CN105061630A - Method for extracting and purifying gentiana polysaccharide - Google Patents
Method for extracting and purifying gentiana polysaccharide Download PDFInfo
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- CN105061630A CN105061630A CN201510594389.1A CN201510594389A CN105061630A CN 105061630 A CN105061630 A CN 105061630A CN 201510594389 A CN201510594389 A CN 201510594389A CN 105061630 A CN105061630 A CN 105061630A
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Abstract
The invention discloses a method for extracting and purifying gentiana polysaccharide and relates to the technical field of deep processing of gentiana. The method comprises four operation steps of a pretreatment step, a degreasing step, a polysaccharide extraction step and a rough gentiana polysaccharide refining step. The method for extracting and purifying the gentiana polysaccharide, disclosed by the invention, is a method of preparing a rough gentiana polysaccharide product by using a water-extraction and alcohol-precipitation method and purifying by using an agarose gel column to prepare the refined gentiana polysaccharide. The extraction method is simple and low in operation cost; the yield of the gentiana polysaccharide obtained by extraction is 12.8 percent, and the yield of the gentiana polysaccharide obtained by adsorption and purification is 33.6 percent.
Description
Technical field
The present invention relates to the deep process technology field of rough gentian, particularly relate to the extracting and purifying method of Gentiana Polysaccharides.
Background technology
Rough gentian is Gentianaceae (Gentianaceae) plant Gentiana manshurica (GentianamanshuricaKitag.), rough gentian (GentianascabraBge.), the dry root and rhizome of G. triflora (GentianatrifloraPall.) or Yunnan rough gentian (GentianarigescensFranch.), rough gentian begins to be loaded in Shennong's Herbal, be classified as middle product, bitter, cold in nature, for clear dampness-heat in the liver and gallbladder, rush down the conventional Chinese medicine of part of the body cavity below the umbilicus, housing the bladder, kidneys and bowels stagnated fire, there is liver protecting, stomach invigorating, reducing blood-fat, anti-inflammatory, anti-hyperthyroidism, blood glucose increasing, anti-oxidant, antimycotic and hypotensive activity.Chemical efficiency composition in rough gentian comprises triterpenes, flavonoid, iridoids, mouth mountain ketone, alkaloids and polysaccharide, is main, lacks the research for Gentiana Polysaccharides purification process for the research of Gentiana Polysaccharides mainly with crude product.
Summary of the invention
The present invention, for above-mentioned the deficiencies in the prior art, provides one and utilizes water extraction and alcohol precipitation method to obtain bark of Radix Gentianae polysaccharide crude, utilize sepharose post to carry out the method for polysaccharide purification afterwards, and method is simple, and running cost is low.
The extracting and purifying method of Gentiana Polysaccharides of the present invention, its operation steps is as follows:
A, pre-treatment: after rough gentian is removed impurity, 55 ~ 65 DEG C of oven dry, are ground to 30 ~ 60 order powder;
B, degreasing:
(1) alcohol degreasing:
The rough gentian powder of a step process is immersed its weight is 5 ~ 15 times, mass concentration is in the ethanol of 85%, stir 1 ~ 4h 60 ~ 80 DEG C of reflux, after cooling, continue stirring 10 ~ 18h under room temperature, remove liquid; Secondary adds its weight 5 ~ 15 times, mass concentration is the ethanol of 85%, stirred at ambient temperature 3 ~ 7 hours, removes liquid;
(2) acetone degreasing:
Rough gentian powder after alcohol degreasing is added its weight 2 ~ 8 times, mass concentration is the acetone soln of 99%, remove liquid after stirring 0.2 ~ 1h, by this powder at room temperature seasoning 10 ~ 18h, obtain degreasing rough gentian powder;
C, Polyose extraction:
Degreasing rough gentian powder b step obtained adds the water of its weight 20 ~ 50 times, and at 90 DEG C, reflux stirs 1 ~ 5h; Get its lysate, under 50 DEG C of conditions, be only concentrated into 1/6 ~ 1/2 of original volume; In this concentrated solution, add ethanol, to its system, alcohol concn is not less than 75%, adjustment temperature to 2 ~ 6 DEG C, leaves standstill 10 ~ 18h, has polysaccharide floss to separate out, filtering liquid, at room temperature seasoning 10 ~ 18h; Successively wash respectively with the dehydrated alcohol of its weight 2 ~ 8 times and the acetone of 2 ~ 8 times, get its throw out seasoning 10 ~ 18h, obtained thick Gentiana Polysaccharides;
D, thick Gentiana Polysaccharides are refined:
(1) low molecular weight impurities is gone:
Thick Gentiana Polysaccharides step c obtained adds the water of its weight 5 ~ 15 times, is separated, gets the dialyzate that its molecular weight is greater than 3500Da with the dialysis of 3500Da dialysis membrane, by its freeze-drying, and obtained removal of impurity Gentiana Polysaccharides;
(2) fractionation by adsorption:
The removal of impurity Gentiana Polysaccharides that (1) step obtains is added its weight to be the water dissolution of 0.01 ~ 0.06 times, to be heated to 60 DEG C of dissolvings; Be separated with DEAE-gelose gel column chromatography, after first rinsing 2h with distilled water coutroi velocity 1.5mL/min, collect desorbed solution 3h; After continuing to rinse 3h with 0.5MNaCl with same flow velocity again, collect desorbed solution 2h; Improving parsing agent NaCl concentration is 1.0M, continues to collect desorbed solution 2h, collects the desorbed solution of above-mentioned three steps, be Gentiana Polysaccharides mixed solution;
(3) refining:
By the Gentiana Polysaccharides mixed solution of (2) step gained, with the dialysis of 3500Da dialysis membrane, get the dialyzate that its molecular weight is greater than 3500Da and carry out freeze-drying, obtained finished product Gentiana Polysaccharides.
As a further improvement on the present invention, its operation steps is as follows:
A, pre-treatment: after rough gentian is removed impurity, 55 ~ 65 DEG C of oven dry, are ground to 40 ~ 50 order powder;
B, degreasing:
(1) alcohol degreasing:
The rough gentian powder of a step process is immersed its weight is 8 ~ 12 times, mass concentration is in the ethanol of 85%, stir 2 ~ 3h 65 ~ 75 DEG C of reflux, after cooling, continue stirring 12 ~ 16h under room temperature, remove liquid; Secondary adds its weight 8 ~ 12 times, mass concentration is the ethanol of 85%, stirred at ambient temperature 4 ~ 5 hours, removes liquid;
(2) acetone degreasing:
Rough gentian powder after alcohol degreasing is added its weight 4 ~ 6 times, mass concentration is the acetone soln of 99%, remove liquid after stirring 0.4 ~ 0.8h, by this powder at room temperature seasoning 12 ~ 16h, obtain degreasing rough gentian powder;
C, Polyose extraction:
Degreasing rough gentian powder b step obtained adds the water of its weight 30 ~ 40 times, and at 90 DEG C, reflux stirs 2 ~ 4h; Get its lysate, under 50 DEG C of conditions, be only concentrated into 1/5 ~ 1/3 of original volume; In this concentrated solution, add ethanol, to its system, ethanol mass concentration is not less than 75%, adjustment temperature to 3 ~ 5 DEG C, leaves standstill 12 ~ 16h, has polysaccharide floss to separate out, filtering liquid, at room temperature seasoning 12 ~ 16h; Successively wash respectively with the dehydrated alcohol of its weight 4 ~ 6 times and the acetone of 4 ~ 6 times, get its throw out seasoning 12 ~ 16h, obtained thick Gentiana Polysaccharides;
D, thick Gentiana Polysaccharides are refined:
(1) low molecular weight impurities is gone:
Thick Gentiana Polysaccharides step c obtained adds the water of its weight 8 ~ 12 times, is separated, gets the dialyzate that its molecular weight is greater than 3500Da with the dialysis of 3500Da dialysis membrane, by its freeze-drying, and obtained removal of impurity Gentiana Polysaccharides;
(2) fractionation by adsorption:
The removal of impurity Gentiana Polysaccharides that (1) step obtains is added its weight to be the water dissolution of 0.02 ~ 0.04 times, to be heated to 60 DEG C of dissolvings; Be separated with DEAE-gelose gel column chromatography, after first rinsing 2h with distilled water coutroi velocity 1.5mL/min, collect desorbed solution 3h; After continuing to rinse 3h with 0.5MNaCl with same flow velocity again, collect desorbed solution 2h; Improving parsing agent NaCl concentration is 1.0M, continues to collect desorbed solution 2h, collects the desorbed solution of above-mentioned three steps, be Gentiana Polysaccharides mixed solution;
(3) refining:
By the Gentiana Polysaccharides mixed solution of (2) step gained, with the dialysis of 3500Da dialysis membrane, get the dialyzate that its molecular weight is greater than 3500Da and carry out freeze-drying, obtained finished product Gentiana Polysaccharides.
As a further improvement on the present invention, in step c, described liquid to be evaporated is that degreasing rough gentian powder carries out more than the 1 time latter incorporated liquid of reflux continuously.
The extracting and purifying method of Gentiana Polysaccharides of the present invention, utilize water extraction and alcohol precipitation method to obtain Gentiana Polysaccharides crude product, recycling sepharose column purification obtains the method for refining Gentiana Polysaccharides, this extracting method is simple, running cost is low, its productive rate of the Gentiana Polysaccharides of gained is 12.8%, and obtaining its productive rate of Gentiana Polysaccharides through adsorption and purification is 33.6%.
Accompanying drawing explanation
The HPSEC color atlas of the thick Gentiana Polysaccharides of Fig. 1;
The HPSEC color atlas of Fig. 2 Gentiana Polysaccharides separated portion I;
The HPSEC color atlas of Fig. 3 Gentiana Polysaccharides separated portion II.
Embodiment
Embodiment 1
The extracting and purifying method of Gentiana Polysaccharides of the present invention, its operation steps is as follows:
A, pre-treatment: after rough gentian is removed impurity, 55 DEG C of oven dry, are ground to 30 order powder;
B, degreasing:
(1) alcohol degreasing:
The rough gentian powder of a step process is immersed its weight is 5 times, mass concentration is in the ethanol of 85%, stir 1h 60 DEG C of reflux, continue under room temperature to stir 10h after cooling, remove liquid; Secondary adds its weight 5 times, mass concentration is the ethanol of 85%, stirred at ambient temperature 3 hours, removes liquid;
(2) acetone degreasing:
Rough gentian powder after alcohol degreasing is added its weight 2 times, mass concentration is the acetone soln of 99%, remove liquid after stirring 0.2h, by this powder at room temperature seasoning 10h, obtain degreasing rough gentian powder;
C, Polyose extraction:
Degreasing rough gentian powder b step obtained adds the water of its weight 20 times, and at 90 DEG C, reflux stirs 1h; Get its lysate, under 50 DEG C of conditions, be only concentrated into 1/6 of original volume; In this concentrated solution, add ethanol, to its system, alcohol concn is not less than 75%, adjustment temperature to 2 DEG C, leaves standstill 10h, has polysaccharide floss to separate out, filtering liquid, at room temperature seasoning 10h; Successively wash respectively with the dehydrated alcohol of its weight 2 times and the acetone of 2 times, get its throw out seasoning 10h, obtained thick Gentiana Polysaccharides;
D, thick Gentiana Polysaccharides are refined:
(1) low molecular weight impurities is gone:
Thick Gentiana Polysaccharides step c obtained adds the water of its weight 5 times, is separated 3d, gets the dialyzate that its molecular weight is greater than 3500Da with the dialysis of 3500Da dialysis membrane, by its freeze-drying, and obtained removal of impurity Gentiana Polysaccharides;
(2) fractionation by adsorption:
The removal of impurity Gentiana Polysaccharides that (1) step obtains is added its weight to be the water dissolution of 0.01 times, to be heated to 60 DEG C of dissolvings; Be separated with DEAE-gelose gel column chromatography, after first rinsing 2h with distilled water coutroi velocity 1.5mL/min, collect desorbed solution 3h; After continuing to rinse 3h with 0.5MNaCl with same flow velocity again, collect desorbed solution 2h; Improving parsing agent NaCl concentration is 1.0M, continues to collect desorbed solution 2h, collects the desorbed solution of above-mentioned three steps, be Gentiana Polysaccharides mixed solution;
(3) refining:
By the Gentiana Polysaccharides mixed solution of (2) step gained, with 3500Da dialysis membrane dialysis 3d, get the dialyzate that its molecular weight is greater than 3500Da and carry out freeze-drying, obtained finished product Gentiana Polysaccharides.
Embodiment 2
The extracting and purifying method of Gentiana Polysaccharides of the present invention, its operation steps is as follows:
A, pre-treatment: after rough gentian is removed impurity, 65 DEG C of oven dry, are ground to 60 order powder;
B, degreasing:
(1) alcohol degreasing:
The rough gentian powder of a step process is immersed its weight is 15 times, mass concentration is in the ethanol of 85%, stir 4h 80 DEG C of reflux, continue under room temperature to stir 18h after cooling, remove liquid; Secondary adds its weight 15 times, mass concentration is the ethanol of 85%, stirred at ambient temperature 7 hours, removes liquid;
(2) acetone degreasing:
Rough gentian powder after alcohol degreasing is added its weight 8 times, mass concentration is the acetone soln of 99%, remove liquid after stirring 1h, by this powder at room temperature seasoning 18h, obtain degreasing rough gentian powder;
C, Polyose extraction:
Degreasing rough gentian powder b step obtained adds the water of its weight 50 times, and at 90 DEG C, reflux stirs 5h; Get its lysate, under 50 DEG C of conditions, be only concentrated into 1/2 of original volume; In this concentrated solution, add ethanol, to its system, alcohol concn is not less than 75%, adjustment temperature to 6 DEG C, leaves standstill 18h, has polysaccharide floss to separate out, filtering liquid, at room temperature seasoning 18h; Successively wash respectively with the dehydrated alcohol of its weight 8 times and the acetone of 8 times, get its throw out seasoning 18h, obtained thick Gentiana Polysaccharides;
D, thick Gentiana Polysaccharides are refined:
(1) low molecular weight impurities is gone:
Thick Gentiana Polysaccharides step c obtained adds the water of its weight 15 times, is separated 3d, gets the dialyzate that its molecular weight is greater than 3500Da with the dialysis of 3500Da dialysis membrane, by its freeze-drying, and obtained removal of impurity Gentiana Polysaccharides;
(2) fractionation by adsorption:
The removal of impurity Gentiana Polysaccharides that (1) step obtains is added its weight to be the water dissolution of 0.06 times, to be heated to 60 DEG C of dissolvings; Be separated with DEAE-gelose gel column chromatography, after first rinsing 2h with distilled water coutroi velocity 1.5mL/min, collect desorbed solution 3h; After continuing to rinse 3h with 0.5MNaCl with same flow velocity again, collect desorbed solution 2h; Improving parsing agent NaCl concentration is 1.0M, continues to collect desorbed solution 2h, collects the desorbed solution of above-mentioned three steps, be Gentiana Polysaccharides mixed solution;
(3) refining:
By the Gentiana Polysaccharides mixed solution of (2) step gained, with 3500Da dialysis membrane dialysis 3d, get the dialyzate that its molecular weight is greater than 3500Da and carry out freeze-drying, obtained finished product Gentiana Polysaccharides.
Embodiment 3
The extracting and purifying method of Gentiana Polysaccharides, its operation steps is as follows:
A, pre-treatment: after rough gentian is removed impurity, 55 DEG C of oven dry, are ground to 40 order powder;
B, degreasing:
(1) alcohol degreasing:
The rough gentian powder of a step process is immersed its weight is 8 times, mass concentration is in the ethanol of 85%, stir 2h 65 DEG C of reflux, continue under room temperature to stir 12h after cooling, remove liquid; Secondary adds its weight 8 times, mass concentration is the ethanol of 85%, stirred at ambient temperature 4 hours, removes liquid;
(2) acetone degreasing:
Rough gentian powder after alcohol degreasing is added its weight 4 times, mass concentration is the acetone soln of 99%, remove liquid after stirring 0.4h, by this powder at room temperature seasoning 12h, obtain degreasing rough gentian powder;
C, Polyose extraction:
Degreasing rough gentian powder b step obtained adds the water of its weight 30 times, and at 90 DEG C, reflux stirs 2h; Get its lysate, under 50 DEG C of conditions, be only concentrated into 1/5 of original volume; In this concentrated solution, add ethanol, to its system, ethanol mass concentration is not less than 75%, adjustment temperature to 3 DEG C, leaves standstill 12h, has polysaccharide floss to separate out, filtering liquid, at room temperature seasoning 12h; Successively wash respectively with the dehydrated alcohol of its weight 4 times and the acetone of 4 times, get its throw out seasoning 12h, obtained thick Gentiana Polysaccharides;
D, thick Gentiana Polysaccharides are refined:
(1) low molecular weight impurities is gone:
Thick Gentiana Polysaccharides step c obtained adds the water of its weight 8 times, is separated 3d, gets the dialyzate that its molecular weight is greater than 3500Da with the dialysis of 3500Da dialysis membrane, by its freeze-drying, and obtained removal of impurity Gentiana Polysaccharides;
(2) fractionation by adsorption:
The removal of impurity Gentiana Polysaccharides that (1) step obtains is added its weight to be the water dissolution of 0.02 times, to be heated to 60 DEG C of dissolvings; Be separated with DEAE-gelose gel column chromatography, after first rinsing 2h with distilled water coutroi velocity 1.5mL/min, collect desorbed solution 3h; After continuing to rinse 3h with 0.5MNaCl with same flow velocity again, collect desorbed solution 2h; Improving parsing agent NaCl concentration is 1.0M, continues to collect desorbed solution 2h, collects the desorbed solution of above-mentioned three steps, be Gentiana Polysaccharides mixed solution;
(3) refining:
By the Gentiana Polysaccharides mixed solution of (2) step gained, with 3500Da dialysis membrane dialysis 3d, get the dialyzate that its molecular weight is greater than 3500Da and carry out freeze-drying, obtained finished product Gentiana Polysaccharides.
Embodiment 4
The extracting and purifying method of Gentiana Polysaccharides, its operation steps is as follows:
A, pre-treatment: after rough gentian is removed impurity, 65 DEG C of oven dry, are ground to 50 order powder;
B, degreasing:
(1) alcohol degreasing:
The rough gentian powder of a step process is immersed its weight is 12 times, mass concentration is in the ethanol of 85%, stir 3h 75 DEG C of reflux, continue under room temperature to stir 16h after cooling, remove liquid; Secondary adds its weight 12 times, mass concentration is the ethanol of 85%, stirred at ambient temperature 5 hours, removes liquid;
(2) acetone degreasing:
Rough gentian powder after alcohol degreasing is added its weight 6 times, mass concentration is the acetone soln of 99%, remove liquid after stirring 0.8h, by this powder at room temperature seasoning 16h, obtain degreasing rough gentian powder;
C, Polyose extraction:
Degreasing rough gentian powder b step obtained adds the water of its weight 40 times, and at 90 DEG C, reflux stirs 4h; Get its lysate, under 50 DEG C of conditions, be only concentrated into 1/3 of original volume; In this concentrated solution, add ethanol, to its system, ethanol mass concentration is not less than 75%, adjustment temperature to 5 DEG C, leaves standstill 16h, has polysaccharide floss to separate out, filtering liquid, at room temperature seasoning 16h; Successively wash respectively with the dehydrated alcohol of its weight 6 times and the acetone of 6 times, get its throw out seasoning 16h, obtained thick Gentiana Polysaccharides;
D, thick Gentiana Polysaccharides are refined:
(1) low molecular weight impurities is gone:
Thick Gentiana Polysaccharides step c obtained adds the water of its weight 12 times, is separated 3d, gets the dialyzate that its molecular weight is greater than 3500Da with the dialysis of 3500Da dialysis membrane, by its freeze-drying, and obtained removal of impurity Gentiana Polysaccharides;
(2) fractionation by adsorption:
The removal of impurity Gentiana Polysaccharides that (1) step obtains is added its weight to be the water dissolution of 0.04 times, to be heated to 60 DEG C of dissolvings; Be separated with DEAE-gelose gel column chromatography, after first rinsing 2h with distilled water coutroi velocity 1.5mL/min, collect desorbed solution 3h; After continuing to rinse 3h with 0.5MNaCl with same flow velocity again, collect desorbed solution 2h; Improving parsing agent NaCl concentration is 1.0M, continues to collect desorbed solution 2h, collects the desorbed solution of above-mentioned three steps, be Gentiana Polysaccharides mixed solution;
(3) refining:
By the Gentiana Polysaccharides mixed solution of (2) step gained, with 3500Da dialysis membrane dialysis 3d, get the dialyzate that its molecular weight is greater than 3500Da and carry out freeze-drying, obtained finished product Gentiana Polysaccharides.
Embodiment 5
The extracting and purifying method of Gentiana Polysaccharides, its operation steps is as follows:
A, pre-treatment: after rough gentian is removed impurity, 50 DEG C of oven dry, are ground to 45 order powder;
B, degreasing:
(1) alcohol degreasing:
The rough gentian powder of a step process is immersed its weight is 10 times, mass concentration is in the ethanol of 85%, stir 2.5h 70 DEG C of reflux, continue under room temperature to stir 14h after cooling, remove liquid; Secondary adds its weight 10 times, mass concentration is the ethanol of 85%, stirred at ambient temperature 4.5 hours, removes liquid;
(2) acetone degreasing:
Rough gentian powder after alcohol degreasing is added its weight 5 times, mass concentration is the acetone soln of 99%, remove liquid after stirring 0.6h, by this powder at room temperature seasoning 14h, obtain degreasing rough gentian powder;
C, Polyose extraction:
Degreasing rough gentian powder b step obtained adds the water of its weight 35 times, and at 90 DEG C, reflux stirs 3h; Get its lysate, under 50 DEG C of conditions, be only concentrated into 1/4 of original volume; In this concentrated solution, add ethanol, to its system, ethanol mass concentration is not less than 75%, adjustment temperature to 4 DEG C, leaves standstill 14h, has polysaccharide floss to separate out, filtering liquid, at room temperature seasoning 14h; Successively wash respectively with the dehydrated alcohol of its weight 5 times and the acetone of 5 times, get its throw out seasoning 14h, obtained thick Gentiana Polysaccharides;
D, thick Gentiana Polysaccharides are refined:
(1) low molecular weight impurities is gone:
Thick Gentiana Polysaccharides step c obtained adds the water of its weight 10 times, is separated 3d, gets the dialyzate that its molecular weight is greater than 3500Da with the dialysis of 3500Da dialysis membrane, by its freeze-drying, and obtained removal of impurity Gentiana Polysaccharides;
(2) fractionation by adsorption:
The removal of impurity Gentiana Polysaccharides that (1) step obtains is added its weight to be the water dissolution of 0.03 times, to be heated to 60 DEG C of dissolvings; Be separated with DEAE-gelose gel column chromatography, after first rinsing 2h with distilled water coutroi velocity 1.5mL/min, collect desorbed solution 3h; After continuing to rinse 3h with 0.5MNaCl with same flow velocity again, collect desorbed solution 2h; Improving parsing agent NaCl concentration is 1.0M, continues to collect desorbed solution 2h, collects the desorbed solution of above-mentioned three steps, be Gentiana Polysaccharides mixed solution;
(3) refining:
By the Gentiana Polysaccharides mixed solution of (2) step gained, with 3500Da dialysis membrane dialysis 3d, get the dialyzate that its molecular weight is greater than 3500Da and carry out freeze-drying, obtained finished product Gentiana Polysaccharides.
Aforesaid method is utilized to be that raw material extracts Gentiana Polysaccharides with rough gentian, wherein d(2) in step separable go out two Gentiana Polysaccharides components, after first it rinse 2h with distilled water coutroi velocity 1.5mL/min, collect desorbed solution 3h, the desorbed solution of this collection step is Gentiana Polysaccharides separated portion I desorbed solution; After continuing to rinse 3h with 0.5MNaCl with same flow velocity again, collect desorbed solution 2h, improving parsing agent NaCl concentration is 1.0M, continue to collect desorbed solution 2h, the desorbed solution of two consecutive steps collections is Gentiana Polysaccharides separated portion II desorbed solution herein, finally by freeze-drying after two parts of desorbed solution dialysis, obtain Gentiana Polysaccharides separated portion I and Gentiana Polysaccharides separated portion II.
Utilize aforesaid method to obtain the productive rate of thick Gentiana Polysaccharides, Gentiana Polysaccharides separated portion I and Gentiana Polysaccharides separated portion II and total reducing sugar amount as shown in table 1, the composition of its Gentiana Polysaccharides, Gentiana Polysaccharides separated portion I and Gentiana Polysaccharides separated portion II is as shown in table 2:
Table 1 thick Gentiana Polysaccharides, Gentiana Polysaccharides separated portion I and Gentiana Polysaccharides separated portion II productive rate
The chemical constitution of table 2 thick Gentiana Polysaccharides, Gentiana Polysaccharides separated portion I and Gentiana Polysaccharides separated portion II
In table 1, table 2,
aoutput=(polysaccharide crude weight
/rough gentian raw material weight) × 100,
boutput=(separated portion weight
/inject the weight of separator column polysaccharide crude) × 100.
Utilize aforesaid method to obtain thick Gentiana Polysaccharides, Gentiana Polysaccharides separated portion I and Gentiana Polysaccharides separated portion II, its molecular weight determination and measurement result as follows:
The measuring method of molecular weight: take 5mg polysaccharide sample dissolution in 2mL distilled water, detection system is injected with after 3 μm of membrane filtrations after 60 DEG C of dissolvings, what adopt is HPSEC-UV-MALLS-RI(Efficient numerical method-UV-detector-multi-angle laser light scattering instrument-differential refraction detector combined system) determining molecular weight, this system comprises pump, syringe, Size Exclusion Chromatograph SEC post (TSKG5000PW; 7.5 × 600mm, TosoBiosep, Mongomeryville, PA, USA), 280nmUV detector, RI detects, and moving phase is 0.15MNaNO
3and 0.02%NaN
3, flow velocity is 0.4mL/min, and interpretation of result adopts ASTRA6.1 software (WyattTechnologyCorp).
Detected result is as shown in Fig. 1 ~ 3 and table 3, Fig. 1 ~ 3 are the HPSEC color atlas of thick Gentiana Polysaccharides, Gentiana Polysaccharides separated portion I and Gentiana Polysaccharides separated portion II respectively, and table 3 is molecular-weight average (Mw) and the rotation radius (Rg) of thick Gentiana Polysaccharides, Gentiana Polysaccharides separated portion I and Gentiana Polysaccharides separated portion II.
The molecular-weight average of table 3 thick Gentiana Polysaccharides, Gentiana Polysaccharides separated portion I and Gentiana Polysaccharides separated portion II (
m w) and rotation radius (
r g)
Claims (3)
1. the extracting and purifying method of Gentiana Polysaccharides, its operation steps is as follows:
A, pre-treatment: after rough gentian is removed impurity, 55 ~ 65 DEG C of oven dry, are ground to 30 ~ 60 order powder;
B, degreasing:
(1) alcohol degreasing:
The rough gentian powder of a step process is immersed its weight is 5 ~ 15 times, mass concentration is in the ethanol of 85%, stir 1 ~ 4h 60 ~ 80 DEG C of reflux, after cooling, continue stirring 10 ~ 18h under room temperature, remove liquid; Secondary adds its weight 5 ~ 15 times, mass concentration is the ethanol of 85%, stirred at ambient temperature 3 ~ 7 hours, removes liquid;
(2) acetone degreasing:
Rough gentian powder after alcohol degreasing is added its weight 2 ~ 8 times, mass concentration is the acetone soln of 99%, remove liquid after stirring 0.2 ~ 1h, by this powder at room temperature seasoning 10 ~ 18h, obtain degreasing rough gentian powder;
C, Polyose extraction:
Degreasing rough gentian powder b step obtained adds the water of its weight 20 ~ 50 times, and at 90 DEG C, reflux stirs 1 ~ 5h; Get its lysate, under 50 DEG C of conditions, be only concentrated into 1/6 ~ 1/2 of original volume; In this concentrated solution, add ethanol, to its system, alcohol concn is not less than 75%, adjustment temperature to 2 ~ 6 DEG C, leaves standstill 10 ~ 18h, has polysaccharide floss to separate out, filtering liquid, at room temperature seasoning 10 ~ 18h; Successively wash respectively with the dehydrated alcohol of its weight 2 ~ 8 times and the acetone of 2 ~ 8 times, get its throw out seasoning 10 ~ 18h, obtained thick Gentiana Polysaccharides;
D, thick Gentiana Polysaccharides are refined:
(1) low molecular weight impurities is gone:
Thick Gentiana Polysaccharides step c obtained adds the water of its weight 5 ~ 15 times, is separated, gets the dialyzate that its molecular weight is greater than 3500Da with the dialysis of 3500Da dialysis membrane, by its freeze-drying, and obtained removal of impurity Gentiana Polysaccharides;
(2) fractionation by adsorption:
The removal of impurity Gentiana Polysaccharides that (1) step obtains is added its weight to be the water dissolution of 0.01 ~ 0.06 times, to be heated to 60 DEG C of dissolvings; Be separated with DEAE-gelose gel column chromatography, after first rinsing 2h with distilled water coutroi velocity 1.5mL/min, collect desorbed solution 3h; After continuing to rinse 3h with 0.5MNaCl with same flow velocity again, collect desorbed solution 2h; Improving parsing agent NaCl concentration is 1.0M, continues to collect desorbed solution 2h, collects the desorbed solution of above-mentioned three steps, be Gentiana Polysaccharides mixed solution;
(3) refining:
By the Gentiana Polysaccharides mixed solution of (2) step gained, with the dialysis of 3500Da dialysis membrane, get the dialyzate that its molecular weight is greater than 3500Da and carry out freeze-drying, obtained finished product Gentiana Polysaccharides.
2. the extracting and purifying method of Gentiana Polysaccharides as claimed in claim 1, its operation steps is as follows:
A, pre-treatment: after rough gentian is removed impurity, 55 ~ 65 DEG C of oven dry, are ground to 40 ~ 50 order powder;
B, degreasing:
(1) alcohol degreasing:
The rough gentian powder of a step process is immersed its weight is 8 ~ 12 times, mass concentration is in the ethanol of 85%, stir 2 ~ 3h 65 ~ 75 DEG C of reflux, after cooling, continue stirring 12 ~ 16h under room temperature, remove liquid; Secondary adds its weight 8 ~ 12 times, mass concentration is the ethanol of 85%, stirred at ambient temperature 4 ~ 5 hours, removes liquid;
(2) acetone degreasing:
Rough gentian powder after alcohol degreasing is added its weight 4 ~ 6 times, mass concentration is the acetone soln of 99%, remove liquid after stirring 0.4 ~ 0.8h, by this powder at room temperature seasoning 12 ~ 16h, obtain degreasing rough gentian powder;
C, Polyose extraction:
Degreasing rough gentian powder b step obtained adds the water of its weight 30 ~ 40 times, and at 90 DEG C, reflux stirs 2 ~ 4h; Get its lysate, under 50 DEG C of conditions, be only concentrated into 1/5 ~ 1/3 of original volume; In this concentrated solution, add ethanol, to its system, ethanol mass concentration is not less than 75%, adjustment temperature to 3 ~ 5 DEG C, leaves standstill 12 ~ 16h, has polysaccharide floss to separate out, filtering liquid, at room temperature seasoning 12 ~ 16h; Successively wash respectively with the dehydrated alcohol of its weight 4 ~ 6 times and the acetone of 4 ~ 6 times, get its throw out seasoning 12 ~ 16h, obtained thick Gentiana Polysaccharides;
D, thick Gentiana Polysaccharides are refined:
(1) low molecular weight impurities is gone:
Thick Gentiana Polysaccharides step c obtained adds the water of its weight 8 ~ 12 times, is separated, gets the dialyzate that its molecular weight is greater than 3500Da with the dialysis of 3500Da dialysis membrane, by its freeze-drying, and obtained removal of impurity Gentiana Polysaccharides;
(2) fractionation by adsorption:
The removal of impurity Gentiana Polysaccharides that (1) step obtains is added its weight to be the water dissolution of 0.02 ~ 0.04 times, to be heated to 60 DEG C of dissolvings; Be separated with DEAE-gelose gel column chromatography, after first rinsing 2h with distilled water coutroi velocity 1.5mL/min, collect desorbed solution 3h; After continuing to rinse 3h with 0.5MNaCl with same flow velocity again, collect desorbed solution 2h; Improving parsing agent NaCl concentration is 1.0M, continues to collect desorbed solution 2h, collects the desorbed solution of above-mentioned three steps, be Gentiana Polysaccharides mixed solution;
(3) refining:
By the Gentiana Polysaccharides mixed solution of (2) step gained, with the dialysis of 3500Da dialysis membrane, get the dialyzate that its molecular weight is greater than 3500Da and carry out freeze-drying, obtained finished product Gentiana Polysaccharides.
3. the extracting and purifying method of Gentiana Polysaccharides as claimed in claim 1, it is characterized in that in step c, described liquid to be evaporated is that degreasing rough gentian powder carries out more than the 1 time latter incorporated liquid of reflux continuously.
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| CN115109168A (en) * | 2022-04-22 | 2022-09-27 | 云南中医药大学 | Gentiana rigescens neutral polysaccharide and preparation method and application thereof |
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Application publication date: 20151118 |