CN103588827A - Separation and purification method for oligomeric trisaccharide in compound red sage root extract - Google Patents
Separation and purification method for oligomeric trisaccharide in compound red sage root extract Download PDFInfo
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Abstract
The invention relates to a separation and purification method for an oligomeric trisaccharide in compound red sage root extract. By adoption of a solid-phase extraction method, saccharides in the compound red sage root extract are separated and purified to obtain an extract of the total saccharides of the compound red sage root extract. Then by adoption of a gel chromatography method, oligosaccharide compounds in the extract of the total saccharides of the compound red sage root extract are separated effectively and an oligomeric trisaccharide alpha-D-glucopyranose-(1->2)-beta-D-fructofuranose-(1->1)-alpha-D-galactopyranose is obtained.
Description
Technical field:
The invention belongs to the field of Chinese medicines, be specifically related to the separation purification method of oligomeric trisaccharide in a kind of compound Salviae Miltiorrhizae medicinal extract.
Background technology:
FUFANG DANSHEN DIWAN is the pill of being made by the red sage root, pseudo-ginseng and borneol, is widely used in clinically prevention, treatment, the first aid of coronary heart diseases and angina pectoris, has now become one of leading brand on domestic cardiovascular market.Compound Salviae Miltiorrhizae medicinal extract is the preparation intermediate of FUFANG DANSHEN DIWAN, is by the red sage root and pseudo-ginseng, the medicinal extract (preparation method of compound Salviae Miltiorrhizae medicinal extract belongs to prior art) obtaining through water extraction, alcohol precipitation, after concentrated.
FUFANG DANSHEN DIWAN listing was progressively set up the perfect processing quality hierarchy of control in more than ten years, with the form of medicine, carried out FDA at present and declared research.But with respect to Western medicine, the current known component of compound Salviae Miltiorrhizae medicinal extract is still lower, improving product processing quality level of control, need to further investigate for more chemical composition of Chinese materia medica wherein, thereby hold more exactly quality product, reduce drug risk, simultaneously for international registration provides support.
Carbohydrate is widely distributed in herbal medicine, is also the chief component composition in Chinese medicinal materials and tcm product, and shared content ratio is very high.Measurement result shows, the carbohydrate content content in compound Salviae Miltiorrhizae medicinal extract reaches approximately 50%, so the research of carbohydrate content is for the total quality control important in inhibiting of compound Salviae Miltiorrhizae medicinal extract.
From compound Salviae Miltiorrhizae medicinal extract, isolated various saccharides composition, obtaining these compositions can understand their effect for analyzing the proportionlity of different sugar constituents in FUFANG DANSHEN DIWAN, simultaneously can be for controlling the quality of product.The invention reside in provide a kind of from compound Salviae Miltiorrhizae medicinal extract the method for separating oligomeric trisaccharide composition, the method is quick, accurately, simple to operate, good separating effect, the product purity obtaining is high, yield is high, the quantitative and qualitative analysis that can be used for carbohydrate content detects.
Summary of the invention:
The present invention is through prolonged and repeated groping, finally obtain the separation purification method of oligomeric trisaccharide in a kind of compound Salviae Miltiorrhizae medicinal extract, and this oligomeric trisaccharide has been carried out to structural confirmation, confirm that this compound is α-D-Glucopyranose-(1 → 2)-beta-D-fructofuranose-(1 → 1)-α-D-galactopyranose.
This separation purification method comprises the steps:
(1) preparation of the total sugar extract of compound Salviae Miltiorrhizae medicinal extract;
(2) the total sugar extract of compound Salviae Miltiorrhizae medicinal extract is crossed polyacrylamide gel chromatography column;
(3) collection of oligomeric trisaccharide composition.
Wherein the preparation method of the total sugar extract of the described compound Salviae Miltiorrhizae medicinal extract of step (1) is:
Compound Salviae Miltiorrhizae medicinal extract adds water ultrasonic dissolution, and upper to processed good solid-phase extraction column, flow velocity is 0.5-1.0mL/min, then water gradation washing, collects sample solution and scrub stream fluid, and evaporate to dryness, obtains the total sugar extract of compound Salviae Miltiorrhizae medicinal extract.
Wherein said solid-phase extraction column adopts Cleanert PS-SPE column extractor, and before using, through processing, treatment process is: by solid-phase extraction column washed with methanol, water rinses again afterwards.
Wherein said compound Salviae Miltiorrhizae medicinal extract is that the red sage root and pseudo-ginseng are through water extraction, alcohol precipitation, the medicinal extract obtaining after concentrated, its preparation method is specially: get respectively 41.06 parts of reds sage root, 8.03 part pseudo-ginseng, pulverize, put into extractor, the buck (pH=7.0) that adds 5 times of amounts of above-mentioned crude drug, extract 2 hours, filter, collect just filtrate, the dregs of a decoction add 4 times of water gagings, decoct 1 hour, filter, mix with the filtrate of extracting for the first time, concentrating under reduced pressure, until liquor capacity (L) is 0.9-1.1 with the ratio of crude drug quality (Kg), add 95% ethanol until contain alcohol amount to 69-71%, standing 12 hours, filter, filtrate concentration and recovery ethanol becomes medicinal extract, relative density is 1.32-1.40.The method belongs to prior art.
Wherein the described polyacrylamide gel chromatography column excessively of step (2) is that the total sugar extract of compound Salviae Miltiorrhizae medicinal extract obtaining is dissolved with ultrapure water, after dissolving, loading is to processed good chromatography column, the column temperature of chromatography column is 35-45 ℃, with ultrapure water, carry out wash-out, every 10min collects 1 pipe elutriant, collects the stream part while being equivalent to 0.65-0.75 column volume.
Wherein, polyacrylamide gel chromatography column is prior art, can obtain with following methods:
Take a certain amount of polyacrylamide gel, pour in ultrapure water, at room temperature profit rises more than 12 hours, vacuum outgas, and dress post arrives the height (100 ~ 120cm) needing, then uses the ultrapure water of 2 ~ 3 times of bed volumes with constant flow velocity balance chromatography column.
Preferably, in step (2), the column temperature of chromatography column is 40 ℃.
Applied sample amount described in step (2) is total sugar extract/100 of 0.075-0.125g compound Salviae Miltiorrhizae medicinal extract gram polyacrylamide gels.
The elution flow rate of chromatography column described in step (2) be 0.0184-0.0367 column volume/hour.
Wherein the collection method of the oligomeric trisaccharide composition described in step (3) is: by HPLC method, elutriant is detected, and the elutriant that collection retention time is 27.5 ± 0.2min, evaporate to dryness, obtains the oligomeric trisaccharide of eluate.
The method that described HPLC detects elutriant is:
Adopt HPLC-ELSD method, HPLC adopts Prevail TM Carbohydrate ES chromatographic column, and chromatographic condition is for take acetonitrile as mobile phase A, and water is Mobile phase B, and gradient elution is set to:
Flow velocity 0.8mL/min, 30 ℃ of column temperatures; Adopt ELSD detector, detector parameters is set to gain 10, air pressure 25psi, and 60 ℃ of drift tubes, Neb heater is 60%.
The structural confirmation method of the oligomeric trisaccharide of eluate of the present invention be high resolution mass spectrum,
1h-NMR,
13c-NMR, DEPT, HMQC, H-HCOSY, HSQC, H-Htcosy, HMBC nuclear magnetic scanning.
ESI-MS(m/z): high resolution mass spectrum records [M+Na] of this product
+the total mass number at peak is 527.1583, with C
18h
32o
16the relative error of calculated value 527.1583 is-0.16ppm to show the molecular formula C of coupling the most being provided by high resolution mass spectrum
18h
32o
16therefore, the chemical formula C of this testing compound
18h
32o
16.
1h-NMR (D
2o, 500MHz): δ 5.19 is the terminal hydrogen signal on glucose, bimodal, and coupling constant is 3.7, illustrates that this sugar is α configuration, is 1 hydrogen (1H, d, J-3.7,1-H); δ 4.64 is the signal of the terminal hydrogen on semi-lactosi, bimodal, illustrates that this sugar is α configuration, is 1 hydrogen (1H, d, J-3.7,1-H).
13c-NMR (D
2o, 125MHz): δ 104.04 is the carbon signal (1C, 2-C) of 2 of fructofuranoses, and δ 99.06 is the carbon signal (1C, 1-C) of 1 of galactopyranose, the carbon signal (1C, 1-C) that δ 91.63 for 1 of Glucopyranose is.
DEPT: determine that by DEPT the carbon of 2 of fructofuranoses is quaternary carbon.
It is relevant that HSQC: δ 91.63 and δ 5.19 have, δ 104.04(1C, 2-C) be hydrogen signal (2H, d, the J that the carbon signal of 2 of fructofuranoses and δ 3.47 and δ 2.72 are 1 of fructofuranose
1a, 1b=11.5,1a-H, 1b-H) have relevant; HMBC: δ 66.68 is the carbon signal (1C, 1-C) and the hydrogen signal (1H, d, J-3.7,1-H) of δ 5.19 for 1 of Glucopyranose of 1 of fructofuranose; δ 99.06 is that the carbon signal (1C, 1-C) of 1 of galactopyranose is hydrogen signal (2H, d, the J of 2 of fructofuranoses with δ 3.47 and δ 3.72
1a, 1b=11.5,1a-H, 1b-H) there is a distant relation.Illustrate that connection site and the order of connection that this is sugared are α-D-Glucopyranose-(1 → 2)-beta-D-fructofuranose-(1 → 1)-α-D-galactopyranose.
1h-NMR(D
2o, 500MHz) with
13c-NMR(D
2o, 125MHz) spectral data and classification ownership in table 1, provide.
Table 1
1h-NMR and 1
3C-NMR data gather
The spectrum data gathering in table 1 is through consulting, consistent with bibliographical information α-D-Glucopyranose-(1 → 2)-beta-D-fructofuranose-(1 → 1)-α-D-galactopyranose.Therefore determine that this compound is α-D-Glucopyranose-(1 → 2)-beta-D-fructofuranose-(1 → 1)-α-D-galactopyranose.
The structure of this oligomeric trisaccharide is shown in Fig. 2.Fig. 3 ~ 10 are shown in by collection of illustrative plates.
Accompanying drawing explanation:
The total sugar extract ELSD-HPLC of Fig. 1 compound Salviae Miltiorrhizae medicinal extract color atlas
The structure of the oligomeric trisaccharide that Fig. 2 separation and purification of the present invention goes out
The oligomeric trisaccharide of Fig. 3 the present invention
1h-NMR collection of illustrative plates
The oligomeric trisaccharide of Fig. 4 the present invention
13c-NMR collection of illustrative plates
The ESI-MS collection of illustrative plates of the oligomeric trisaccharide of Fig. 5 the present invention
The DEPT collection of illustrative plates of the oligomeric trisaccharide of Fig. 6 the present invention
The H-Hcosy collection of illustrative plates of the oligomeric trisaccharide of Fig. 7 the present invention
The H-Htocsy collection of illustrative plates of the oligomeric trisaccharide of Fig. 8 the present invention
The HSQC collection of illustrative plates of the oligomeric trisaccharide of Fig. 9 the present invention
The HMBC collection of illustrative plates of the oligomeric trisaccharide of Figure 10 the present invention
Embodiment:
Below by specific embodiment, further illustrate the present invention, following embodiment is that the simple modifications that essence according to the present invention is carried out the present invention all belongs to the scope of protection of present invention for the present invention rather than limitation of the present invention are described.
Embodiment 1
1, instrument
AKTA prime protein purification system (GE of GE Medical Group); High pressure water-cooling sandwich chromatography column (2.5cm * 100cm, Shanghai Chu Bai laboratory equipment company limited); Waters 2695 high performance liquid chromatographs (U.S. Waters company); Waters 2420ELSD detector (U.S. Waters company); Bio-Gel P-2 gathers propionic acid amide gel (U.S. Bio-Red company); Milli-Q Academic ultra-pure-water treatment system (U.S. Millipore company); Solid-phase extraction column (Cleanert PS-SPE, 500mg/6mL, Agela Science and Technology Ltd.); Thermostat water bath (Ningbo Tian Heng instrument plant).
2, reagent and reagent
Acetonitrile (chromatographically pure, Merck company); Methyl alcohol (analytical pure, Concord, Tianjin Science and Technology Ltd.); Ultrapure water (Milli-Q ultra-pure-water treatment system makes, specific conductivity 18.2M Ω cm).
3, the preparation of the total sugar extract of compound Salviae Miltiorrhizae medicinal extract
Solid-phase extraction column (Cleanert PS-SPE, 500mg/6mL) is used to 10mL washed with methanol, with 10mL water, rinse again afterwards, can be standby after flushing.
Precision takes 0.4g compound Salviae Miltiorrhizae medicinal extract, adds 10mL water, ultrasonic it is dissolved completely.Upper to processed good solid-phase extraction column (Cleanert PS-SPE, 500mg/6mL), the about 1mL/min of flow velocity, water gradation washing again, collects sample solution and the about 45mL of scrub stream fluid, evaporate to dryness, weigh, obtain altogether the total sugar extract of 0.2g compound Salviae Miltiorrhizae medicinal extract, standby.
4, the preparation of polyacrylamide gel chromatography column
Take the polyacrylamide gel (Bio-Gel P-2) of 200g, slowly pour in ultrapure water, at room temperature profit rises more than 12 hours.Upper strata fine particle is toppled over away, repeatedly for several times, till the fine particle without suspending.
By the poly-propionic acid amide gel vacuum outgas of handling well, by automatic sedimentation method, fill post to 100cm afterwards.Use again the ultrapure water of 2～3 times of bed volumes with constant flow velocity balance chromatography column, can be for later separation.
5, loading, wash-out, collection elutriant
After the 0.2g obtaining is processed, sample dissolves with the ultrapure water of 0.5mL, and after dissolving, loading is to processed good chromatography column, and chromatography column temperature will remain at 40 ℃.
With ultrapure water with 0.2mL/min(0.0245 column volume/hour) speed carry out wash-out, every 10min collects 1 pipe elutriant, stream part when collection is equivalent to 0.65-0.75 column volume (collecting stream part of 159-184 pipe, 318-368ml), carries out HPLC and inspects.
6, the detection of elutriant
Chromatographic condition: adopt Prevail TM Carbohydrate ES chromatographic column, take acetonitrile as mobile phase A, water is Mobile phase B, and gradient is set to:
Flow velocity is 0.8mL/min, 30 ℃ of column temperatures; Adopt WATERS 2420ELSD detector, parameter is set to gain 10, air pressure 25psi, and 60 ℃ of drift tubes, Neb heater is 60%.
What collection was obtained respectively manages elutriant, and according to above-mentioned chromatographic condition, directly sample introduction successively, detects recording responses value.
7, the collection of oligomeric trisaccharide
To only contain retention time is the elutriant merging of 27.5 ± 0.2min chromatographic peak, and evaporate to dryness, obtains unknown compound, standby.
8, the structural confirmation of oligomeric trisaccharide
Unknown compound is soluble in water, rare alcohol, ethanol.Molish reaction is positive.Illustrate that this compound may be carbohydrate.
By high resolution mass spectrum,
1h-NMR,
13the methods such as C-NMR, DEPT, HMQC, H-HCOSY, HSQC, H-Htcosy, HMBC nuclear magnetic scanning, can determine that this unknown compound is: α-D-Glucopyranose-(1 → 2)-beta-D-fructofuranose-(1 → 1)-α-D-galactopyranose.
1, instrument
AKTA prime protein purification system (GE of GE Medical Group); High pressure water-cooling sandwich chromatography column (5.0cm * 100cm, the GE of GE Medical Group); Waters 2695 high performance liquid chromatographs (U.S. Waters company); Waters 2420ELSD detector (U.S. Waters company); Bio-Gel P-2 gathers propionic acid amide gel (U.S. Bio-Red company); Milli-Q Academic ultra-pure-water treatment system (U.S. Millipore company); Solid-phase extraction column (Cleanert PS-SPE, 500mg/6mL, Agela Science and Technology Ltd.); Thermostat water bath (Ningbo Tian Heng instrument plant).
2, reagent and reagent
Acetonitrile (chromatographically pure, Merck company); Methyl alcohol (analytical pure, Concord, Tianjin Science and Technology Ltd.); Ultrapure water (Milli-Q ultra-pure-water treatment system makes, specific conductivity 18.2M Ω cm).
3, the preparation of the total sugar extract of compound Salviae Miltiorrhizae medicinal extract
Solid-phase extraction column (Cleanert PS-SPE, 500mg/6mL) is used to 10mL washed with methanol, with 10mL water, rinse again afterwards, can be standby after flushing.
Precision takes 1.6g compound Salviae Miltiorrhizae medicinal extract, adds 50mL water, ultrasonic it is dissolved completely.Upper to processed good 4 solid-phase extraction columns (Cleanert PS-SPE, 500mg/6mL), the about 1mL/min of flow velocity, water gradation washing again, merges and collects sample solution and the about 45mL of scrub stream fluid, evaporate to dryness, weigh, obtain altogether the total sugar extract of 0.8g compound Salviae Miltiorrhizae medicinal extract, standby.
4, the preparation of polyacrylamide gel chromatography column
Take the polyacrylamide gel (Bio-Gel P-2) of 800g, slowly pour in ultrapure water, at room temperature profit rises more than 12 hours.Upper strata fine particle is toppled over away, repeatedly for several times, till the fine particle without suspending.
By the poly-propionic acid amide gel vacuum outgas of handling well, by automatic sedimentation method, fill post to 100cm afterwards.Use again the ultrapure water of 2～3 times of bed volumes with constant flow velocity balance chromatography column, can be for later separation.
5, loading, wash-out, collection elutriant
After the 0.8g obtaining is processed, sample dissolves with the ultrapure water of 2.0mL, and after dissolving, loading is to processed good chromatography column, and chromatography column temperature will remain at 35 ℃.
With ultrapure water with 0.8mL/min(0.0245 column volume/hour) speed carry out wash-out, every 10min collects 1 pipe elutriant, stream part when collection is equivalent to 0.65-0.75 column volume (collecting stream part of 159-184 pipe, 1272 ~ 1472ml), carries out HPLC and inspects.
6, the detection of elutriant
Chromatographic condition: adopt Prevail TM Carbohydrate ES chromatographic column, take acetonitrile as mobile phase A, water is Mobile phase B, and gradient is set to:
Flow velocity is 0.8mL/min, 30 ℃ of column temperatures; Adopt WATERS 2420ELSD detector, parameter is set to gain 10, air pressure 25psi, and 60 ℃ of drift tubes, Neb heater is 60%.
What collection was obtained respectively manages elutriant, and according to above-mentioned chromatographic condition, directly sample introduction successively, detects recording responses value.
7, the collection of oligomeric trisaccharide
To only contain retention time is the elutriant merging of 27.5 ± 0.2min chromatographic peak, and evaporate to dryness, obtains unknown compound, standby.
8, the structural confirmation of oligomeric trisaccharide
Unknown compound is soluble in water, rare alcohol, ethanol.Molish reaction is positive.Illustrate that this compound may be carbohydrate.
By high resolution mass spectrum,
1h-NMR,
13the methods such as C-NMR, DEPT, HMQC, H-HCOSY, HSQC, H-Htcosy, HMBC nuclear magnetic scanning, can determine that this unknown compound is: α-D-Glucopyranose-(1 → 2)-beta-D-fructofuranose-(1 → 1)-α-D-galactopyranose.
Embodiment 3
1, instrument
AKTA prime protein purification system (GE of GE Medical Group); High pressure water-cooling sandwich chromatography column (2.5cm * 120cm, U.S. Bio-Red company); Waters 2695 high performance liquid chromatographs (U.S. Waters company); Waters 2420ELSD detector (U.S. Waters company); Bio-Gel P-2 gathers propionic acid amide gel (U.S. Bio-Red company); Milli-Q Academic ultra-pure-water treatment system (U.S. Millipore company); Solid-phase extraction column (Cleanert PS-SPE, 500mg/6mL, Agela Science and Technology Ltd.); Thermostat water bath (Ningbo Tian Heng instrument plant).
2, reagent and reagent
Acetonitrile (chromatographically pure, Merck company); Methyl alcohol (analytical pure, Concord, Tianjin Science and Technology Ltd.); Ultrapure water (Milli-Q ultra-pure-water treatment system makes, specific conductivity 18.2M Ω cm).
3, the preparation of the total sugar extract of compound Salviae Miltiorrhizae medicinal extract
Solid-phase extraction column (Cleanert PS-SPE, 500mg/6mL) is used to 10mL washed with methanol, with 10mL water, rinse again afterwards, can be standby after flushing.
Precision takes 0.4g compound Salviae Miltiorrhizae medicinal extract, adds 10mL water, ultrasonic it is dissolved completely.Upper to processed good solid-phase extraction column (Cleanert PS-SPE, 500mg/6mL), the about 0.8mL/min of flow velocity, water gradation washing again, collects sample solution and the about 45mL of scrub stream fluid, evaporate to dryness, weigh, obtain altogether the total sugar extract of 0.2g compound Salviae Miltiorrhizae medicinal extract, standby.
4, the preparation of polyacrylamide gel chromatography column
Take the polyacrylamide gel (Bio-Gel P-2) of 240g, slowly pour in ultrapure water, at room temperature profit rises more than 12 hours.Upper strata fine particle is toppled over away, repeatedly for several times, till the fine particle without suspending.
By the poly-propionic acid amide gel vacuum outgas of handling well, by automatic sedimentation method, fill post to 120cm afterwards.Use again the ultrapure water of 2～3 times of bed volumes with constant flow velocity balance chromatography column, can be for later separation.
5, loading, wash-out, collection elutriant
After the 0.2g obtaining is processed, sample dissolves with the ultrapure water of 0.5mL, and after dissolving, loading is to processed good chromatography column, and chromatography column temperature will remain at 45 ℃.
With ultrapure water with 0.2mL/min(0.0245 volume/hour) speed carry out wash-out, every 10min collects 1 pipe elutriant, collects the stream part (collecting stream part of 198-216 pipe, 396-432ml) while being equivalent to 0.65-0.75 column volume, carries out HPLC and inspects.
6, the detection of elutriant
Chromatographic condition: adopt Prevail TM Carbohydrate ES chromatographic column, take acetonitrile as mobile phase A, water is Mobile phase B, and gradient is set to:
Flow velocity is 0.8mL/min, 30 ℃ of column temperatures; Adopt WATERS 2420ELSD detector, parameter is set to gain 10, air pressure 25psi, and 60 ℃ of drift tubes, Neb heater is 60%.
What collection was obtained respectively manages elutriant, and according to above-mentioned chromatographic condition, directly sample introduction successively, detects recording responses value.
7, the collection of oligomeric trisaccharide
To only contain retention time is the elutriant merging of 27.5 ± 0.2min chromatographic peak, and evaporate to dryness, obtains unknown compound, standby.
8, the structural confirmation of oligomeric trisaccharide
Unknown compound is soluble in water, rare alcohol, ethanol.Molish reaction is positive.Illustrate that this compound may be carbohydrate.
By high resolution mass spectrum,
1h-NMR,
13the methods such as C-NMR, DEPT, HMQC, H-HCOSY, HSQC, H-Htcosy, HMBC nuclear magnetic scanning, can determine that this unknown compound is: α-D-Glucopyranose-(1 → 2)-beta-D-fructofuranose-(1 → 1)-α-D-galactopyranose.
Embodiment 4
1, instrument
AKTA prime protein purification system (GE of GE Medical Group); High pressure water-cooling sandwich chromatography column (2.5cm * 100cm, Shanghai Chu Bai laboratory equipment company limited); Waters 2695 high performance liquid chromatographs (U.S. Waters company); Waters 2420ELSD detector (U.S. Waters company); Bio-Gel P-2 gathers propionic acid amide gel (U.S. Bio-Red company); Milli-Q Academic ultra-pure-water treatment system (U.S. Millipore company); Solid-phase extraction column (Cleanert PS-SPE, 500mg/6mL, Agela Science and Technology Ltd.); Thermostat water bath (Ningbo Tian Heng instrument plant).
2, reagent and reagent
Acetonitrile (chromatographically pure, Merck company); Methyl alcohol (analytical pure, Concord, Tianjin Science and Technology Ltd.); Ultrapure water (Milli-Q ultra-pure-water treatment system makes, specific conductivity 18.2M Ω cm).
3, the preparation of the total sugar extract of compound Salviae Miltiorrhizae medicinal extract
Solid-phase extraction column (Cleanert PS-SPE, 500mg/6mL) is used to 10mL washed with methanol, with 10mL water, rinse again afterwards, can be standby after flushing.
Precision takes 0.5g compound Salviae Miltiorrhizae medicinal extract, adds 10mL water, ultrasonic it is dissolved completely.Upper to processed good solid-phase extraction column (Cleanert PS-SPE, 500mg/6mL), the about 0.5mL/min of flow velocity, water gradation washing again, collects sample solution and the about 45mL of scrub stream fluid, evaporate to dryness, weigh, obtain altogether the total sugar extract of 0.25g compound Salviae Miltiorrhizae medicinal extract, standby.
4, the preparation of polyacrylamide gel chromatography column
Take the polyacrylamide gel (Bio-Gel P-2) of 200g, slowly pour in ultrapure water, at room temperature profit rises more than 12 hours.Upper strata fine particle is toppled over away, repeatedly for several times, till the fine particle without suspending.
By the poly-propionic acid amide gel vacuum outgas of handling well, by automatic sedimentation method, fill post to 100cm afterwards.Use again the ultrapure water of 2～3 times of bed volumes with constant flow velocity balance chromatography column, can be for later separation.
5, loading, wash-out, collection elutriant
After the 0.25g obtaining is processed, sample dissolves with the ultrapure water of 0.5mL, and after dissolving, loading is to processed good chromatography column, and chromatography column temperature will remain at 40 ℃.
With ultrapure water with 0.15mL/min(0.0184 volume/hour) speed carry out wash-out, every 10min collects 1 pipe elutriant, collects the stream part (collecting stream part of 212-245 pipe, 318-368ml) while being equivalent to 0.65-0.75 column volume, carries out HPLC and inspects.
6, the detection of elutriant
Chromatographic condition: adopt Prevail TM Carbohydrate ES chromatographic column, take acetonitrile as mobile phase A, water is Mobile phase B, and gradient is set to:
Flow velocity is 0.8mL/min, 30 ℃ of column temperatures; Adopt WATERS 2420ELSD detector, parameter is set to gain 10, air pressure 25psi, and 60 ℃ of drift tubes, Neb heater is 60%.
What collection was obtained respectively manages elutriant, and according to above-mentioned chromatographic condition, directly sample introduction successively, detects recording responses value.
7, the collection of oligomeric trisaccharide
To only contain retention time is the elutriant merging of 27.5 ± 0.2min chromatographic peak, and evaporate to dryness, obtains unknown compound, standby.
8, the structural confirmation of oligomeric trisaccharide
Unknown compound is soluble in water, rare alcohol, ethanol.Molish reaction is positive.Illustrate that this compound may be carbohydrate.
By high resolution mass spectrum,
1h-NMR,
13the methods such as C-NMR, DEPT, HMQC, H-HCOSY, HSQC, H-Htcosy, HMBC nuclear magnetic scanning, can determine that this unknown compound is: α-D-Glucopyranose-(1 → 2)-beta-D-fructofuranose-(1 → 1)-α-D-galactopyranose.
Embodiment 5
1, instrument
AKTA prime protein purification system (GE of GE Medical Group); High pressure water-cooling sandwich chromatography column (2.5cm * 100cm, Shanghai Chu Bai laboratory equipment company limited); Waters 2695 high performance liquid chromatographs (U.S. Waters company); Waters 2420ELSD detector (U.S. Waters company); Bio-Gel P-2 gathers propionic acid amide gel (U.S. Bio-Red company); Milli-Q Academic ultra-pure-water treatment system (U.S. Millipore company); Solid-phase extraction column (Cleanert PS-SPE, 500mg/6mL, Agela Science and Technology Ltd.); Thermostat water bath (Ningbo Tian Heng instrument plant).
2, reagent and reagent
Acetonitrile (chromatographically pure, Merck company); Methyl alcohol (analytical pure, Concord, Tianjin Science and Technology Ltd.); Ultrapure water (Milli-Q ultra-pure-water treatment system makes, specific conductivity 18.2M Ω cm).
3, the preparation of the total sugar extract of compound Salviae Miltiorrhizae medicinal extract
Solid-phase extraction column (Cleanert PS-SPE, 500mg/6mL) is used to 10mL washed with methanol, with 10mL water, rinse again afterwards, can be standby after flushing.
Precision takes 0.3g compound Salviae Miltiorrhizae medicinal extract, adds 10mL water, ultrasonic it is dissolved completely.Upper to processed good solid-phase extraction column (Cleanert PS-SPE, 500mg/6mL), the about 0.6mL/min of flow velocity, water gradation washing again, collects sample solution and the about 40mL of scrub stream fluid, evaporate to dryness, weigh, obtain altogether the total sugar extract of 0.15g compound Salviae Miltiorrhizae medicinal extract, standby.
4, the preparation of polyacrylamide gel chromatography column
Take the polyacrylamide gel (Bio-Gel P-2) of 200g, slowly pour in ultrapure water, at room temperature profit rises more than 12 hours.Upper strata fine particle is toppled over away, repeatedly for several times, till the fine particle without suspending.
By the poly-propionic acid amide gel vacuum outgas of handling well, by automatic sedimentation method, fill post to 100cm afterwards.Use again the ultrapure water of 2～3 times of bed volumes with constant flow velocity balance chromatography column, can be for later separation.
5, loading, wash-out, collection elutriant
After the 0.15g obtaining is processed, sample dissolves with the ultrapure water of 0.5mL, and after dissolving, loading is to processed good chromatography column, and chromatography column temperature will remain at 40 ℃.
With ultrapure water with 0.3mL/min(0.0367 volume/hour) speed carry out wash-out, every 10min collects 1 pipe elutriant, collects the stream part (collecting stream part of 106-123 pipe, 318-368ml) while being equivalent to 0.65-0.75 column volume, carries out HPLC and inspects.
6, the detection of elutriant
Chromatographic condition: adopt Prevail TM Carbohydrate ES chromatographic column, take acetonitrile as mobile phase A, water is Mobile phase B, and gradient is set to:
Flow velocity is 0.8mL/min, 30 ℃ of column temperatures; Adopt WATERS 2420ELSD detector, parameter is set to gain 10, air pressure 25psi, and 60 ℃ of drift tubes, Neb heater is 60%.
What collection was obtained respectively manages elutriant, and according to above-mentioned chromatographic condition, directly sample introduction successively, detects recording responses value.
7, the collection of oligomeric trisaccharide
To only contain retention time is the elutriant merging of 27.5 ± 0.2min chromatographic peak, and evaporate to dryness, obtains unknown compound, standby.
8, the structural confirmation of oligomeric trisaccharide
Unknown compound is soluble in water, rare alcohol, ethanol.Molish reaction is positive.Illustrate that this compound may be carbohydrate.
By high resolution mass spectrum,
1h-NMR,
13the methods such as C-NMR, DEPT, HMQC, H-HCOSY, HSQC, H-Htcosy, HMBC nuclear magnetic scanning, can determine that this unknown compound is: α-D-Glucopyranose-(1 → 2)-beta-D-fructofuranose-(1 → 1)-α-D-galactopyranose.
Claims (10)
1. a separation purification method for oligomeric trisaccharide in compound Salviae Miltiorrhizae medicinal extract, comprises the steps:
(1) preparation of the total sugar extract of compound Salviae Miltiorrhizae medicinal extract;
(2) the total sugar extract of compound Salviae Miltiorrhizae medicinal extract is crossed polyacrylamide gel chromatography column;
(3) collection of oligomeric trisaccharide composition.
2. the method for claim 1, wherein the preparation method of the total sugar extract of the described compound Salviae Miltiorrhizae medicinal extract of step (1) is:
Compound Salviae Miltiorrhizae medicinal extract adds water ultrasonic dissolution, and upper to processed good solid-phase extraction column, flow velocity is 0.5-1.0mL/min, then water gradation washing, collects sample solution and scrub stream fluid, and evaporate to dryness, obtains the total sugar extract of compound Salviae Miltiorrhizae medicinal extract.
3. method as claimed in claim 2, wherein said solid-phase extraction column adopts Cleanert PS-SPE column extractor, and before using, through processing, treatment process is: by solid-phase extraction column washed with methanol, water flushing more afterwards.
4. the method for claim 1, wherein the described polyacrylamide gel chromatography column excessively of step (2) is that the total sugar extract of compound Salviae Miltiorrhizae medicinal extract obtaining is dissolved with ultrapure water, after dissolving, loading is to processed good chromatography column, the column temperature of chromatography column is 35-45 ℃, with ultrapure water, carry out wash-out, collect the stream part while being equivalent to 0.65-0.75 column volume.
5. method as claimed in claim 4, wherein, polyacrylamide gel chromatography column obtains with following methods:
Take polyacrylamide gel, pour in ultrapure water, at room temperature profit rises more than 12 hours, vacuum outgas, and dress post, dress post height is 100-120cm, then uses the ultrapure water of 2 ~ 3 times of bed volumes with constant flow velocity balance chromatography column.
6. method as claimed in claim 4, wherein the column temperature of chromatography column is 40 ℃.
7. method as claimed in claim 4, wherein applied sample amount is total sugar extract/100 of 0.075-0.125g compound Salviae Miltiorrhizae medicinal extract gram polyacrylamide gels.
8. method as claimed in claim 4, the elution flow rate of wherein said chromatography column be 0.0184-0.0367 column volume/hour.
9. the method for claim 1, wherein the collection method of the described oligomeric trisaccharide composition of step (3) is: with HPLC method, elutriant is detected, the elutriant that collection retention time is 27.5 ± 0.2min, evaporate to dryness, obtains eluate.
10. method as claimed in claim 9, the method that wherein said HPLC detects elutriant is:
Adopt HPLC-ELSD method, HPLC adopts Prevail TM Carbohydrate ES chromatographic column, and chromatographic condition is for take acetonitrile as mobile phase A, and water is Mobile phase B, and gradient elution is set to:
Flow velocity 0.8mL/min, 30 ℃ of column temperatures; Adopt ELSD detector, detector parameters is set to gain 10, air pressure 25psi, and 60 ℃ of drift tubes, Neb heater is 60%.
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| CN107722074A (en) * | 2017-09-26 | 2018-02-23 | 江南大学 | A kind of calcium containing compound isolated from vinegar egg juice and its application |
| CN116626208A (en) * | 2023-07-21 | 2023-08-22 | 东莞理工学院 | Liquid chromatography-mass spectrometry combined detection method for galactooligosaccharide and application thereof |
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| CN107722074B (en) * | 2017-09-26 | 2020-01-24 | 江南大学 | Calcium-containing compound separated from vinegar egg liquid and application thereof |
| CN116626208A (en) * | 2023-07-21 | 2023-08-22 | 东莞理工学院 | Liquid chromatography-mass spectrometry combined detection method for galactooligosaccharide and application thereof |
| CN116626208B (en) * | 2023-07-21 | 2023-10-27 | 东莞理工学院 | Liquid chromatography-mass spectrometry combined detection method for galactooligosaccharide and application thereof |
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