvent I-organic solvent II-water ternary mixed solvent.
In step 5), step 6), the step 7) in the acetonitrile-water mixed solvent volumetric concentration of acetonitrile be preferably 10～30%.
Filtrate is in sample on the ratio of mixture/1g C18 silica gel of 0.02～0.06g centella asiatica glucoside A and centella asiatica glucoside B in the step 7).
C18 column regeneration process is for being that the acetonitrile-water mixed solvent of 80～95% acetonitrile-water and 10～30% washes with volumetric concentration successively in the step 9).
Vacuum concentration in the step 8), crystallization, process of vacuum drying are as follows: the centella asiatica glucoside A of collection and centella asiatica glucoside B elutriant are being lower than 50 ℃ of following vacuum concentration to doing respectively, dissolve with small amount of methanol, the ethyl acetate crystallization that adds 8 times of amounts (volume ratio) again, filter the back and be lower than 50 ℃ of following vacuum-dryings, get centella asiatica glucoside A and centella asiatica glucoside B product respectively.
The present invention adopts dry method and wet method two-step chromatography to prepare the pure product of centella asiatica glucoside A and centella asiatica glucoside B, have that technological process is simple, process cost is low, centella asiatica glucoside A and centella asiatica glucoside B product yield and purity advantages of higher, centella asiatica glucoside A and the centella asiatica glucoside B pure product exploitation that can be used for new drug of the purity for preparing more than 95%.
Description of drawings
Accompanying drawing is a kind of process flow diagram for preparing centella asiatica glucoside A and centella asiatica glucoside B from asiatic centella total saponins simultaneously.
Embodiment
The preprocessing process of C18 silica gel among the present invention: C18 silica gel need add the methyl alcohol soaked overnight before use fully launches the bonding on the matrix mutually, to give full play to its separating power.
The preparation process of raw material asiatic centella total saponins: get Herba Centellae hay 500g, add 6000g respectively, 5000g, 3500g aqueous ethanolic solution soak and extract three times, and wherein the alcoholic acid volume percent is 80% in the aqueous ethanolic solution, extracting solution merges, concentrating under reduced pressure is removed ethanol, gets concentrated solution 2.9L, concentrated solution dilute with water after-filtration, get asiatic centella total saponins aqueous solution 7.5L behind the constant volume, asiatic centella total saponins content is 1.5g/L after testing; Pack into through pretreated macroporous adsorbent resin HPD100 in internal diameter is the chromatography column of 4cm, resin column is high to be 20cm (aspect ratio is 5), and the bed volume of resin column is 251mL; Concentrated solution is crossed chromatography column with the flow velocity of 1.5BV/h, penetrates to effluent liquid moderate snow grass total saponins, adds concentrated solution 7000mL altogether; First water washing resin post with 4 bed volume, then with the flow velocity wash-out asiatic centella total saponins of alcohol-water mixed flow with 2BV/h, the middle mutually alcoholic acid volume percent of alcohol-water mixed flow is 30%, elutriant merges after TLC analyzes, after concentrated, crystallization, vacuum-drying, get product asiatic centella total saponins 12.3g again, the resin column purge process rate of recovery is 89.5%, and the asiatic centella total saponins mass content is 80.3% in the product after testing.Above process repeats 10 times, gets asiatic centella total saponins 125.3g altogether, and the asiatic centella total saponins mass content is 80.7% in the product after testing.
Analysis condition used among the present invention is as follows:
Thin-layer chromatography (TLC) condition is as follows: silica gel G 254 thin plates, developping agent are propyl carbinol-ethyl acetate-water=4: 1: 5 (getting upper solution uses), and developer is 10% vitriol oil ethanolic soln.
High performance liquid chromatography (HPLC) analysis condition: Agilent1100 liquid-phase chromatographic analysis system, chromatographic column are Phenomenex C-18column Synergi
TMFusion-RP 80A (4 μ, 150mm * 4.60mm ID), moving phase acetonitrile-water (20: 80), flow velocity 0.4mL/min, 20 ℃ of column temperatures, ultraviolet detection wavelength 205nm, purity check usable floor area normalization method.
Embodiment 1
Get the 3g asiatic centella total saponins and add in the flask, add the 100mL dissolve with methanol, add 60g silica gel (mixing the sample ratio is 0.05) again, stir, must mix sample silica gel after the evaporated in vacuo with rotatory evaporator; Add silica gel in internal diameter is the chromatography column of 4cm, the silica gel height after dress is real is 12cm, then adds and mixes sample silica gel, and the sample silica gel height of mixing after dress is real is 4.0cm (aspect ratio is 3, and silica gel is 3 with mixing sample silica gel aspect ratio, and total bed volume is 201ml); With ethyl acetate-methanol-water (volume ratio is 15: 8: 2) ternary mixed solvent is moving phase, with 1.7mL/min (the flow velocity wash-out of 0.5 bed volume/h); Elutriant is merged into three cuts after TLC analyzes, cut 1 is a centella asiatica glucoside solution, and cut 2 is the mixing solutions of centella asiatica glucoside, centella asiatica glucoside A, centella asiatica glucoside B, and cut 3 is the mixing solutions of centella asiatica glucoside A and centella asiatica glucoside B; The mixture of 429mg centella asiatica glucoside A and centella asiatica glucoside B will be got after cut 3 vacuum concentration, the vacuum-drying; With the mixture of gained with the 2mL volumetric concentration be 10% acetonitrile-water mixed solvent dissolving, after filtering filtrate; Internal diameter is 2cm, highly is that the chromatography column of 8cm (filling C18 silica gel amount is 14g for aspect ratio 4, bed volume 25mL) adopts wet method dress post, is that 10% acetonitrile-water mixed solvent washes pillar with volumetric concentration; Get sample on the 0.3mL filtrate (applied sample amount is mixture/1g C18 silica gel of 0.005 g centella asiatica glucoside A and centella asiatica glucoside B), volumetric concentration is 10% acetonitrile-water mixed solvent with 0.2mL/min (the flow velocity wash-out of 0.5 bed volume/h); Elutriant is merged into three cuts after HPLC analyzes, cut 4 is a centella asiatica glucoside B solution, cut 5 is the mixing solutions of centella asiatica glucoside B and centella asiatica glucoside A, cut 6 is a centella asiatica glucoside A solution, cut 4 gets centella asiatica glucoside B18mg respectively after vacuum concentration, crystallization, vacuum-drying, purity is 96.6%, and cut 6 gets centella asiatica glucoside A32mg respectively after vacuum concentration, crystallization, vacuum-drying, and purity is 98.3%; C18 regeneration back is reusable.
Embodiment 2
Get the 3g asiatic centella total saponins and add in the flask, add the 100mL dissolve with methanol, add 43g silica gel (mixing the sample ratio is 0.07) again, stir, must mix sample silica gel after the evaporated in vacuo with rotatory evaporator; Add silica gel in internal diameter is the chromatography column of 4cm, the silica gel height after dress is real is 16cm, then adds and mixes sample silica gel, and the sample silica gel height of mixing after dress is real is 4.0cm (aspect ratio is 4, and silica gel is 4 with mixing sample silica gel aspect ratio, and total bed volume is 251ml); With ethyl acetate, alcohol and water (volume ratio is 65: 29: 6) ternary mixed solvent is moving phase, with 4.2mL/min (the flow velocity wash-out of 1 bed volume/h); Elutriant is merged into three cuts after TLC analyzes, cut 1 is a centella asiatica glucoside solution, and cut 2 is the mixing solutions of centella asiatica glucoside, centella asiatica glucoside A, centella asiatica glucoside B, and cut 3 is the mixing solutions of centella asiatica glucoside A and centella asiatica glucoside B; The mixture of 638mg centella asiatica glucoside A and centella asiatica glucoside B will be got after cut 3 vacuum concentration, the vacuum-drying; With the mixture of gained with the 3mL volumetric concentration be 15% acetonitrile-water mixed solvent dissolving, after filtering filtrate; Internal diameter is 2cm, highly is that the chromatography column of 12cm (filling C18 silica gel amount is 21g for aspect ratio 6, bed volume 38mL) adopts wet method dress post, is that 15% acetonitrile-water mixed solvent washes pillar with volumetric concentration; Get sample on the 1.0mL filtrate (applied sample amount is mixture/1g C18 silica gel of 0.01g centella asiatica glucoside A and centella asiatica glucoside B), volumetric concentration is 15% acetonitrile-water mixed solvent with 0.6mL/min (the flow velocity wash-out of 1 bed volume/h); Elutriant is merged into three cuts after HPLC analyzes, cut 4 is a centella asiatica glucoside B solution, cut 5 is the mixing solutions of centella asiatica glucoside B and centella asiatica glucoside A, cut 6 is a centella asiatica glucoside A solution, cut 4 gets centella asiatica glucoside B51mg respectively after vacuum concentration, crystallization, vacuum-drying, purity is 93.1%, and cut 6 gets centella asiatica glucoside A88mg respectively after vacuum concentration, crystallization, vacuum-drying, and purity is 94.7%; C18 regeneration back is reusable.
Embodiment 3
Get the 3g asiatic centella total saponins and add in the flask, add the 100mL dissolve with methanol, add 43g silica gel (mixing the sample ratio is 0.07) again, stir, must mix sample silica gel after the evaporated in vacuo with rotatory evaporator; Add silica gel in internal diameter is the chromatography column of 4cm, the silica gel height after dress is real is 20cm, then adds and mixes sample silica gel, and the sample silica gel height of mixing after dress is real is 4.0cm (aspect ratio is 5, and silica gel is 5 with mixing sample silica gel aspect ratio, and total bed volume is 301ml); With chloroform-methanol-water (volume ratio is 35: 13: 2) ternary mixed solvent is moving phase, with 7.5 mL/min (the flow velocity wash-out of 1.5 bed volume/h); Elutriant is merged into three cuts after TLC analyzes, cut 1 is a centella asiatica glucoside solution, and cut 2 is the mixing solutions of centella asiatica glucoside, centella asiatica glucoside A, centella asiatica glucoside B, and cut 3 is the mixing solutions of centella asiatica glucoside A and centella asiatica glucoside B; The mixture of 675mg centella asiatica glucoside A and centella asiatica glucoside B will be got after cut 3 vacuum concentration, the vacuum-drying; With the mixture of gained with the 3mL volumetric concentration be 20% acetonitrile-water mixed solvent dissolving, after filtering filtrate; Internal diameter is 2cm, highly is that the chromatography column of 16cm (filling C18 silica gel amount is 28g for aspect ratio 8, bed volume 50mL) adopts wet method dress post, is that 20% acetonitrile-water mixed solvent washes pillar with volumetric concentration; Get sample on the 2.5mL filtrate (applied sample amount is mixture/1g C18 silica gel of 0.02g centella asiatica glucoside A and centella asiatica glucoside B), volumetric concentration is 20% acetonitrile-water mixed solvent with 1.3mL/min (the flow velocity wash-out of 1.5 bed volume/h); Elutriant is merged into three cuts after HPLC analyzes, cut 4 is a centella asiatica glucoside B solution, cut 5 is the mixing solutions of centella asiatica glucoside B and centella asiatica glucoside A, cut 6 is a centella asiatica glucoside A solution, cut 4 gets centella asiatica glucoside B143mg respectively after vacuum concentration, crystallization, vacuum-drying, purity is 95.1%, and cut 6 gets centella asiatica glucoside A245mg respectively after vacuum concentration, crystallization, vacuum-drying, and purity is 96.8%; C18 regeneration back is reusable.
Embodiment 4
Get the 5g asiatic centella total saponins and add in the flask, add the 100mL dissolve with methanol, add 56g silica gel (mixing the sample ratio is 0.09) again, stir, must mix sample silica gel after the evaporated in vacuo with rotatory evaporator; Add silica gel in internal diameter is the chromatography column of 4cm, the silica gel height after dress is real is 20cm, then adds and mixes sample silica gel, and the sample silica gel height of mixing after dress is real is 4.0cm (aspect ratio is 5, and silica gel is 5 with mixing sample silica gel aspect ratio, and total bed volume is 301ml); With chloroform-alcohol-water (volume ratio is 35: 13: 2) ternary mixed solvent is moving phase, with 10.0mL/min (the flow velocity wash-out of 2 bed volume/h); Elutriant is merged into three cuts after TLC analyzes, cut 1 is a centella asiatica glucoside solution, and cut 2 is the mixing solutions of centella asiatica glucoside, centella asiatica glucoside A, centella asiatica glucoside B, and cut 3 is the mixing solutions of centella asiatica glucoside A and centella asiatica glucoside B; The mixture of 916mg centella asiatica glucoside A and centella asiatica glucoside B will be got after cut 3 vacuum concentration, the vacuum-drying; With the mixture of gained with the 4mL volumetric concentration be 25% acetonitrile-water mixed solvent dissolving, after filtering filtrate; Internal diameter is 2cm, highly is that the chromatography column of 20cm (filling C18 silica gel amount is 35g for aspect ratio 10, bed volume 63mL) adopts wet method dress post, is that 25% acetonitrile-water mixed solvent washes pillar with volumetric concentration; Get sample on the 0.2mL filtrate (applied sample amount is mixture/1g C18 silica gel of 0.001g centella asiatica glucoside A and centella asiatica glucoside B), volumetric concentration is 25% acetonitrile-water mixed solvent with 2.1mL/min (the flow velocity wash-out of 2 bed volume/h); Elutriant is merged into three cuts after HPLC analyzes, cut 4 is a centella asiatica glucoside B solution, cut 5 is the mixing solutions of centella asiatica glucoside B and centella asiatica glucoside A, cut 6 is a centella asiatica glucoside A solution, cut 4 gets centella asiatica glucoside B8mg respectively after vacuum concentration, crystallization, vacuum-drying, purity is 91.6%, and cut 6 gets centella asiatica glucoside A14mg respectively after vacuum concentration, crystallization, vacuum-drying, and purity is 93.2%; C18 regeneration back is reusable.
Embodiment 5
Get the 5g asiatic centella total saponins and add in the flask, add the 100mL dissolve with methanol, add 56g silica gel (mixing the sample ratio is 0.09) again, stir, must mix sample silica gel after the evaporated in vacuo with rotatory evaporator; Add silica gel in internal diameter is the chromatography column of 4cm, the silica gel height after dress is real is 24cm, then adds and mixes sample silica gel, and the sample silica gel height of mixing after dress is real is 4.0cm (aspect ratio is 6, and silica gel is 6 with mixing sample silica gel aspect ratio, and total bed volume is 352ml); With methylene chloride-methanol-water (volume ratio is 75: 21: 4) ternary mixed solvent is moving phase, with 14.7mL/min (the flow velocity wash-out of 2.5 bed volume/h); Elutriant is merged into three cuts after TLC analyzes, cut 1 is a centella asiatica glucoside solution, and cut 2 is the mixing solutions of centella asiatica glucoside, centella asiatica glucoside A, centella asiatica glucoside B, and cut 3 is the mixing solutions of centella asiatica glucoside A and centella asiatica glucoside B; The mixture of 868mg centella asiatica glucoside A and centella asiatica glucoside B will be got after cut 3 vacuum concentration, the vacuum-drying; With the mixture of gained with the 4mL volumetric concentration be 30% acetonitrile-water mixed solvent dissolving, after filtering filtrate; Internal diameter is 2cm, highly is that the chromatography column of 24cm (filling C18 silica gel amount is 42g for aspect ratio 12, bed volume 75mL) adopts wet method dress post, is that 30% acetonitrile-water mixed solvent washes pillar with volumetric concentration; Get sample on the 2.9mL filtrate (applied sample amount is mixture/1g C18 silica gel of 0.015g centella asiatica glucoside A and centella asiatica glucoside B), volumetric concentration is 30% acetonitrile-water mixed solvent with 3.1mL/min (the flow velocity wash-out of 2.5 bed volume/h); Elutriant is merged into three cuts after HPLC analyzes, cut 4 is a centella asiatica glucoside B solution, cut 5 is the mixing solutions of centella asiatica glucoside B and centella asiatica glucoside A, cut 6 is a centella asiatica glucoside A solution, cut 4 gets centella asiatica glucoside B140mg respectively after vacuum concentration, crystallization, vacuum-drying, purity is 88.9%, and cut 6 gets centella asiatica glucoside A241mg respectively after vacuum concentration, crystallization, vacuum-drying, and purity is 90.5%; C18 regeneration back is reusable.
Embodiment 6
Get the 5g asiatic centella total saponins and add in the flask, add the 100mL dissolve with methanol, add 45g silica gel (mixing the sample ratio is 0.11) again, stir, must mix sample silica gel after the evaporated in vacuo with rotatory evaporator; Add silica gel in internal diameter is the chromatography column of 4cm, the silica gel height after dress is real is 24cm, then adds and mixes sample silica gel, and the sample silica gel height of mixing after dress is real is 4.0cm (aspect ratio is 6, and silica gel is 6 with mixing sample silica gel aspect ratio, and total bed volume is 352ml); With dichloromethane-ethanol-water (volume ratio is 40: 9: 1) ternary mixed solvent is moving phase, with 17.6mL/min (the flow velocity wash-out of 3 bed volume/h); Elutriant is merged into three cuts after TLC analyzes, cut 1 is a centella asiatica glucoside solution, and cut 2 is the mixing solutions of centella asiatica glucoside, centella asiatica glucoside A, centella asiatica glucoside B, and cut 3 is the mixing solutions of centella asiatica glucoside A and centella asiatica glucoside B; The mixture of 943mg centella asiatica glucoside A and centella asiatica glucoside B will be got after cut 3 vacuum concentration, the vacuum-drying; With the mixture of gained with the 4mL volumetric concentration be 15% acetonitrile-water mixed solvent dissolving, after filtering filtrate; Internal diameter is 2cm, highly is that the chromatography column of 12cm (filling C18 silica gel amount is 21g for aspect ratio 6, bed volume 38mL) adopts wet method dress post, is that 15% acetonitrile-water mixed solvent washes pillar with volumetric concentration; Get sample on the 2.2mL filtrate (applied sample amount is mixture/1g C18 silica gel of 0.025g centella asiatica glucoside A and centella asiatica glucoside B), volumetric concentration is 15% acetonitrile-water mixed solvent with 1.9mL/min (the flow velocity wash-out of 3 bed volume/h); Elutriant is merged into three cuts after HPLC analyzes, cut 4 is a centella asiatica glucoside B solution, cut 5 is the mixing solutions of centella asiatica glucoside B and centella asiatica glucoside A, cut 6 is a centella asiatica glucoside A solution, cut 4 gets centella asiatica glucoside B123mg respectively after vacuum concentration, crystallization, vacuum-drying, purity is 91.0%, and cut 6 gets centella asiatica glucoside A210mg respectively after vacuum concentration, crystallization, vacuum-drying, and purity is 92.6%; C18 regeneration back is reusable.
Embodiment 7
Get the 5g asiatic centella total saponins and add in the flask, add the 100mL dissolve with methanol, add 45g silica gel (mixing the sample ratio is 0.11) again, stir, must mix sample silica gel after the evaporated in vacuo with rotatory evaporator; Add silica gel in internal diameter is the chromatography column of 4cm, the silica gel height after dress is real is 24cm, then adds and mixes sample silica gel, and the sample silica gel height of mixing after dress is real is 4.0cm (aspect ratio is 6, and silica gel is 6 with mixing sample silica gel aspect ratio, and total bed volume is 352ml); With ethyl acetate-methanol-water (volume ratio is 15: 8: 2) ternary mixed solvent is moving phase, with 2.9mL/min (the flow velocity wash-out of 0.5 bed volume/h); Elutriant is merged into three cuts after TLC analyzes, cut 1 is a centella asiatica glucoside solution, and cut 2 is the mixing solutions of centella asiatica glucoside, centella asiatica glucoside A, centella asiatica glucoside B, and cut 3 is the mixing solutions of centella asiatica glucoside A and centella asiatica glucoside B; The mixture of 931mg centella asiatica glucoside A and centella asiatica glucoside B will be got after cut 3 vacuum concentration, the vacuum-drying; With the mixture of gained with the 5mL volumetric concentration be 20% acetonitrile-water mixed solvent dissolving, after filtering filtrate; Internal diameter is 2cm, highly is that the chromatography column of 16cm (filling C18 silica gel amount is 28g for aspect ratio 8, bed volume 50mL) adopts wet method dress post, is that 20% acetonitrile-water mixed solvent washes pillar with volumetric concentration; Get sample on the 4.5mL filtrate (applied sample amount is mixture/1g C18 silica gel of 0.03g centella asiatica glucoside A and centella asiatica glucoside B), volumetric concentration is 20% acetonitrile-water mixed solvent with 0.4mL/min (the flow velocity wash-out of 0.5 bed volume/h); Elutriant is merged into three cuts after HPLC analyzes, cut 4 is a centella asiatica glucoside B solution, cut 5 is the mixing solutions of centella asiatica glucoside B and centella asiatica glucoside A, cut 6 is a centella asiatica glucoside A solution, cut 4 gets centella asiatica glucoside B225mg respectively after vacuum concentration, crystallization, vacuum-drying, purity is 97.6%, and cut 6 gets centella asiatica glucoside A386mg respectively after vacuum concentration, crystallization, vacuum-drying, and purity is 99.3%; C18 regeneration back is reusable.
Embodiment 8
Get the 6g asiatic centella total saponins and add in the flask, add the 100mL dissolve with methanol, add 46g silica gel (mixing the sample ratio is 0.13) again, stir, must mix sample silica gel after the evaporated in vacuo with rotatory evaporator; Add silica gel in internal diameter is the chromatography column of 4cm, the silica gel height after dress is real is 28cm, then adds and mixes sample silica gel, and the sample silica gel height of mixing after dress is real is 4.0cm (aspect ratio is 7, and silica gel is 7 with mixing sample silica gel aspect ratio, and total bed volume is 402ml); With ethyl acetate, alcohol and water (volume ratio is 65: 29: 6) ternary mixed solvent is moving phase, with 6.7mL/min (the flow velocity wash-out of 1 bed volume/h); Elutriant is merged into three cuts after TLC analyzes, cut 1 is a centella asiatica glucoside solution, and cut 2 is the mixing solutions of centella asiatica glucoside, centella asiatica glucoside A, centella asiatica glucoside B, and cut 3 is the mixing solutions of centella asiatica glucoside A and centella asiatica glucoside B; The mixture of 1254mg centella asiatica glucoside A and centella asiatica glucoside B will be got after cut 3 vacuum concentration, the vacuum-drying; With the mixture of gained with the 5mL volumetric concentration be 25% acetonitrile-water mixed solvent dissolving, after filtering filtrate; Internal diameter is 2cm, highly is that the chromatography column of 20cm (filling C18 silica gel amount is 35g for aspect ratio 10, bed volume 63mL) adopts wet method dress post, is that 25% acetonitrile-water mixed solvent washes pillar with volumetric concentration; Get sample on the 4.2mL filtrate (applied sample amount is mixture/1g C18 silica gel of 0.03g centella asiatica glucoside A and centella asiatica glucoside B), volumetric concentration is 25% acetonitrile-water mixed solvent with 1.0mL/min (the flow velocity wash-out of 1 bed volume/h); Elutriant is merged into three cuts after HPLC analyzes, cut 4 is a centella asiatica glucoside B solution, cut 5 is the mixing solutions of centella asiatica glucoside B and centella asiatica glucoside A, cut 6 is a centella asiatica glucoside A solution, cut 4 gets centella asiatica glucoside B254mg respectively after vacuum concentration, crystallization, vacuum-drying, purity is 92.6%, and cut 6 gets centella asiatica glucoside A435mg respectively after vacuum concentration, crystallization, vacuum-drying, and purity is 94.2%; C18 regeneration back is reusable.
Embodiment 9
Get the 6g asiatic centella total saponins and add in the flask, add the 100mL dissolve with methanol, add 40g silica gel (mixing the sample ratio is 0.15) again, stir, must mix sample silica gel after the evaporated in vacuo with rotatory evaporator; Add silica gel in internal diameter is the chromatography column of 4cm, the silica gel height after dress is real is 28cm, then adds and mixes sample silica gel, and the sample silica gel height of mixing after dress is real is 4.0cm (aspect ratio is 7, and silica gel is 7 with mixing sample silica gel aspect ratio, and total bed volume is 402ml); With chloroform-methanol-water (volume ratio is 35: 13: 2) ternary mixed solvent is moving phase, with 10.0mL/min (the flow velocity wash-out of 1.5 bed volume/h); Elutriant is merged into three cuts after TLC analyzes, cut 1 is a centella asiatica glucoside solution, and cut 2 is the mixing solutions of centella asiatica glucoside, centella asiatica glucoside A, centella asiatica glucoside B, and cut 3 is the mixing solutions of centella asiatica glucoside A and centella asiatica glucoside B; The mixture of 1608mg centella asiatica glucoside A and centella asiatica glucoside B will be got after cut 3 vacuum concentration, the vacuum-drying; With the mixture of gained with the 8mL volumetric concentration be 15% acetonitrile-water mixed solvent dissolving, after filtering filtrate; Internal diameter is 2cm, highly is that the chromatography column of 12cm (filling C18 silica gel amount is 21g for aspect ratio 6, bed volume 38mL) adopts wet method dress post, is that 15% acetonitrile-water mixed solvent washes pillar with volumetric concentration; Get sample on the 3.6mL filtrate (applied sample amount is mixture/1g C18 silica gel of 0.035g centella asiatica glucoside A and centella asiatica glucoside B), volumetric concentration is 15% acetonitrile-water mixed solvent with 0.9mL/min (the flow velocity wash-out of 1.5 bed volume/h); Elutriant is merged into three cuts after HPLC analyzes, cut 4 is a centella asiatica glucoside B solution, cut 5 is the mixing solutions of centella asiatica glucoside B and centella asiatica glucoside A, cut 6 is a centella asiatica glucoside A solution, cut 4 gets centella asiatica glucoside B170mg respectively after vacuum concentration, crystallization, vacuum-drying, purity is 90.5%, and cut 6 gets centella asiatica glucoside A291mg respectively after vacuum concentration, crystallization, vacuum-drying, and purity is 92.1%; C18 regeneration back is reusable.
Embodiment 10
Get the 8g asiatic centella total saponins and add in the flask, add the 100mL dissolve with methanol, add 53g silica gel (mixing the sample ratio is 0.15) again, stir, must mix sample silica gel after the evaporated in vacuo with rotatory evaporator; Add silica gel in internal diameter is the chromatography column of 4cm, the silica gel height after dress is real is 32cm, then adds and mixes sample silica gel, and the sample silica gel height of mixing after dress is real is 4.6cm (aspect ratio is 8, and silica gel is 7 with mixing sample silica gel aspect ratio, and total bed volume is 459ml); With chloroform-alcohol-water (volume ratio is 35: 13: 2) ternary mixed solvent is moving phase, with 15.3mL/min (the flow velocity wash-out of 2 bed volume/h); Elutriant is merged into three cuts after TLC analyzes, cut 1 is a centella asiatica glucoside solution, and cut 2 is the mixing solutions of centella asiatica glucoside, centella asiatica glucoside A, centella asiatica glucoside B, and cut 3 is the mixing solutions of centella asiatica glucoside A and centella asiatica glucoside B; The mixture of 1745mg centella asiatica glucoside A and centella asiatica glucoside B will be got after cut 3 vacuum concentration, the vacuum-drying; With the mixture of gained with the 8mL volumetric concentration be 20% acetonitrile-water mixed solvent dissolving, after filtering filtrate; Internal diameter is 2cm, highly is that the chromatography column of 16cm (filling C18 silica gel amount is 28g for aspect ratio 8, bed volume 50mL) adopts wet method dress post, is that 20% acetonitrile-water mixed solvent washes pillar with volumetric concentration; Get sample on the 5.8mL filtrate (applied sample amount is mixture/lg C18 silica gel of 0.045g centella asiatica glucoside A and centella asiatica glucoside B), volumetric concentration is 20% acetonitrile-water mixed solvent with 1.7mL/min (the flow velocity wash-out of 2 bed volume/h); Elutriant is merged into three cuts after HPLC analyzes, cut 4 is a centella asiatica glucoside B solution, cut 5 is the mixing solutions of centella asiatica glucoside B and centella asiatica glucoside A, cut 6 is a centella asiatica glucoside A solution, cut 4 gets centella asiatica glucoside B274mg respectively after vacuum concentration, crystallization, vacuum-drying, purity is 87.8%, and cut 6 gets centella asiatica glucoside A470mg respectively after vacuum concentration, crystallization, vacuum-drying, and purity is 89.4%; C18 regeneration back is reusable.
Embodiment 11
Get the 8g asiatic centella total saponins and add in the flask, add the 100mL dissolve with methanol, add 47g silica gel (mixing the sample ratio is 0.17) again, stir, must mix sample silica gel after the evaporated in vacuo with rotatory evaporator; Add silica gel in internal diameter is the chromatography column of 4cm, the silica gel height after dress is real is 32cm, then adds and mixes sample silica gel, and the sample silica gel height of mixing after dress is real is 4.6cm (aspect ratio is 8, and silica gel is 7 with mixing sample silica gel aspect ratio, and total bed volume is 459ml); With methylene chloride-methanol-water (volume ratio is 75: 21: 4) ternary mixed solvent is moving phase, with 19.1mL/min (the flow velocity wash-out of 2.5 bed volume/h); Elutriant is merged into three cuts after TLC analyzes, cut 1 is a centella asiatica glucoside solution, and cut 2 is the mixing solutions of centella asiatica glucoside, centella asiatica glucoside A, centella asiatica glucoside B, and cut 3 is the mixing solutions of centella asiatica glucoside A and centella asiatica glucoside B; The mixture of 1770mg centella asiatica glucoside A and centella asiatica glucoside B will be got after cut 3 vacuum concentration, the vacuum-drying; With the mixture of gained with the 8mL volumetric concentration be 25% acetonitrile-water mixed solvent dissolving, after filtering filtrate; Internal diameter is 2cm, highly is that the chromatography column of 20cm (filling C18 silica gel amount is 35g for aspect ratio 10, bed volume 63mL) adopts wet method dress post, is that 25% acetonitrile-water mixed solvent washes pillar with volumetric concentration; Get sample on the 6.3mL filtrate (applied sample amount is mixture/1g C18 silica gel of 0.04g centella asiatica glucoside A and centella asiatica glucoside B), volumetric concentration is 25% acetonitrile-water mixed solvent with 2.6mL/min (the flow velocity wash-out of 2.5 bed volume/h); Elutriant is merged into three cuts after HPLC analyzes, cut 4 is a centella asiatica glucoside B solution, cut 5 is the mixing solutions of centella asiatica glucoside B and centella asiatica glucoside A, cut 6 is a centella asiatica glucoside A solution, cut 4 gets centella asiatica glucoside B316mg respectively after vacuum concentration, crystallization, vacuum-drying, purity is 89.5%, and cut 6 gets centella asiatica glucoside A541mg respectively after vacuum concentration, crystallization, vacuum-drying, and purity is 91.0%; C18 regeneration back is reusable.
Embodiment 12
Get the 8g asiatic centella total saponins and add in the flask, add the 100mL dissolve with methanol, add 40g silica gel (mixing the sample ratio is 0.2) again, stir, must mix sample silica gel after the evaporated in vacuo with rotatory evaporator; Add silica gel in internal diameter is the chromatography column of 4cm, the silica gel height after dress is real is 36cm, then adds and mixes sample silica gel, and the sample silica gel height of mixing after dress is real is 4.5cm (aspect ratio is 9, and silica gel is 8 with mixing sample silica gel aspect ratio, and total bed volume is 509ml); With dichloromethane-ethanol-water (volume ratio is 40: 9: 1) ternary mixed solvent is moving phase, with 25.4mL/min (the flow velocity wash-out of 3 bed volume/h); Elutriant is merged into three cuts after TLC analyzes, cut 1 is a centella asiatica glucoside solution, and cut 2 is the mixing solutions of centella asiatica glucoside, centella asiatica glucoside A, centella asiatica glucoside B, and cut 3 is the mixing solutions of centella asiatica glucoside A and centella asiatica glucoside B; The mixture of 2170mg centella asiatica glucoside A and centella asiatica glucoside B will be got after cut 3 vacuum concentration, the vacuum-drying; With the mixture of gained with the 8mL volumetric concentration be 20% acetonitrile-water mixed solvent dissolving, after filtering filtrate; Internal diameter is 2cm, highly is that the chromatography column of 20cm (filling C18 silica gel amount is 35g for aspect ratio 10, bed volume 63mL) adopts wet method dress post, is that 20% acetonitrile-water mixed solvent washes pillar with volumetric concentration; Get sample on the 6.4mL filtrate (applied sample amount is mixture/1g C18 silica gel of 0.05g centella asiatica glucoside A and centella asiatica glucoside B), volumetric concentration is 20% acetonitrile-water mixed solvent with 3.1mL/min (the flow velocity wash-out of 3 bed volume/h); Elutriant is merged into three cuts after HPLC analyzes, cut 4 is a centella asiatica glucoside B solution, cut 5 is the mixing solutions of centella asiatica glucoside B and centella asiatica glucoside A, cut 6 is a centella asiatica glucoside A solution, cut 4 gets centella asiatica glucoside B395mg respectively after vacuum concentration, crystallization, vacuum-drying, purity is 89.5%, and cut 6 gets centella asiatica glucoside A676mg respectively after vacuum concentration, crystallization, vacuum-drying, and purity is 91.0%; C18 regeneration back is reusable.
Embodiment 13
Get the 6g asiatic centella total saponins and add in the flask, add the 100mL dissolve with methanol, add 75g silica gel (mixing the sample ratio is 0.08) again, stir, must mix sample silica gel after the evaporated in vacuo with rotatory evaporator; Add silica gel in internal diameter is the chromatography column of 5.5cm, the silica gel height after dress is real is 27.5cm, then adds and mixes sample silica gel, and the sample silica gel height of mixing after dress is real is 3.9cm (aspect ratio is 5, and silica gel is 7 with mixing sample silica gel aspect ratio, and total bed volume is 746ml); With chloroform-methanol-water (volume ratio is 35: 13: 2) ternary mixed solvent is moving phase, with 24.9mL/min (the flow velocity wash-out of 2 bed volume/h); Elutriant is merged into three cuts after TLC analyzes, cut 1 is a centella asiatica glucoside solution, and cut 2 is the mixing solutions of centella asiatica glucoside, centella asiatica glucoside A, centella asiatica glucoside B, and cut 3 is the mixing solutions of centella asiatica glucoside A and centella asiatica glucoside B; The mixture of 1528mg centella asiatica glucoside A and centella asiatica glucoside B will be got after cut 3 vacuum concentration, the vacuum-drying; With the mixture of gained with the 3mL volumetric concentration be 20% acetonitrile-water mixed solvent dissolving, after filtering filtrate; Internal diameter is 2cm, highly is that the chromatography column of 12cm (filling C18 silica gel amount is 21g for aspect ratio 6, bed volume 38mL) adopts wet method dress post, is that 20% acetonitrile-water mixed solvent washes pillar with volumetric concentration; Get sample on the 2.5mL filtrate (applied sample amount is mixture/1g C18 silica gel of 0.06g centella asiatica glucoside A and centella asiatica glucoside B), volumetric concentration is 20% acetonitrile-water mixed solvent with 1.3mL/min (the flow velocity wash-out of 2 bed volume/h); Elutriant is merged into three cuts after HPLC analyzes, cut 4 is a centella asiatica glucoside B solution, cut 5 is the mixing solutions of centella asiatica glucoside B and centella asiatica glucoside A, cut 6 is a centella asiatica glucoside A solution, cut 4 gets centella asiatica glucoside B318mg respectively after vacuum concentration, crystallization, vacuum-drying, purity is 94.6%, and cut 6 gets centella asiatica glucoside A545mg respectively after vacuum concentration, crystallization, vacuum-drying, and purity is 96.3%; C18 regeneration back is reusable.
Embodiment 14
Get the 8g asiatic centella total saponins and add in the flask, add the 100mL dissolve with methanol, add 80g silica gel (mixing the sample ratio is 0.1) again, stir, must mix sample silica gel after the evaporated in vacuo with rotatory evaporator; Add silica gel in internal diameter is the chromatography column of 5.5cm, the silica gel height after dress is real is 27.5cm, then adds and mixes sample silica gel, and the sample silica gel height of mixing after dress is real is 3.9cm (aspect ratio is 5, and silica gel is 7 with mixing sample silica gel aspect ratio, and total bed volume is 746ml); With ethyl acetate, alcohol and water (volume ratio is 65: 29: 6) ternary mixed solvent is moving phase, with 18.7mL/min (the flow velocity wash-out of 1.5 bed volume/h); Elutriant is merged into three cuts after TLC analyzes, cut 1 is a centella asiatica glucoside solution, and cut 2 is the mixing solutions of centella asiatica glucoside, centella asiatica glucoside A, centella asiatica glucoside B, and cut 3 is the mixing solutions of centella asiatica glucoside A and centella asiatica glucoside B; The mixture of 1871mg centella asiatica glucoside A and centella asiatica glucoside B will be got after cut 3 vacuum concentration, the vacuum-drying; With the mixture of gained with the 4mL volumetric concentration be 20% acetonitrile-water mixed solvent dissolving, after filtering filtrate; Internal diameter is 2cm, highly is that the chromatography column of 12cm (filling C18 silica gel amount is 21g for aspect ratio 6, bed volume 38mL) adopts wet method dress post, is that 20% acetonitrile-water mixed solvent washes pillar with volumetric concentration; Get sample on the 3.1mL filtrate (applied sample amount is mixture/1gC18 silica gel of 0.07g centella asiatica glucoside A and centella asiatica glucoside B), volumetric concentration is 20% acetonitrile-water mixed solvent with 0.9mL/min (the flow velocity wash-out of 1.5 bed volume/h); Elutriant is merged into three cuts after HPLC analyzes, cut 4 is a centella asiatica glucoside B solution, cut 5 is the mixing solutions of centella asiatica glucoside B and centella asiatica glucoside A, cut 6 is a centella asiatica glucoside A solution, cut 4 gets centella asiatica glucoside B347mg respectively after vacuum concentration, crystallization, vacuum-drying, purity is 91.6%, and cut 6 gets centella asiatica glucoside A595mg respectively after vacuum concentration, crystallization, vacuum-drying, and purity is 93.2%; C18 regeneration back is reusable.
Embodiment 15
Get the 12g asiatic centella total saponins and add in the flask, add the 100mL dissolve with methanol, add 100g silica gel (mixing the sample ratio is 0.12) again, stir, must mix sample silica gel after the evaporated in vacuo with rotatory evaporator; Add silica gel in internal diameter is the chromatography column of 5.5cm, the silica gel height after dress is real is 33cm, then adds and mixes sample silica gel, and the sample silica gel height of mixing after dress is real is 5.5cm (aspect ratio is 6, and silica gel is 6 with mixing sample silica gel aspect ratio, and total bed volume is 914ml); With ethyl acetate, alcohol and water (volume ratio is 65: 29: 6) ternary mixed solvent is moving phase, with 22.9mL/min (the flow velocity wash-out of 1.5 bed volume/h); Elutriant is merged into three cuts after TLC analyzes, cut 1 is a centella asiatica glucoside solution, and cut 2 is the mixing solutions of centella asiatica glucoside, centella asiatica glucoside A, centella asiatica glucoside B, and cut 3 is the mixing solutions of centella asiatica glucoside A and centella asiatica glucoside B; The mixture of 3243mg centella asiatica glucoside A and centella asiatica glucoside B will be got after cut 3 vacuum concentration, the vacuum-drying; With the mixture of gained with the 4mL volumetric concentration be 25% acetonitrile-water mixed solvent dissolving, after filtering filtrate; Internal diameter is 2cm, highly is that the chromatography column of 12cm (filling C18 silica gel amount is 21g for aspect ratio 6, bed volume 38mL) adopts wet method dress post, is that 25% acetonitrile-water mixed solvent washes pillar with volumetric concentration; Get sample on the 2.1mL filtrate (applied sample amount is mixture/1gC18 silica gel of 0.08g centella asiatica glucoside A and centella asiatica glucoside B), volumetric concentration is 25% acetonitrile-water mixed solvent with 1.3mL/min (the flow velocity wash-out of 2 bed volume/h); Elutriant is merged into three cuts after HPLC analyzes, cut 4 is a centella asiatica glucoside B solution, cut 5 is the mixing solutions of centella asiatica glucoside B and centella asiatica glucoside A, cut 6 is a centella asiatica glucoside A solution, cut 4 gets centella asiatica glucoside B365mg respectively after vacuum concentration, crystallization, vacuum-drying, purity is 87.8%, and cut 6 gets centella asiatica glucoside A626mg respectively after vacuum concentration, crystallization, vacuum-drying, and purity is 89.4%; C18 regeneration back is reusable.
Embodiment 16
Get the 12g asiatic centella total saponins and add in the flask, add the 100mL dissolve with methanol, add 100g silica gel (mixing the sample ratio is 0.12) again, stir, must mix sample silica gel after the evaporated in vacuo with rotatory evaporator; Add silica gel in internal diameter is the chromatography column of 5.5cm, the silica gel height after dress is real is 33cm, then adds and mixes sample silica gel, and the sample silica gel height of mixing after dress is real is 5.5cm (aspect ratio is 6, and silica gel is 6 with mixing sample silica gel aspect ratio, and total bed volume is 914ml); With chloroform-methanol-water (volume ratio is 35: 13: 2) ternary mixed solvent is moving phase, with 30.5mL/min (the flow velocity wash-out of 2 bed volume/h); Elutriant is merged into three cuts after TLC analyzes, cut 1 is a centella asiatica glucoside solution, and cut 2 is the mixing solutions of centella asiatica glucoside, centella asiatica glucoside A, centella asiatica glucoside B, and cut 3 is the mixing solutions of centella asiatica glucoside A and centella asiatica glucoside B; The mixture of 3109mg centella asiatica glucoside A and centella asiatica glucoside B will be got after cut 3 vacuum concentration, the vacuum-drying; With the mixture of gained with the 4mL volumetric concentration be 25% acetonitrile-water mixed solvent dissolving, after filtering filtrate; Internal diameter is 2cm, highly is that the chromatography column of 16cm (filling C18 silica gel amount is 28g for aspect ratio 8, bed volume 50mL) adopts wet method dress post, is that 25% acetonitrile-water mixed solvent washes pillar with volumetric concentration; Get sample on the 3.2mL filtrate (applied sample amount is mixture/1g C18 silica gel of 0.09g centella asiatica glucoside A and centella asiatica glucoside B), volumetric concentration is 25% acetonitrile-water mixed solvent with 1.3mL/min (the flow velocity wash-out of 1.5 bed volume/h); Elutriant is merged into three cuts after HPLC analyzes, cut 4 is a centella asiatica glucoside B solution, cut 5 is the mixing solutions of centella asiatica glucoside B and centella asiatica glucoside A, cut 6 is a centella asiatica glucoside A solution, cut 4 gets centella asiatica glucoside B521mg respectively after vacuum concentration, crystallization, vacuum-drying, purity is 85.6%, and cut 6 gets centella asiatica glucoside A893mg respectively after vacuum concentration, crystallization, vacuum-drying, and purity is 87.1%; C18 regeneration back is reusable.
Embodiment 17
Get the 10g asiatic centella total saponins and add in the flask, add the 100mL dissolve with methanol, add 77g silica gel (mixing the sample ratio is 0.13) again, stir, must mix sample silica gel after the evaporated in vacuo with rotatory evaporator; Add silica gel in internal diameter is the chromatography column of 4.5cm, the silica gel height after dress is real is 31.5cm, then adds and mixes sample silica gel, and the sample silica gel height of mixing after dress is real is 6.3cm (aspect ratio is 7, and silica gel is 5 with mixing sample silica gel aspect ratio, and total bed volume is 601m1); With chloroform-methanol-water (volume ratio is 35: 13: 2) ternary mixed solvent is moving phase, with 20.0mL/min (the flow velocity wash-out of 2 bed volume/h); Elutriant is merged into three cuts after TLC analyzes, cut 1 is a centella asiatica glucoside solution, and cut 2 is the mixing solutions of centella asiatica glucoside, centella asiatica glucoside A, centella asiatica glucoside B, and cut 3 is the mixing solutions of centella asiatica glucoside A and centella asiatica glucoside B; The mixture of 2722mg centella asiatica glucoside A and centella asiatica glucoside B will be got after cut 3 vacuum concentration, the vacuum-drying; With the mixture of gained with the 6mL volumetric concentration be 20% acetonitrile-water mixed solvent dissolving, after filtering filtrate; Internal diameter is 2cm, highly is that the chromatography column of 12cm (filling C18 silica gel amount is 21g for aspect ratio 6, bed volume 38mL) adopts wet method dress post, is that 20% acetonitrile-water mixed solvent washes pillar with volumetric concentration; Get sample on the 4.6mL filtrate (applied sample amount is mixture/1g C18 silica gel of 0.1g centella asiatica glucoside A and centella asiatica glucoside B), volumetric concentration is 20% acetonitrile-water mixed solvent with 1.3mL/min (the flow velocity wash-out of 2 bed volume/h); Elutriant is merged into three cuts after HPLC analyzes, cut 4 is a centella asiatica glucoside B solution, cut 5 is the mixing solutions of centella asiatica glucoside B and centella asiatica glucoside A, cut 6 is a centella asiatica glucoside A solution, cut 4 gets centella asiatica glucoside B417mg respectively after vacuum concentration, crystallization, vacuum-drying, purity is 84.0%, and cut 6 gets centella asiatica glucoside A715mg respectively after vacuum concentration, crystallization, vacuum-drying, and purity is 85.4%; C18 regeneration back is reusable.
Embodiment 18
Get the 8g asiatic centella total saponins and add in the flask, add the 100mL dissolve with methanol, add 53g silica gel (mixing the sample ratio is 0.15) again, stir, must mix sample silica gel after the evaporated in vacuo with rotatory evaporator; Add silica gel in internal diameter is the chromatography column of 4cm, the silica gel height after dress is real is 28cm, then adds and mixes sample silica gel, and the sample silica gel height of mixing after dress is real is 5.6cm (aspect ratio is 7, and silica gel is 5 with mixing sample silica gel aspect ratio, and total bed volume is 422ml); With ethyl acetate-methanol-water (volume ratio is 15: 8: 2) ternary mixed solvent is moving phase, with 10.6mL/min (the flow velocity wash-out of 1.5 bed volume/h); Elutriant is merged into three cuts after TLC analyzes, cut 1 is a centella asiatica glucoside solution, and cut 2 is the mixing solutions of centella asiatica glucoside, centella asiatica glucoside A, centella asiatica glucoside B, and cut 3 is the mixing solutions of centella asiatica glucoside A and centella asiatica glucoside B; The mixture of 2071mg centella asiatica glucoside A and centella asiatica glucoside B will be got after cut 3 vacuum concentration, the vacuum-drying; With the mixture of gained with the 8mL volumetric concentration be 25% acetonitrile-water mixed solvent dissolving, after filtering filtrate; Internal diameter is 2cm, highly is that the chromatography column of 16cm (filling C18 silica gel amount is 28g for aspect ratio 8, bed volume 50mL) adopts wet method dress post, is that 25% acetonitrile-water mixed solvent washes pillar with volumetric concentration; Get sample on the 6.5mL filtrate (applied sample amount is mixture/1g C18 silica gel of 0.06g centella asiatica glucoside A and centella asiatica glucoside B), volumetric concentration is 25% acetonitrile-water mixed solvent with 1.7mL/min (the flow velocity wash-out of 2 bed volume/h); Elutriant is merged into three cuts after HPLC analyzes, cut 4 is a centella asiatica glucoside B solution, cut 5 is the mixing solutions of centella asiatica glucoside B and centella asiatica glucoside A, cut 6 is a centella asiatica glucoside A solution, cut 4 gets centella asiatica glucoside B410mg respectively after vacuum concentration, crystallization, vacuum-drying, purity is 93.1%, and cut 6 gets centella asiatica glucoside A703mg respectively after vacuum concentration, crystallization, vacuum-drying, and purity is 94.7%; C18 regeneration back is reusable.
Claims (10)
1. one kind prepares the method for centella asiatica glucoside A and centella asiatica glucoside B simultaneously from asiatic centella total saponins, and the step of method is as follows:
1) asiatic centella total saponins adds the silica gel mixing and stirring after with dissolve with methanol, must mix sample silica gel behind the evaporate to dryness, and the mass ratio of asiatic centella total saponins and silica gel is 0.05～0.2: 1;
2) add silica gel in the chromatography column in regular turn and mix sample silica gel, adopt dry column-packing, the aspect ratio of dry method chromatography column is 3～9, and silica gel is 3～8 with the aspect ratio of mixing sample silica gel;
3) be moving phase with organic solvent I-organic solvent II-water ternary mixed solvent, with the flow velocity wash-out of 0.5～3 bed volume/h;
4) elutriant is merged into three cuts after TLC analyzes, and cut 1 is a centella asiatica glucoside solution, and cut 2 is the mixing solutions of centella asiatica glucoside, centella asiatica glucoside A, centella asiatica glucoside B, and cut 3 is the mixing solutions of centella asiatica glucoside A and centella asiatica glucoside B;
5) after using the dissolving of acetonitrile-water mixed solvent, filtration after cut 3 vacuum concentration, the vacuum-drying, get filtrate;
6) C18 silica gel adopts wet method dress post, and the aspect ratio of pillar is 4～12, washes pillar mutually with the acetonitrile-water mixed solvent;
7) filtrate is in sample on the ratio of mixture/1g C18 silica gel of 0.01～0.1g centella asiatica glucoside A and centella asiatica glucoside B, and the acetonitrile-water mixed solvent is with the flow velocity wash-out of 0.5～3 bed volume/h;
8) elutriant is merged into three cuts after HPLC analyzes, cut 4 is a centella asiatica glucoside B solution, cut 5 is the mixing solutions of centella asiatica glucoside B and centella asiatica glucoside A, cut 6 is a centella asiatica glucoside A solution, and cut 4 gets centella asiatica glucoside B and centella asiatica glucoside A product respectively with cut 6 after vacuum concentration, crystallization, vacuum-drying;
9) the C18 post can be gone up sample once more after acetonitrile-water mixed solvent flushing regeneration.
2. according to claim 1ly a kind ofly prepare the method for centella asiatica glucoside A and centella asiatica glucoside B simultaneously, it is characterized in that in the described step 1) that ratio with 0.08～0.15g asiatic centella total saponins/1g silica gel adds silica gel and stirs from asiatic centella total saponins.
3. according to claim 1ly a kind ofly prepare the method for centella asiatica glucoside A and centella asiatica glucoside B simultaneously, it is characterized in that organic solvent I is chloroform or methylene dichloride or ethyl acetate in the described step 3) from asiatic centella total saponins.
4. according to claim 1ly a kind ofly prepare the method for centella asiatica glucoside A and centella asiatica glucoside B simultaneously, it is characterized in that organic solvent II is methyl alcohol or ethanol in the described step 3) from asiatic centella total saponins.
5. according to claim 1ly a kind ofly prepare the method for centella asiatica glucoside A and centella asiatica glucoside B simultaneously from asiatic centella total saponins, the volumetric concentration that it is characterized in that organic solvent I in described step 3) organic solvent I-organic solvent II-water ternary mixed solvent is 60～80%.
6. according to claim 1ly a kind ofly prepare the method for centella asiatica glucoside A and centella asiatica glucoside B simultaneously from asiatic centella total saponins, the volumetric concentration that it is characterized in that organic solvent II in described step 3) organic solvent I-organic solvent II-water ternary mixed solvent is 18～38%.
7. according to claim 1ly a kind ofly prepare the method for centella asiatica glucoside A and centella asiatica glucoside B simultaneously from asiatic centella total saponins, the volumetric concentration that it is characterized in that water in described step 3) organic solvent I-organic solvent II-water ternary mixed solvent is 2～10%.
8. according to claim 1ly a kind ofly prepare the method for centella asiatica glucoside A and centella asiatica glucoside B simultaneously, it is characterized in that in described step 5), step 6), the step 7) that the volumetric concentration of acetonitrile is 10～30% in the acetonitrile-water mixed solvent from asiatic centella total saponins.
9. according to claim 1ly a kind ofly prepare the method for centella asiatica glucoside A and centella asiatica glucoside B simultaneously, it is characterized in that filtrate is in sample on the ratio of mixture/1g C18 silica gel of 0.02～0.06g centella asiatica glucoside A and centella asiatica glucoside B in the described step 7) from asiatic centella total saponins.
10. according to claim 1ly a kind ofly prepare the method for centella asiatica glucoside A and centella asiatica glucoside B simultaneously, it is characterized in that C18 column regeneration process in the described step 9) is for being that 80～95% acetonitrile-water and 10～30% acetonitrile-water mixed solvent wash with volumetric concentration successively from asiatic centella total saponins.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101463064B (en) * | 2009-01-15 | 2011-03-23 | 浙江大学 | Method for preparing asiatic acid, madecassic acid and domino asiatic acid at the same time from Centella asiatica total aglycone |
| CN101429225B (en) * | 2008-12-18 | 2011-03-23 | 浙江大学 | Method for producing asiatic acid with asiaticoside acid hydrolysis |
| CN101724006B (en) * | 2009-12-22 | 2012-05-23 | 浙江大学 | Method for separating asiaticoside-B, hydroxyl asiaticoside and asiaticoside |
| CN102477063A (en) * | 2010-11-24 | 2012-05-30 | 上海医药工业研究院 | Method for preparing high-purity madecassoside and asiaticoside B |
| CN109879926A (en) * | 2019-02-27 | 2019-06-14 | 江西省药品检验检测研究院 | Triterpene glycosides compound and its extraction separation method in Longtube Ground Ivy Herb |
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2007
- 2007-12-05 CN CN200710164474XA patent/CN101182346B/en not_active Expired - Fee Related
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101429225B (en) * | 2008-12-18 | 2011-03-23 | 浙江大学 | Method for producing asiatic acid with asiaticoside acid hydrolysis |
| CN101463064B (en) * | 2009-01-15 | 2011-03-23 | 浙江大学 | Method for preparing asiatic acid, madecassic acid and domino asiatic acid at the same time from Centella asiatica total aglycone |
| CN101724006B (en) * | 2009-12-22 | 2012-05-23 | 浙江大学 | Method for separating asiaticoside-B, hydroxyl asiaticoside and asiaticoside |
| CN102477063A (en) * | 2010-11-24 | 2012-05-30 | 上海医药工业研究院 | Method for preparing high-purity madecassoside and asiaticoside B |
| CN109879926A (en) * | 2019-02-27 | 2019-06-14 | 江西省药品检验检测研究院 | Triterpene glycosides compound and its extraction separation method in Longtube Ground Ivy Herb |
| CN109879926B (en) * | 2019-02-27 | 2021-07-23 | 江西省药品检验检测研究院 | Triterpene glycoside compounds in Glechomae herba and extraction and separation method thereof |
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