CN1876669A - High purity pharmaceutical stevioside preparation method - Google Patents

High purity pharmaceutical stevioside preparation method Download PDF

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Publication number
CN1876669A
CN1876669A CN 200610010278 CN200610010278A CN1876669A CN 1876669 A CN1876669 A CN 1876669A CN 200610010278 CN200610010278 CN 200610010278 CN 200610010278 A CN200610010278 A CN 200610010278A CN 1876669 A CN1876669 A CN 1876669A
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time
stevioside
column chromatography
elutriant
wash
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CN 200610010278
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CN100395254C (en
Inventor
郝再彬
原益山
苍晶
杨丹
王贵民
一井真比古
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HARBIN LUYE TECHNOLOGY DEVELOPMENT Co Ltd
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HARBIN LUYE TECHNOLOGY DEVELOPMENT Co Ltd
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Abstract

The invention relates the preparing method of sweet chrysanthemum glycoside, comprising Al2O3 chromatography purification, compression and silica gel column chromatography purification. The first chromatography uses Al2O3 as fixed phase, removing amylaceum, barley sugar, protein and coloring matter, and collecting eluent. The second chromatography uses silica gel as fixed phase, removing impurity and collecting eluent to concentrate. The sweet chrysanthemum content is up to 95-98%. The invention has the advantages of high JECFA requirement and good application value.

Description

The preparation method of high purity pharmaceutical stevioside
One, technical field
What the present invention relates to is a kind of method of purification, specifically a kind of method of purification of stevioside.
Two, background technology
Purpose and meaning: sweeting agent mainly contains three major types in the industrial production:
The first kind is to be the chemical industry sintetics: asccharin, Sodium Cyclamate, aspartame etc.;
Second class is the natural plant sugar of chemical modification, as Xylitol, mannitol;
The 3rd class is a natural plant sugar.Natural plant sugar includes energy metabolism (sucrose, maltose, fructose, glucose etc.) again, noenergy metabolism (Radix Glycyrrhizae glucosides, stevioside etc.).
Along with improving constantly of human living standard, people tend to the higher natural product of safety in utilization more: Radix Glycyrrhizae glucosides and stevioside, therefore Radix Glycyrrhizae glucosides and stevioside have more wide application prospect in the life in future.
Stevioside has following characteristics:
1, stevioside sugariness height, relevant with glucosides concentration, be approximately 250～450 times of sucrose, sweet taste approaches sucrose, can replace asccharin and part sucrose; Stevioside mainly contains 8 kinds of one-tenth and is grouped into, and wherein Stevioside and Rebaudioside composition reach more than 90%, and the Rebaudioside sweet taste approaches sucrose most.
2, low in calories, its heat is approximately 1/300 of sucrose, good stability, and it does not participate in metabolism in human body.
3, avirulent characteristics, a large amount of experimentation on animalies has proved the stevioside nontoxicity, beginning in 1970, Japan will be from sweet Stevia extract in the blade stevioside as sweeting agent in industry widespread uses such as food, healthcare products, medicines.
4, reporting in addition that stevioside has diabetes, pediatric saprodontia, obesity, hypertension, heart trouble necessarily prevents and treats function and auxiliary curative effect.
In August, 2004, JECFA has approved the tentative case that qualities of stevia extract uses in the 63rd meeting.Should tentative case allow stevioside to use, think that the safety picked-up stevioside amount of per day for adults was 2mg/kg as sweeting agent.The enforcement that should try case means: the application of stevioside will go to the world from some areas.Must be more than 95% but JECFA thinks as the stevioside purity of sweeting agent; The stevioside purity of producing in China has only 80% at present, does not also reach the requirement of this tentative case.
Three, summary of the invention
The object of the present invention is to provide a kind of purity of stevioside can being reached more than 95%, make the concentration of stevioside can reach the preparation method of the high purity stevioside of medicinal standard.
The object of the present invention is achieved like this:
1, Al 2O 3Column chromatography purification
(1) Al 2O 3Pre-treatment: the Al that takes by weighing certainweight 2O 3, the HCL through 5% is washed till neutrality with distilled water after soaking 12h, wet method dress post, and blade diameter length ratio is 1: 10;
(2) go up sample: taking by weighing the purity that dries to constant weight under 80 ℃ is 80% stevioside coarse dry powder, and the coarse dry powder height is Al 2O 31/5 of height;
(3) wash-out and qualitative detection: the moving phase first time of column chromatography is acetone with the first time: sherwood oil=carry out wash-out at 1: 1, and the natural elution flow rate or the liquid level discrepancy in elevation are 1m, elutriant are carried out sulfuric acid colour developing qualitative detection, till redfree; Use instead with column chromatography for the first time the second time moving phase promptly 100% ethanol carry out wash-out, collect with automatic Fraction Collector simultaneously; Elutriant is carried out sulfuric acid colour developing qualitative detection, till not having a pink;
2. concentrate: collect the moving phase elutriant second time of column chromatography for the first time, concentrate;
3. silica gel column chromatography purifying
(1) dress post: take by weighing the silica gel of certainweight, the back wet method dress post that is soaked in water, blade diameter length ratio is 1: 10;
(2) go up sample: with sample on the concentrated liquid in the 2nd step, applied sample amount is 1/10 of a silicagel column height;
(3) wash-out: the moving phase of column chromatography is that 20% ethanol carries out wash-out with the second time, the nature elution flow rate or the liquid level discrepancy in elevation are 1m, elutriant is carried out sulfuric acid colour developing qualitative detection, no pink appears from pink and till, collect this section elutriant and obtain product of the present invention.
Product of the present invention can come qualitative and quantitative analysis by such method:
(1) qualitative detection the method----silica gel thin-layer chromatography of stevioside: with the boric acid solution of the 0.1mol/L system silica-gel plate of sizing mixing, expansion system: (propyl carbinol: acetate: ether: water=9: 6: 3: 1); Developer: 50% sulfuric acid.Present method can distinguish two kinds of principal monomer compositions (St and RA), maltose, the glucose in the stevioside.
(2) detection by quantitative of stevioside has 5 kinds of method references.
A. spectrophotometer visible light method.The patent of Hao Zaibin application: the method and the special agent of rapid determination stevioside content, number of patent application: 200510010277.3; Patent publication No.: CN1731144A; Publicity on February 8 in 2006 is seen Gazette of Patent for Invention 2006,22 (06): 311;
B. portable injection chemiluminescence method.Yang Dan, Hao Zaibin (2005), chemical engineer 04:23～25;
C. the Tripotassium iron hexacyanide-luminol,3-aminophthalic acid cyclic hydrazide portable injection chemiluminescence method.The patent (applying for) of Hao Zaibin application;
D. high pressure liquid chromatography uv detection method.Slope is herded (2004) such as Cheng Hui.Food sanitation will (Japanese).45(2)：81～86；
E. method such as capillary electrophoresis uv detection method.Shao Hanjuan, Hu Yonggang (2001).BULLETIN OF BOTANY Vol., 18 (1): 113～117.
The definition of high purity pharmaceutical stevioside: highly purified content is 95%-98%; The tentative case moderate purity that reaches the qualities of stevia extract use of JECFA approval simultaneously must be in the requirement more than 95%.[annotate: the tentative case of qualities of stevia extract use has been approved by the Expert C-on Food Additive (JECFA) under in August, 2004 World Health Organization and United Nations's grain tissue (WHO/FAO) in the 63rd meeting, purity must in the requirement more than 95%].
Medicinal: as to meet the requirement of Pharmacopoeia of People's Republic of China.
The present invention based on principle be: adsorption chromatography is exactly to utilize this adsorptive power of sorbent material can be subjected to solvent to influence the character that changes, after material in sample is adsorbed agent absorption, with suitable elutriant flushing, change the adsorptive power of sorbent material, make the material desorb that is adsorbed, move forward with elutriant.Through the process of desorb of repeatedly absorption-desorb-absorption again-again, material can move along the working direction of elutriant, and its translational speed depends on that sorbent material is to the adsorptive power of this material under the prevailing condition.If sorbent material is to the high adsorption capacity of material, the speed that this material moves forward is slow; Otherwise, if a little less than the adsorptive power, its translational speed is fast.Because sorbent material is to the adsorptive power difference of component in the sample, thus in elution process a component just can be owing to translational speed is different separating gradually, the ultimate principle of Here it is adsorption chromatography.
Can make the content of stevioside bring up to 95%--98% by 80% through method of the present invention, play the purpose of retrofit, the high purity stevioside of producing for China moves towards the world market and lays a good foundation.This is a stevioside big producing country for changing China, rather than produces the situation of power; For the stevioside of China industrial future, have certain theoretical significance and actual application value.
Chromatography is with Al for the first time 2O 3Be stationary phase,, can remove macromolecular impurity such as glucose, maltose, protein, pigment, concentrate behind the collection part elutriant and carry out the chromatography second time with the chromatographic flow phase wash-out first time.For the second time chromatography is stationary phase with silica gel, with chromatographic flow phase wash-out for the second time, can remove other impurity such as grade, collects to concentrate behind the part elutriant and carries out after testing that stevioside content can reach 95%--98%.
The method for preparing the high purity stevioside of the present invention can make the content of stevioside reach 98%, has reached the requirement of JECFA, has certain theoretical significance and higher actual application value.
Four, description of drawings
Fig. 1 is the structural formula of stevioside, is St when wherein R is H, is RA when R is glucose;
Fig. 2 is the color reaction of several sugar, wherein: 1.St; 2.RA; 3. maltose; 4. glucose;
The thin-layer chromatogram of several sugar of Fig. 3, wherein: 1.St; 2.RA; 3. glucose; 4. maltose.
Five, embodiment
Below the present invention is done more detailed description:
1. material: stevioside meal.
2. plant and instrument:
BT124S ten thousand/electronic balance;
Glass chromatography column (diameter 5.0cm * long 80.0cm);
Glass test tube;
SBSZ100 numerical control meter drips automatic Fraction Collector;
The UV-1600 ultraviolet-visible pectrophotometer.
3. reagent:
(1) reagent used of column chromatography: column chromatography: Al for the first time 2O 3, for the first time moving phase (acetone: sherwood oil=1: 1), moving phase (100% ethanol) for the second time; Column chromatography for the second time: silica gel (100 μ m), moving phase (20% ethanol).
(2) reagent used of qualitative detection: stevioside standard substance (RA of the st of 300 times of sucrose sweetness and 450 times of sucrose sweetness), glucose, maltose, propyl carbinol, acetate, ether, water, 50% sulfuric acid.
4.Al 2O 3The column chromatography purification stevioside
(1) Al 2O 3Pre-treatment: the Al that takes by weighing certainweight 2O 3, the HCL through 5% is washed till neutrality with distilled water after soaking 12h, wet method dress post, and blade diameter length ratio is 1: 10.
(2) go up sample: taking by weighing the purity that dries to constant weight under 80 is 80% stevioside sample (coarse dry powder), and the coarse dry powder height is Al 2O 31/5 of height.
(3) wash-out and qualitative detection: (acetone: sherwood oil=1: 1) carry out wash-out, the natural elution flow rate or the liquid level discrepancy in elevation are 1m, elutriant are carried out sulfuric acid colour developing qualitative detection (Fig. 2), till redfree with the moving phase first time of column chromatography for the first time.Use instead with the moving phase second time (100% ethanol) of column chromatography for the first time and carry out wash-out, collect with automatic Fraction Collector simultaneously.Elutriant is carried out sulfuric acid colour developing qualitative detection (Fig. 2), till not having a pink.
5. concentrate: collect the moving phase second time (100% ethanol) elutriant of column chromatography for the first time, concentrate.
6. silica gel column chromatography purifying stevioside
(1) dress post: take by weighing the silica gel of certainweight, the back wet method dress post that is soaked in water, blade diameter length ratio is 1: 10.
(2) go up sample: with sample on the concentrated liquid in the 5th step, applied sample amount is 1/10 of a silicagel column height.
(3) wash-out: carry out wash-out with the moving phase (20% ethanol) of column chromatography for the second time, the natural elution flow rate or the liquid level discrepancy in elevation are 1m, and elutriant is carried out sulfuric acid colour developing qualitative detection (Fig. 2), no pink appears from pink and till.Collect this section elutriant.
7. qualitative and quantitative analysis:
(1) silicon thin-layer chromatography qualitative detection stevioside: with the boric acid solution of the 0.1mol/L system silica-gel plate of sizing mixing, expansion system: (propyl carbinol: acetate: ether: water=9: 6: 3: 1); Developer: 50% sulfuric acid.Present method can distinguish (Fig. 3) with two kinds of principal monomer compositions (St and RA), maltose, the glucose in the stevioside.
(2) spectrophotometry detection by quantitative stevioside is (referring to the patent of Hao Zaibin application: the method and the special agent of rapid determination stevioside content, number of patent application: 200510010277.3; Patent publication No.: CN1731144A; Publicity on February 8 in 2006 is seen Gazette of Patent for Invention 2006,22 (06): 311)

Claims (1)

  1. A kind of preparation method of high purity pharmaceutical stevioside is characterized in that::
    1.1 Al 2O 3Column chromatography purification
    1.1.1 Al 2O 3Pre-treatment: the Al that takes by weighing certainweight 2O 3, the HCL through 5% is washed till neutrality with distilled water after soaking 12h, wet method dress post, and blade diameter length ratio is 1: 10;
    1.1.2 last sample: taking by weighing the purity that dries to constant weight under 80 ℃ is 80% stevioside coarse dry powder, and the coarse dry powder height is Al 2O 31/5 of height;
    1.1.3 wash-out and qualitative detection: the moving phase first time of column chromatography is acetone with the first time: sherwood oil=carry out wash-out at 1: 1, and the natural elution flow rate or the liquid level discrepancy in elevation are 1m, elutriant are carried out sulfuric acid colour developing qualitative detection, till redfree; Use instead with column chromatography for the first time the second time moving phase promptly 100% ethanol carry out wash-out, collect with automatic Fraction Collector simultaneously; Elutriant is carried out sulfuric acid colour developing qualitative detection, till not having a pink;
    1.2 concentrate: collect the moving phase elutriant second time of column chromatography for the first time, concentrate;
    1.3. silica gel column chromatography purifying
    1.3.1 dress post: take by weighing the silica gel of certainweight, the back wet method dress post that is soaked in water, blade diameter length ratio is 1: 10;
    1.3.2 last sample: with sample on the concentrated liquid in the 2nd step, applied sample amount is 1/10 of a silicagel column height;
    1.3.3 wash-out: the moving phase of column chromatography is that 20% ethanol carries out wash-out with the second time, the nature elution flow rate or the liquid level discrepancy in elevation are 1m, elutriant is carried out sulfuric acid colour developing qualitative detection, no pink appears from pink and till, collect this section elutriant and obtain product of the present invention.
CNB2006100102782A 2006-07-12 2006-07-12 High purity pharmaceutical stevioside preparation method Expired - Fee Related CN100395254C (en)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012042508A1 (en) * 2010-10-01 2012-04-05 Shanghai Yongyou Bioscience Inc. Separation and purification of stevioside and rebaudioside a
CN103667343A (en) * 2013-12-06 2014-03-26 安徽大学 Agrobacterium rhizogenes-mediated transgene Stevia rebaudiana genetic transformation method
CN109293712A (en) * 2018-09-30 2019-02-01 晨光生物科技集团股份有限公司 The industrialized utilization method and its steviol glycoside and chlorogenic acid of a kind of STEVIA REBAUDIANA

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS5241275A (en) * 1975-09-27 1977-03-30 Ajinomoto Kk Production of sweetening agent
JPS52110872A (en) * 1976-03-16 1977-09-17 Kaoru Koudaka Purifying method of sugar solution extracted from dried stevia leaf
JPS53113065A (en) * 1977-03-11 1978-10-03 Nikken Chemicals Co Ltd Obtaining method of novel sweetening substance

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012042508A1 (en) * 2010-10-01 2012-04-05 Shanghai Yongyou Bioscience Inc. Separation and purification of stevioside and rebaudioside a
CN103667343A (en) * 2013-12-06 2014-03-26 安徽大学 Agrobacterium rhizogenes-mediated transgene Stevia rebaudiana genetic transformation method
CN109293712A (en) * 2018-09-30 2019-02-01 晨光生物科技集团股份有限公司 The industrialized utilization method and its steviol glycoside and chlorogenic acid of a kind of STEVIA REBAUDIANA

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