CN104610410A - Extraction process of ginseng saponin Rd - Google Patents
Extraction process of ginseng saponin Rd Download PDFInfo
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Abstract
The invention discloses an extraction process of ginseng saponin Rd. The extraction process comprises the following steps: S1, grinding ginseng saponin Rd-containing herbal materials into herbal powder, performing reflux extraction by using 70-95% ethanol, recycling the ethanol, and concentrating to obtain a total extract; S2, taking the total extract, enabling the total extract to pass through D101 macroporous adsorption resin, eluting by using 40-80% ethanol, recycling the ethanol, and performing deconperssion concentration to obtain total saponin; S3, taking the total saponin, enabling the total saponin to pass through a silica gel chromatography column, performing isocratic elution by using a mixture of n-butanol, ethyl acetate and water, performing thin layer chromatography, combining fractions to obtain crude ginseng saponin Rd; S4, taking the crude ginseng saponin Rd, enabling the crude ginseng saponin Rd to pass through an Rp-C18 silica gel column, performing isocratic elution by using a mixture of methanol and water, performing thin layer chromatography, combining required components, separating out precipitate to obtain the ginseng saponin Rd. By the extraction process, the ginseng saponin Rd separating and purifying effects can be improved, use of a toxic organic reagent is avoided, the operation is simple and convenient and a production process is easy to control; therefore, the extraction process is suitable for industrial production.
Description
Technical field
The present invention relates to the extraction process of a kind of Ginsenoside Rd, belong to ginsenoside extractive technique field.
Background technology
Ginsenoside (Ginsenoside) is a kind of steroid compound, and triterpenoid saponin is mainly present in Panax medicinal material.Ginsenoside is considered to be the activeconstituents in ginseng, thus becomes the target of research, and isolation identification goes out more than 40 and plants ginsenoside monomer at present.Because ginsenoside have impact on multiple metabolic pathway, so its usefulness is also complicated, and the usefulness of various ginsenoside is difficult to separate.And bioactivity research is carried out to monomer be easy to illustrate its definite pharmacological action, therefore the research of ginsenoside monomer compound is got more and more.Rg in ginsenoside monomer compound
1, Rb
1, Rc equivalent is higher, and Rd, Rg
3, Rh
2etc. belonging to rare saponin(e, but pharmacologically active is better than main ginsenoside.Wherein, Ginsenoside Rd is one of principal mode absorbed in enteron aisle after main saponin(e metabolism, all has good pharmacological action to neural system, cardiovascular and cerebrovascular and renal function.
The technique of existing separation and Extraction Ginsenoside Rd mainly adopts method as shown in Figures 2 and 3, eluent adopts chloroform-methanol-water system, leaching process introduces toxic reagent chloroform, and adopt gradient elution, make separation purity not high, operate loaded down with trivial details, production process is wayward, is not suitable for suitability for industrialized production.
Summary of the invention
The object of the invention is to, provide the extraction process of a kind of Ginsenoside Rd, can improve the separation and purification effect of Ginsenoside Rd, avoid the use of toxicity organic reagent, easy and simple to handle, production process is easy to control, and suitability for industrialized is produced.
For solving the problems of the technologies described above, the present invention adopts following technical scheme: the extraction process of a kind of Ginsenoside Rd, comprises the following steps:
S1, becomes medicinal powder by the pulverizing medicinal materials containing Ginsenoside Rd's composition, adopts 70% ~ 95% alcohol reflux, and filter, filtrate recycling ethanol is also concentrated into the medicinal extract that relative density is 1.15 ~ 1.26, obtains general extractive;
S2, gets general extractive, adds 2 times amount purified water, stirs evenly, upper D101 macroporous adsorptive resins, first uses purified water wash-out, continues with 40% ~ 80% ethanol elution, collect ethanol eluate, reclaim ethanol and be evaporated to the clear cream that relative density is 1.10 ~ 1.20, obtaining total saponins;
S3, get total saponins, be adsorbed on 2 times amount silica gel, evaporate into dry, sieve, added in silica gel column chromatography, with propyl carbinol-ethyl acetate-water=2 ﹕ 3 ﹕ 5 isocratic elution, Fractional Collections, merge Ginsenoside Rd through thin-layer chromatography inspection and flow part, decompression and solvent recovery, to dry, obtains Ginsenoside Rd's crude product;
S4, get Ginsenoside Rd's crude product, adds purified water with dissolve with methanol, stir evenly, upper Rp-C18 reversed phase chromatography silicagel column, with methanol-water=75 ﹕ 25 isocratic elution, Fractional Collections, required component is merged after thin-layer chromatography checks, decompression and solvent recovery, leaves standstill, and separates out precipitation, upper liquid returns reversed phase chromatography silicagel column and is separated, and precipitates dry Ginsenoside Rd.
In the extraction process of aforesaid Ginsenoside Rd, described step S1 is specially: the pulverizing medicinal materials containing Ginsenoside Rd's composition is become 40 ~ 60 object medicinal powder, adds 2 times amount 85% ethanol, cold soaking 15 ~ 18h, filter, the dregs of a decoction add 1.8 times amount 85% ethanol, refluxing extraction 3 times, each 2 hours, filter, merging filtrate, reclaims ethanol and is concentrated into the medicinal extract that relative density is 1.24 ~ 1.26, obtaining general extractive.
In the extraction process of aforesaid Ginsenoside Rd, described step S2 comprises:
S21, dress post: with the fully moistening D101 macroporous adsorbent resin of anticipating of ethanol, stirs and gets rid of bubble, dress post, soaks 12h, and adding water after mixing with 95% alcohol flushing to effluent liquid is not muddiness, continue with purified water displacement ethanol wherein to without alcohol taste, for subsequent use;
S22, loading: get general extractive, adds 2 times amount purified water, stirs evenly, and slowly adds in the D101 macroporous adsorptive resins of anticipating, and makes all to enter post bed, standing adsorption 12h;
S23, wash-out: first use 5L/kg
silica gelpurified water wash-out, water elution liquid discards; Continue with 60% ethanol elution being no less than purified water consumption, collect 60% ethanol eluate, reclaim ethanol and be evaporated to the clear cream that relative density is 1.10 ~ 1.20, obtaining total saponins.
In the extraction process of aforesaid Ginsenoside Rd, described step S3 comprises:
S31, upper prop sample preparation: get total saponins, is adsorbed on the silica gel of 2 times amount, evaporates into dry, crosses 60 mesh sieves, for subsequent use;
S32, dress post: the column chromatography silica gel getting total saponins 10 times amount, with the upper solution wet method dress post of propyl carbinol-ethyl acetate-water=2 ﹕ 3 ﹕ 5, repeatedly uses eluent, makes silica gel fully sink closely;
S33, loading and wash-out: in silica gel column chromatography, reserved certain elutriant makes it flood silica gel, is slowly added in chromatography column by upper prop sample, with the upper solution isocratic elution of propyl carbinol-ethyl acetate-water=2 ﹕ 3 ﹕ 5, Fractional Collections, often flowing part collecting amount is 1.5L/kg
silica gel, after thin-layer chromatography inspection, merge Ginsenoside Rd flow part, 65 ~ 70 DEG C of decompression and solvent recoveries, to dry, obtain Ginsenoside Rd's crude product.
In the extraction process of aforesaid Ginsenoside Rd, described step S4 comprises:
S41, upper prop sample preparation: get Ginsenoside Rd's crude product, after the ratio dissolve with methanol in 2.5mL methyl alcohol/g Ginsenoside Rd crude product, adds the purified water of methyl alcohol volume 1/3, stirs evenly, for subsequent use;
S42, dress post: take reversed phase chromatography silica gel Rp-C18 in the ratio of 1kg silica gel/30g Ginsenoside Rd crude product, fill post with methanol wet;
S43, loading and wash-out: by upper prop sample wet method upper prop, with methanol-water=75 ﹕ 25 isocratic elution, Fractional Collections, often flowing part collecting amount is 0.5L/kg
silica gel, after thin-layer chromatography checks, merge required component, 75 ~ 80 DEG C of decompression and solvent recoveries are to 1.5 ~ 2 times of upper prop sample volume, and leave standstill 15 ~ 18h, separate out precipitation, upper liquid returns reversed phase chromatography silicagel column and is separated, and is deposited in 40 DEG C of vacuum-dryings, obtains Ginsenoside Rd.
In the extraction process of aforesaid Ginsenoside Rd, described thin-layer chromatography inspection is specially: often flow part and get 10mL respectively, evaporate to dryness, residue adds 1mL dissolve with methanol, as need testing solution; Separately get appropriate Ginsenoside Rd's reference substance, add methyl alcohol and make the solution of every 1mL containing 2mg, product solution in contrast; Tlc according to " Chinese Pharmacopoeia " version in 2010 annex VI B is tested, draw described need testing solution 5 ~ 10 μ L and reference substance solution 5 μ L, put respectively on same gel GF 254 plate, using Zheng Ding Chun – Yi Suan Yi Zhi – water=4: the upper solution of 3: 5 is as developping agent, launch, take out, dry, spray, with 10% vitriol oil-ethanolic soln, is heated to spot development in 105 DEG C clear.
In the extraction process of aforesaid Ginsenoside Rd, the described medicinal material containing Ginsenoside Rd's composition is pseudo-ginseng, ginseng or Radix Panacis Quinquefolii.
For solving the problem existing for prior art, applicant has carried out perfect research to Ginsenoside Rd's extraction process, specific as follows:
1, the shaker test of concentration densities
Medicinal material 20Kg containing Ginsenoside Rd's composition is ground into medicinal powder, adopt 70% ~ 95% alcohol reflux, filter, filtrate recycling ethanol is also concentrated into the medicinal extract that relative density I is respectively 1.08,1.10,1.12,1.15,1.18,1.20,1.23,1.26,1.30, obtains general extractive; Get general extractive, add 2 times amount purified water, stir evenly, upper D101 macroporous adsorptive resins, first use purified water wash-out, continue and use 60% ethanol elution, collect ethanol eluate, reclaim ethanol and be evaporated to the clear cream that relative density II is respectively 1.03,1.06,1.08,1.10,1.12,1.15,1.18,1.20, obtaining total saponins.
Table 1
As seen from the above table, medicinal material is adopted 70% ~ 95% alcohol reflux, filter, filtrate recycling ethanol to be concentrated into relative density I be the medicinal extract of 1.15 ~ 1.26, obtain general extractive; Get general extractive, add 2 times amount purified water, stir evenly, upper D101 macroporous adsorptive resins, first uses purified water wash-out, continues and uses 60% ethanol elution, collect ethanol eluate, reclaim ethanol and be also evaporated to the clear cream that relative density II is 1.10 ~ 1.20, the effect obtaining total saponins is better, especially when relative density I be 1.24 ~ 1.26, relative density II be 1.10 ~ 1.20 time for best.
2, normal phase silicagel column separation condition is perfect
Technical study data shows, and to the separation of Ginsenoside Rd's normal phase silicagel column, what moving phase adopted has: A. chloroform-methanol-water; B. chloroform-methanol-vinyl acetic monomer-water; C. n-Butanol acetic acid ethyl ester-water; D. vinyl acetic monomer-methyl alcohol equal solvent system.Wherein, during with propyl carbinol-ethyl acetate-water (4: 1: 5) for developping agent, better, because the ratio of propyl carbinol is large, difficult volatilization, concentrates difficulty slightly greatly, therefore is not used Ginsenoside Rd's separating effect relative to other solvent systemss.
But consider the safeguard protection of the operability of suitability for industrialized production, environment and personnel, finally still select hypotoxic propyl carbinol-ethyl acetate-water system to make elutriant, carry out isocratic elution.For solvent recovery problem, solve by the amount ratio reducing propyl carbinol in moving phase.
For screening suitable elutriant to be separated the Ginsenoside Rd containing impurity, the present invention gets ginsenoside Rg respectively
1, ginsenoside Re, Ginsenoside Rd, ginsenoside Rb
1, Panax Notoginseng saponin R
1reference substance is appropriate, adds methyl alcohol respectively and makes every 1mL respectively containing the solution of 2mg.Test according to tlc (" Chinese Pharmacopoeia " version in 2010 annex VI B), draw above-mentioned solution 5 μ L respectively, point is on same silica gel g thin-layer plate, respectively with developping agent 1: propyl carbinol-ethyl acetate-water (4: 3: 5, V/V) upper liquid is developping agent, developping agent 2: propyl carbinol-ethyl acetate-water (2: 3: 5, V/V) upper liquid is developping agent, developping agent 3: chloroform-methanol-water (6.5: 3.5: 1, V/V) subnatant is developping agent, launch, take out, dry, spray is with 10% vitriol oil-ethanolic soln, spot development is heated to clear in 105 DEG C, the results are shown in Table 2.
Table 2
As shown in Table 1, with above-mentioned three kinds of developping agents for elutriant carries out silica gel column chromatography test, three kinds of elutriant consumption difference are little as a result.The Rd impurity that developping agent 1 and 3 obtains is more; The obtained Rd impurity of developping agent 2 is less, is more conducive to next step and is separated, therefore select developping agent 2 namely: the upper liquid of propyl carbinol-ethyl acetate-water (2: 3: 5) is as the elutriant of silica gel column chromatography.
Test-results shows, be that elutriant carries out isocratic elution with the upper liquid of propyl carbinol-ethyl acetate-water (2: 3: 5), Ginsenoside Rd's separating effect is best, easy control easy and simple to handle.
3, Rp-C18 post separation condition is perfect
According to the condition of this product HPLC assay, respectively with methanol-water (55:45), methanol-water (65:35), methanol-water (75:25), methanol-water (85:15) is moving phase isocratic elution, gradient elution is carried out with chloroform-methanol-water (83:16:1) → chloroform-methanol-water (76:22:2) → chloroform-methanol-water (68.5:27.5:4) → chloroform-methanol-water (65:30:5) → chloroform-methanol-water (61:33:6) → chloroform-methanol-water (54:38.5:7.5) → methyl alcohol, test-results shows, when adopting methanol-water (75:25) for moving phase, Ginsenoside Rd and other impurity peaks resolution better, therefore, select methanol-water (75:25) isocratic elution.
Below by test, extraction process is verified, specific as follows:
1, major equipment and solvent (see table 3)
Table 3
Except methyl alcohol is chromatographically pure, all the other reagent are analytical pure.
2, inspection after construction
The Ginsenoside Rd's sample (20081028-1 ~ 20081028-5) obtained by extraction process of the present invention is tested, the results detailed in Table 4.
Table 4
Compared with prior art, eluent chloroform-methanol-water used for purification on normal-phase silica gel chromatographic step in existing extraction process is changed into propyl carbinol-ethyl acetate-water by the present invention, and gradient elution is changed into isocratic elution, also carry out perfect to purification step, the display of trial production result, the technique after improvement and existing technics comparing have the following advantages:
(1) change gradient elution into isocratic elution, production process is more easily controlled, suitability for industrialized is produced;
(2) organic solvent using this kind of toxicity of chloroform large is avoided in technological process;
(3) the Ginsenoside Rd's content produced as a trial all reaches more than 95%, can improve the separation and purification effect of Ginsenoside Rd.
Accompanying drawing explanation
Fig. 1 is the process flow sheet of the embodiment of the present invention;
Fig. 2, Fig. 3 are the schemas of existing Ginsenoside Rd's extraction process.
Embodiment
The embodiment of the present invention 1: the extraction process of a kind of Ginsenoside Rd, as shown in Figure 1, comprises the following steps:
1, the medicinal material (such as pseudo-ginseng, ginseng, Radix Panacis Quinquefolii etc.) containing Ginsenoside Rd's composition is ground into 50 object medicinal powder, add 2 times amount 85% ethanol, cold soaking 16h, filter, the dregs of a decoction add 1.8 times amount 85% ethanol, refluxing extraction 3 times, each 2 hours, filter, merging filtrate, reclaim ethanol and be concentrated into the medicinal extract that relative density is 1.24, obtaining general extractive.
2, total saponins is extracted
(1) fill post: with the fully moistening D101 macroporous adsorbent resin of anticipating of ethanol, stir and get rid of bubble, dress post, soak 12h, to add water after mixing not in muddiness with 95% alcohol flushing to effluent liquid, continue with purified water displacement ethanol wherein to without alcohol taste, for subsequent use;
(2) loading: get general extractive, adds 2 times amount purified water, stirs evenly, and slowly adds in the D101 macroporous adsorptive resins of anticipating, and makes all to enter post bed, standing adsorption 12h;
(3) wash-out: first use 5L/kg
silica gelpurified water wash-out, water elution liquid discards; Continue with 60% ethanol elution being no less than purified water consumption, collect 60% ethanol eluate, reclaim ethanol and be evaporated to the clear cream that relative density is 1.20, obtaining total saponins, for subsequent use after macroporous resin column 5BV 95% ethanol elution.
3, Ginsenoside Rd's crude product is extracted
(1) upper prop sample preparation: get total saponins, is adsorbed on the silica gel of 2 times amount, evaporates into dry, crosses 60 mesh sieves, for subsequent use;
(2) fill post: the column chromatography silica gel getting total saponins 10 times amount, with the upper solution wet method dress post of propyl carbinol-ethyl acetate-water=2 ﹕ 3 ﹕ 5, repeatedly use eluent, make silica gel fully sink closely;
(3) loading and wash-out: in silica gel column chromatography, reserved a certain amount of elutriant makes it flood silica gel and (namely ensures that in silicagel column, liquid level is higher than silica gel top, separating effect is affected) to avoid occurring silica gel tomography because of post inner drying, upper prop sample is slowly added in chromatography column, with the upper solution isocratic elution of propyl carbinol-ethyl acetate-water=2 ﹕ 3 ﹕ 5, Fractional Collections, often flowing part collecting amount is 1.5L/kg
silica gel, after thin-layer chromatography inspection, merge Ginsenoside Rd flow part, 68 DEG C of decompression and solvent recoveries, to dry, obtain Ginsenoside Rd's crude product.
4, purified ginsenoside Rd
(1) upper prop sample preparation: get Ginsenoside Rd's crude product, after the ratio dissolve with methanol in 2.5mL methyl alcohol/g Ginsenoside Rd crude product, adds the purified water of methyl alcohol volume 1/3, stirs evenly, for subsequent use;
(2) post is filled: take reversed phase chromatography silica gel Rp-C18 in the ratio of 1kg silica gel/30g Ginsenoside Rd crude product, fill post with methanol wet;
(3) loading and wash-out: by upper prop sample wet method upper prop, with methanol-water=75 ﹕ 25 isocratic elution, Fractional Collections, often flowing part collecting amount is 0.5L/kg
silica gel, after thin-layer chromatography checks, merge required component, 78 DEG C of decompression and solvent recoveries are to 1.8 times of upper prop sample volume, leave standstill 16h, separate out precipitation, upper liquid returns reversed phase chromatography silicagel column and is separated, be deposited in 40 DEG C of vacuum-dryings, obtain Ginsenoside Rd, for subsequent use after reversed phase chromatography post 6L methanol-eluted fractions.
Described thin-layer chromatography inspection is specially: often flow part and get 10mL respectively, evaporate to dryness, residue adds 1mL dissolve with methanol, as need testing solution; Separately get appropriate Ginsenoside Rd's reference substance, add methyl alcohol and make the solution of every 1mL containing 2mg, product solution in contrast; Tlc according to " Chinese Pharmacopoeia " version in 2010 annex VI B is tested, draw described need testing solution 8 μ L and reference substance solution 5 μ L, put respectively on same gel GF 254 plate, using Zheng Ding Chun – Yi Suan Yi Zhi – water=4: the upper solution of 3: 5 is as developping agent, launch, take out, dry, spray, with 10% vitriol oil-ethanolic soln, is heated to spot development in 105 DEG C clear.
The embodiment of the present invention 2: the extraction process of a kind of Ginsenoside Rd, as shown in Figure 1, comprises the following steps:
1, the medicinal material (such as pseudo-ginseng, ginseng, Radix Panacis Quinquefolii etc.) containing Ginsenoside Rd's composition is ground into 40 object medicinal powder, add 2 times amount 70% ethanol, cold soaking 15h, filter, the dregs of a decoction add 1.8 times amount 70% ethanol, refluxing extraction 3 times, each 2 hours, filter, merging filtrate, reclaim ethanol and be concentrated into the medicinal extract that relative density is 1.15, obtaining general extractive.
2, total saponins is extracted
(1) fill post: with the fully moistening D101 macroporous adsorbent resin of anticipating of ethanol, stir and get rid of bubble, dress post, soak 12h, to add water after mixing not in muddiness with 95% alcohol flushing to effluent liquid, continue with purified water displacement ethanol wherein to without alcohol taste, for subsequent use;
(2) loading: get general extractive, adds 2 times amount purified water, stirs evenly, and slowly adds in the D101 macroporous adsorptive resins of anticipating, and makes all to enter post bed, standing adsorption 12h;
(3) wash-out: first use 5L/kg
silica gelpurified water wash-out, water elution liquid discards; Continue with 40% ethanol elution being no less than purified water consumption, collect 40% ethanol eluate, reclaim ethanol and be evaporated to the clear cream that relative density is 1.10, obtaining total saponins, for subsequent use after macroporous resin column 5BV 95% ethanol elution.
3, Ginsenoside Rd's crude product is extracted
(1) upper prop sample preparation: get total saponins, is adsorbed on the silica gel of 2 times amount, evaporates into dry, crosses 60 mesh sieves, for subsequent use;
(2) fill post: the column chromatography silica gel getting total saponins 10 times amount, with the upper solution wet method dress post of propyl carbinol-ethyl acetate-water=2 ﹕ 3 ﹕ 5, repeatedly use eluent, make silica gel fully sink closely;
(3) loading and wash-out: in silica gel column chromatography, reserved a certain amount of elutriant to make in silicagel column liquid level higher than silica gel top (affecting separating effect to avoid occurring silica gel tomography because of post inner drying), upper prop sample is slowly added in chromatography column, with the upper solution isocratic elution of propyl carbinol-ethyl acetate-water=2 ﹕ 3 ﹕ 5, Fractional Collections, often flowing part collecting amount is 1.5L/kg
silica gel, after thin-layer chromatography inspection, merge Ginsenoside Rd flow part, 65 DEG C of decompression and solvent recoveries, to dry, obtain Ginsenoside Rd's crude product.
4, purified ginsenoside Rd
(1) upper prop sample preparation: get Ginsenoside Rd's crude product, after the ratio dissolve with methanol in 2.5mL methyl alcohol/g Ginsenoside Rd crude product, adds the purified water of methyl alcohol volume 1/3, stirs evenly, for subsequent use;
(2) post is filled: take reversed phase chromatography silica gel Rp-C18 in the ratio of 1kg silica gel/30g Ginsenoside Rd crude product, fill post with methanol wet;
(3) loading and wash-out: by upper prop sample wet method upper prop, with methanol-water=75 ﹕ 25 isocratic elution, Fractional Collections, often flowing part collecting amount is 0.5L/kg
silica gel, after thin-layer chromatography checks, merge required component, 75 DEG C of decompression and solvent recoveries are to 1.5 times of upper prop sample volume, and leave standstill 15h, separate out precipitation, upper liquid returns reversed phase chromatography silicagel column and is separated, and is deposited in 40 DEG C of vacuum-dryings, obtains Ginsenoside Rd.
Described thin-layer chromatography inspection is specially: often flow part and get 10mL respectively, evaporate to dryness, residue adds 1mL dissolve with methanol, as need testing solution; Separately get appropriate Ginsenoside Rd's reference substance, add methyl alcohol and make the solution of every 1mL containing 2mg, product solution in contrast; Tlc according to " Chinese Pharmacopoeia " version in 2010 annex VI B is tested, draw described need testing solution 5 μ L and reference substance solution 5 μ L, put respectively on same gel GF 254 plate, using Zheng Ding Chun – Yi Suan Yi Zhi – water=4: the upper solution of 3: 5 is as developping agent, launch, take out, dry, spray, with 10% vitriol oil-ethanolic soln, is heated to spot development in 105 DEG C clear.
The embodiment of the present invention 3: the extraction process of a kind of Ginsenoside Rd, as shown in Figure 1, comprises the following steps:
1, the medicinal material (such as pseudo-ginseng, ginseng, Radix Panacis Quinquefolii etc.) containing Ginsenoside Rd's composition is ground into 60 object medicinal powder, add 2 times amount 95% ethanol, cold soaking 18h, filter, the dregs of a decoction add 1.8 times amount 95% ethanol, refluxing extraction 3 times, each 2 hours, filter, merging filtrate, reclaim ethanol and be concentrated into the medicinal extract that relative density is 1.26, obtaining general extractive.
2, total saponins is extracted
(1) fill post: with the fully moistening D101 macroporous adsorbent resin of anticipating of ethanol, stir and get rid of bubble, dress post, soak 12h, to add water after mixing not in muddiness with 95% alcohol flushing to effluent liquid, continue with purified water displacement ethanol wherein to without alcohol taste, for subsequent use;
(2) loading: get general extractive, adds 2 times amount purified water, stirs evenly, and slowly adds in the D101 macroporous adsorptive resins of anticipating, and makes all to enter post bed, standing adsorption 12h;
(3) wash-out: first use 5L/kg
silica gelpurified water wash-out, water elution liquid discards; Continue with 80% ethanol elution being no less than purified water consumption, collect 80% ethanol eluate, reclaim ethanol and be evaporated to the clear cream that relative density is 1.18, obtaining total saponins, for subsequent use after macroporous resin column 5BV 95% ethanol elution.
3, Ginsenoside Rd's crude product is extracted
(1) upper prop sample preparation: get total saponins, is adsorbed on the silica gel of 2 times amount, evaporates into dry, crosses 60 mesh sieves, for subsequent use;
(2) fill post: the column chromatography silica gel getting total saponins 10 times amount, with the upper solution wet method dress post of propyl carbinol-ethyl acetate-water=2 ﹕ 3 ﹕ 5, repeatedly use eluent, make silica gel fully sink closely;
(3) loading and wash-out: in silica gel column chromatography, reserved a certain amount of elutriant makes it flood silica gel and (namely ensures that in silicagel column, liquid level is higher than silica gel top, separating effect is affected) to avoid occurring silica gel tomography because of post inner drying, upper prop sample is slowly added in chromatography column, with the upper solution isocratic elution of propyl carbinol-ethyl acetate-water=2 ﹕ 3 ﹕ 5, Fractional Collections, often flowing part collecting amount is 1.5L/kg
silica gel, after thin-layer chromatography inspection, merge Ginsenoside Rd flow part, 70 DEG C of decompression and solvent recoveries, to dry, obtain Ginsenoside Rd's crude product.
4, purified ginsenoside Rd
(1) upper prop sample preparation: get Ginsenoside Rd's crude product, after the ratio dissolve with methanol in 2.5mL methyl alcohol/g Ginsenoside Rd crude product, adds the purified water of methyl alcohol volume 1/3, stirs evenly, for subsequent use;
(2) post is filled: take reversed phase chromatography silica gel Rp-C18 in the ratio of 1kg silica gel/30g Ginsenoside Rd crude product, fill post with methanol wet;
(3) loading and wash-out: by upper prop sample wet method upper prop, with methanol-water=75 ﹕ 25 isocratic elution, Fractional Collections, often flowing part collecting amount is 0.5L/kg
silica gel, after thin-layer chromatography checks, merge required component, 80 DEG C of decompression and solvent recoveries are to 2 times of upper prop sample volume, and leave standstill 18h, separate out precipitation, upper liquid returns reversed phase chromatography silicagel column and is separated, and is deposited in 40 DEG C of vacuum-dryings, obtains Ginsenoside Rd.
Claims (7)
1. a Ginsenoside Rd's extraction process, is characterized in that, comprises the following steps:
S1, becomes medicinal powder by the pulverizing medicinal materials containing Ginsenoside Rd's composition, adopts 70% ~ 95% alcohol reflux, and filter, filtrate recycling ethanol is also concentrated into the medicinal extract that relative density is 1.15 ~ 1.26, obtains general extractive;
S2, gets general extractive, adds 2 times amount purified water, stirs evenly, upper D101 macroporous adsorptive resins, first uses purified water wash-out, continues with 40% ~ 80% ethanol elution, collect ethanol eluate, reclaim ethanol and be evaporated to the clear cream that relative density is 1.10 ~ 1.20, obtaining total saponins;
S3, get total saponins, be adsorbed on 2 times amount silica gel, evaporate into dry, sieve, added in silica gel column chromatography, with propyl carbinol-ethyl acetate-water=2 ﹕ 3 ﹕ 5 isocratic elution, Fractional Collections, merge Ginsenoside Rd through thin-layer chromatography inspection and flow part, decompression and solvent recovery, to dry, obtains Ginsenoside Rd's crude product;
S4, get Ginsenoside Rd's crude product, adds purified water with dissolve with methanol, stir evenly, upper Rp-C18 reversed phase chromatography silicagel column, with methanol-water=75 ﹕ 25 isocratic elution, Fractional Collections, required component is merged after thin-layer chromatography checks, decompression and solvent recovery, leaves standstill, and separates out precipitation, upper liquid returns reversed phase chromatography silicagel column and is separated, and precipitates dry Ginsenoside Rd.
2. the extraction process of Ginsenoside Rd according to claim 1, it is characterized in that: described step S1 is specially: the pulverizing medicinal materials containing Ginsenoside Rd's composition is become 40 ~ 60 object medicinal powder, add 2 times amount 85% ethanol, cold soaking 15 ~ 18h, filter, the dregs of a decoction add 1.8 times amount 85% ethanol, refluxing extraction 3 times, each 2 hours, filter, merging filtrate, reclaims ethanol and is concentrated into the medicinal extract that relative density is 1.24 ~ 1.26, obtaining general extractive.
3. the extraction process of Ginsenoside Rd according to claim 1, is characterized in that: described step S2 comprises:
S21, dress post: with the fully moistening D101 macroporous adsorbent resin of anticipating of ethanol, stirs and gets rid of bubble, dress post, soaks 12h, and adding water after mixing with 95% alcohol flushing to effluent liquid is not muddiness, continue with purified water displacement ethanol wherein to without alcohol taste, for subsequent use;
S22, loading: get general extractive, adds 2 times amount purified water, stirs evenly, and slowly adds in the D101 macroporous adsorptive resins of anticipating, and makes all to enter post bed, standing adsorption 12h;
S23, wash-out: first use 5L/kg
silica gelpurified water wash-out, water elution liquid discards; Continue with 60% ethanol elution being no less than purified water consumption, collect 60% ethanol eluate, reclaim ethanol and be evaporated to the clear cream that relative density is 1.10 ~ 1.20, obtaining total saponins.
4. the extraction process of Ginsenoside Rd according to claim 1, is characterized in that: described step S3 comprises:
S31, upper prop sample preparation: get total saponins, is adsorbed on the silica gel of 2 times amount, evaporates into dry, crosses 60 mesh sieves, for subsequent use;
S32, dress post: the column chromatography silica gel getting total saponins 10 times amount, with the upper solution wet method dress post of propyl carbinol-ethyl acetate-water=2 ﹕ 3 ﹕ 5, repeatedly uses eluent, makes silica gel fully sink closely;
S33, loading and wash-out: slowly added in chromatography column by upper prop sample, with the upper solution isocratic elution of propyl carbinol-ethyl acetate-water=2 ﹕ 3 ﹕ 5, Fractional Collections, often flowing part collecting amount is 1.5L/kg
silica gel, after thin-layer chromatography inspection, merge Ginsenoside Rd flow part, 65 ~ 70 DEG C of decompression and solvent recoveries, to dry, obtain Ginsenoside Rd's crude product.
5. the extraction process of Ginsenoside Rd according to claim 1, is characterized in that: described step S4 comprises:
S41, upper prop sample preparation: get Ginsenoside Rd's crude product, after the ratio dissolve with methanol in 2.5mL methyl alcohol/g Ginsenoside Rd crude product, adds the purified water of methyl alcohol volume 1/3, stirs evenly, for subsequent use;
S42, dress post: take reversed phase chromatography silica gel Rp-C18 in the ratio of 1kg silica gel/30g Ginsenoside Rd crude product, fill post with methanol wet;
S43, loading and wash-out: by upper prop sample wet method upper prop, with methanol-water=75 ﹕ 25 isocratic elution, Fractional Collections, often flowing part collecting amount is 0.5L/kg
silica gel, after thin-layer chromatography checks, merge required component, 75 ~ 80 DEG C of decompression and solvent recoveries are to 1.5 ~ 2 times of upper prop sample volume, and leave standstill 15 ~ 18h, separate out precipitation, upper liquid returns reversed phase chromatography silicagel column and is separated, and is deposited in 40 DEG C of vacuum-dryings, obtains Ginsenoside Rd.
6. the extraction process of the Ginsenoside Rd according to claim 1 or 4 or 5, is characterized in that: described thin-layer chromatography inspection is specially: often flow part and get 10mL respectively, evaporate to dryness, residue adds 1mL dissolve with methanol, as need testing solution; Separately get appropriate Ginsenoside Rd's reference substance, add methyl alcohol and make the solution of every 1mL containing 2mg, product solution in contrast; Tlc according to " Chinese Pharmacopoeia " version in 2010 annex VI B is tested, draw described need testing solution 5 ~ 10 μ L and reference substance solution 5 μ L, put respectively on same gel GF 254 plate, using Zheng Ding Chun – Yi Suan Yi Zhi – water=4: the upper solution of 3: 5 is as developping agent, launch, take out, dry, spray, with 10% vitriol oil-ethanolic soln, is heated to spot development in 105 DEG C clear.
7. the extraction process of the Ginsenoside Rd according to any one of claim 1-5, is characterized in that: the described medicinal material containing Ginsenoside Rd's composition is pseudo-ginseng, ginseng or Radix Panacis Quinquefolii.
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| CN105131070A (en) * | 2015-09-21 | 2015-12-09 | 成都艾比科生物科技有限公司 | Method for directly extracting panasadiol saponins from pseudo-ginseng |
| CN105942519A (en) * | 2016-04-27 | 2016-09-21 | 周飞燕 | Health-care food capable of reducing blood lipid and added with ginseng extract |
| CN106220704A (en) * | 2016-07-21 | 2016-12-14 | 西安岳达生物科技股份有限公司 | A kind of method prepared by ginsenoside's RD high-purity technical |
| CN110156863A (en) * | 2018-02-12 | 2019-08-23 | 吉林紫鑫参工堂生物科技有限公司 | A kind of ginsenoside Rd's automatic control isolation and purification method |
| CN110156863B (en) * | 2018-02-12 | 2022-12-13 | 吉林紫鑫参工堂生物科技有限公司 | Method for automatically controlling, separating and purifying ginsenoside Rd |
| CN110025564A (en) * | 2019-05-10 | 2019-07-19 | 吉林省一向科技有限责任公司 | A kind of anti-aging skin care compositions and preparation method thereof |
| CN111072747A (en) * | 2019-12-26 | 2020-04-28 | 王传涛 | Ginsenoside and ultrasonic extraction method thereof |
| CN111349136A (en) * | 2020-01-17 | 2020-06-30 | 朱业君 | Method for extracting high-purity ginsenoside Rh2 |
| CN113648681A (en) * | 2021-08-27 | 2021-11-16 | 湖南华康生物科技股份有限公司 | Method for extracting and separating ginsenoside from Korean ginseng |
| CN114736262A (en) * | 2022-04-11 | 2022-07-12 | 云南现代民族药工程技术研究中心 | Extraction and separation method of pseudo-ginseng medicinal material |
| CN117017921A (en) * | 2023-10-08 | 2023-11-10 | 吉林农业大学 | Ginsenoside liposome and preparation method thereof |
| CN117017921B (en) * | 2023-10-08 | 2024-03-15 | 吉林农业大学 | Ginsenoside liposome and preparation method thereof |
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