CN104479046A - Method for increasing yield of chondroitin sulfate - Google Patents
Method for increasing yield of chondroitin sulfate Download PDFInfo
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- CN104479046A CN104479046A CN201410813443.2A CN201410813443A CN104479046A CN 104479046 A CN104479046 A CN 104479046A CN 201410813443 A CN201410813443 A CN 201410813443A CN 104479046 A CN104479046 A CN 104479046A
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Abstract
The invention discloses an extraction process for preparing high-purity and high-yield chondroitin sulfate and belongs to the chemical field. The extraction process mainly comprises extraction with acetone, purification with ethanol and the like and solves the problem that oversulfated chondroitin sulfate is mixed in the chondroitin sulfate production process at present. The cost is saved, the purity of the chondroitin sulfate is increased, and the process is simple, easy to operate and suitable for industrial production.
Description
Technical field
This law illustrates and relates to a kind of method extracting synthesis bulk drug, and belong to chemical field, specifically one prepares high-purity high-yield fine work chondroitin sulfate.
Background technology
Chondroitin sulfate is the sodium salt of the CSSO3 extracted in the intestinal mucosa by pig or ox, and belong to mucopolysaccharide material, the form in animal body mainly with chondroitin sulfate-protein complex exists.Medically, chondroitin sulfate all has blood coagulation resisting function in vivo and in vitro, can extend clotting time, prothrombin time and thrombin time.There is anticoagulation clinically, reducing blood-fat, protection endotheliocyte, antiplatelet gather and discharge, short fibrinolytic, suppress aortic smooth muscle cell proliferation, reduce the effect such as blood viscosity and anti-inflammatory.
China is chondroitin sulfate production of raw medicine state maximum in the world, and the method for raw materials medicine chondroitin sulfate mainly contains two kinds: biological enzyme, chemical cleavage method.Biological enzyme mainly produces chondroitin sulfate with enzyme liberating, and method is gentle, but cost is very high, is not suitable for batch production: another kind of chemical cleavage method, output is many, but can affect the activity of chondroitin sulfate in process, is not suitable with medicinal.This technique has drawn the advantage of above two kinds of techniques, and multiple extraction, extraction and ion-exchange techniques, method is gentle, and extracted amount is relatively large, and purity is higher, and can remove the impurity of chondroitin sulfate to greatest extent, such as chondroitin polysulfate.
Summary of the invention
Object of the present invention in order to improve the deficiencies in the prior art, and provides a kind of technique preparing high-purity high-yield chondroitin sulfate; The invention provides a kind of easy high-purity sulfuric acid chrondroitin technology of preparing, obtained chondroitin sulfate is tired all higher than 180U/mg, higher than the 170U/mg that Chinese Pharmacopoeia requires, and qualified according to the detection of Chinese Pharmacopoeia chondroitin sulfate examination criteria.
Technical scheme of the present invention is: a kind of technique preparing high-purity sulfuric acid chrondroitin, and its step is as follows:
(1) crude product chondroitin sulfate is dissolved in tap water, stir 5-10 hour, it is all dissolved;
(2) by lysate in step (1), adjust PH to 7.0-8.0 with hydrochloric acid soln, add stomach en-when being then warming up between 50-55 DEG C, then add pancreatin, insulation 2-4 hour;
(3) enzymolysis solution in step (2) was warming up to 85-90 DEG C in 30 ~ 40 minutes, then etc. static 10-30 minute, stirs, when being cooled to 50-55 DEG C, PH to 10.0-12.0 is adjusted with sodium hydroxide solution, static 20-30 hour, layering, siphon supernatant, filter, stay supernatant liquor to be precipitated, lower sediment puts centrifuge, stays upper strata centrifugate after centrifugal;
(4) treat that in step (3), supernatant liquor and centrifugate temperature are down to 20-30 DEG C, add sodium-chlor, stirring and dissolving, adjust PH to 10.0-12.0 with hydrochloric acid, stir while adding concentration is 75-80% ethanolic soln, precipitation 10-12 hour;
(5) get lower sediment thing purified water in step (4) to dissolve, with sodium hydroxide solution, solution is adjusted PH to 10.0-12.0, then add hydrogen peroxide oxidation 10-12 hour;
(6) solution in step (5) is added sodium-chlor, after stirring and dissolving, with hydrochloric acid soln, solution is adjusted PH to 6.0-7.0, add ethanolic soln, precipitation 10-12 hour;
(7) get lower sediment thing purified water in step (6) to dissolve, adjust PH to 10.0-12.0 with sodium hydroxide solution, then add volume 3-5% hydrogen peroxide, oxidation 10-12 hour;
(8) solution in step (7) is added sodium-chlor, adjust PH to 10.0-12.0 with hydrochloric acid soln, stir while add the ethanolic soln that concentration is 95 ~ 97%, precipitation 10-12 hour;
(9) get lower sediment thing purified water in step (8) to dissolve, adjust PH to 10.0-12.0 with sodium hydroxide solution, then add hydrogen peroxide, oxidation 10-12 hour;
(solution in step (9) is added sodium-chlor by 10, adjust PH to 6.0-7.0 with hydrochloric acid soln, be then refrigerated to-10 DEG C--5 DEG C, stir while add acetone, 24-30 hour is incubated in-5 DEG C-0 DEG C, abandoning supernatant, chondroitin polysulfate is taken away by acetone, by purified water by precipitate dissolves, add sodium-chlor, adjust PH to 6.0-7.0 with hydrochloric acid soln, stir while add ethanol, precipitation 10-12 hour;
(11) after being dissolved by purified water by throw out in step (10), with micron membrane filter board and frame machine micro-filtration, with alcohol settling, then add sodium chloride solution, quiescent setting 10-12 hour, taking precipitate, with dissolve with ethanol, leave standstill 24-30 hour;
Discard upper liquid, stainless steel core rod is put in the product, drain with vacuum pump is logical;
(12) product in step (11) is placed in vacuum drying oven, dries 70-80 hour, obtain product.
beneficial effect:
(1) the present invention in conjunction with heating and oxidation in conjunction with multiple physical extracting method and purifying obtain tiring higher than 180U/mg, yield higher than 80% high-purity sulfuric acid chrondroitin.(2) add hydrogen peroxide by intensification and can effectively control chondroitin sulfate Oxidative inactivation, and achieve oxidation with decolouring and synchronously carry out.(3) polarity difference of foundation chondroitin sulfate and analog thereof, with acetone extract in sodium chloride solution, is using alcohol settling wash-out, is finally obtaining chondroitin sulfate product.The extraction by propanone that technique adopts, chondroitin polysulfate impurity is extracted, and chondroitin sulfate product has been stayed in low salt solutions, with alcohol settling, drastically increase yield, the chondroitin sulfate product finally obtained reaches Chinese Pharmacopoeia standard, and through surveying not containing chondroitin polysulfate composition.
Embodiment
embodiment 1
50g crude product chondroitin sulfate is dissolved in tap water, stirs 5 hours, it is all dissolved; Adjust PH to 7.0 with hydrochloric acid soln, add 5g/ hundred million unit stomach en-when being then warming up between 50 DEG C, then add 10g/ hundred million unit pancreatin, be incubated 2 hours; Then etc. enzymolysis solution was warming up to 85 DEG C in 30 minutes, static 10 minutes, stirs, when being cooled to 50, PH to 10.0 is adjusted, static 20 hours, layering, siphon supernatant with 2mol sodium hydroxide solution, filter, stay supernatant liquor to be precipitated, lower sediment puts centrifuge, stays upper strata centrifugate after centrifugal; Treat that supernatant liquor and centrifugate temperature are down to 20 DEG C, add 25g/L sodium-chlor, stirring and dissolving, adjust PH to 10.0 with hydrochloric acid, stir while adding concentration is 75% ethanolic soln, precipitate 10 hours; Take off a layer throw out purified water to dissolve, with sodium hydroxide solution, solution is adjusted PH to 10.0, then add 3% hydrogen peroxide oxidation 10 hours; Solution is added 20g/L sodium-chlor, after stirring and dissolving, with hydrochloric acid soln, solution is adjusted PH to 6.0, add ethanolic soln, precipitate 10 hours; Take off a layer throw out purified water to dissolve, adjust PH to 10.0-with sodium hydroxide solution, then add volume 3% hydrogen peroxide, be oxidized 10 hours; Solution is added 20g/L sodium-chlor, adjust PH to 10.0 with hydrochloric acid soln, stir while add the ethanolic soln that concentration is 95%, precipitate 10 hours; Take off a layer throw out purified water to dissolve, adjust PH to 10.0 with sodium hydroxide solution, then add 3% hydrogen peroxide, be oxidized 10 hours; Solution is added 20g/L sodium-chlor, adjust PH to 6.0 with hydrochloric acid soln, be then refrigerated to-10 DEG C, stir while add acetone, in-5 DEG C, be incubated 24 hours, abandoning supernatant, by purified water by precipitate dissolves, adds sodium-chlor, adjust PH to 6.0 with hydrochloric acid soln, stir while add ethanol, precipitate 10 hours; After being dissolved by purified water by throw out, with micron membrane filter board and frame machine micro-filtration, with alcohol settling, then add 20g/L sodium chloride solution, quiescent setting 10 hours, taking precipitate, with dissolve with ethanol, leave standstill 24 hours; Discard upper liquid, stainless steel core rod is put in the product, drain with vacuum pump is logical; Be placed on by product in vacuum drying oven, dry 70, it tires as 183U/mg after testing to obtain product, and the rate of recovery is 80.1% chondroitin sulfate be 0.09mg/mL dermatan sulfate is 0.01mg/mL, and indices all meets Chinese Pharmacopoeia requirement.
embodiment 2
Crude product chondroitin sulfate is dissolved in tap water, stirs 5-10 hour, it is all dissolved; Lysate hydrochloric acid soln is adjusted PH to 6.5, adds 7g/ hundred million unit stomach en-when being then warming up between 52, then add 15g/ hundred million unit pancreatin, be incubated 3 hours; Then etc. enzymolysis solution was warming up to 87 DEG C in 35 minutes, static 20 minutes, stirs, when being cooled to 52 DEG C, PH to 11.0 is adjusted, static 25 hours, layering, siphon supernatant with 4mol sodium hydroxide solution, filter, stay supernatant liquor to be precipitated, lower sediment puts centrifuge, stays upper strata centrifugate after centrifugal; Treat that supernatant liquor and centrifugate temperature are down to 25 DEG C, add 25g/L25g/L sodium-chlor, stirring and dissolving, adjust PH to 11.0 with hydrochloric acid, stir while adding concentration is 77% ethanolic soln, precipitation 10-12 hour; Take off a layer throw out purified water to dissolve, with sodium hydroxide solution, solution is adjusted PH to 10.0-12.0, then add 4% hydrogen peroxide oxidation 10-12 hour; Solution is added 25g/L sodium-chlor, after stirring and dissolving, with hydrochloric acid soln, solution is adjusted PH to 6.5, add ethanolic soln, precipitate 11 hours; Take off a layer throw out purified water to dissolve, adjust PH to 11.0 with sodium hydroxide solution, then add volume 4% hydrogen peroxide, be oxidized 11 hours; Solution is added 25g/L sodium-chlor, adjust PH to 11.0 with hydrochloric acid soln, stir while add the ethanolic soln that concentration is 96%, precipitate 11 hours; Take off a layer throw out purified water to dissolve, adjust PH to 10.0-12.0 with sodium hydroxide solution, then add 4% hydrogen peroxide, be oxidized 11 hours; Solution is added 25g/L sodium-chlor, adjust PH to 6.5 with hydrochloric acid soln, be then refrigerated to-7, stir while add acetone, in-3, be incubated 24-30 hour, abandoning supernatant, by purified water by precipitate dissolves, add sodium-chlor, adjust PH to 6.5 with hydrochloric acid soln, stir while add ethanol, precipitate 11 hours; After being dissolved by purified water by throw out, with micron membrane filter board and frame machine micro-filtration, with alcohol settling, then add 25g/L sodium chloride solution, quiescent setting 10-12 hour, taking precipitate, with dissolve with ethanol, leave standstill 27 hours; Discard upper liquid, stainless steel core rod is put in the product, drain with vacuum pump is logical; Product is placed in vacuum drying oven, dries 75 hours, obtain product.After testing, it tires as 209U/mg to obtain product, and the rate of recovery is 89% chondroitin sulfate be 0.0001mg/mL dermatan sulfate is 0.0005mg/mL, and indices all meets Chinese Pharmacopoeia requirement.
embodiment 3
Crude product chondroitin sulfate is dissolved in tap water, stirs 10 hours, it is all dissolved; Adjust PH to 8.0 with hydrochloric acid soln, add 10g/ hundred million unit stomach en-when being then warming up between 50-55 DEG C, then add 20g/ hundred million unit pancreatin, be incubated 4 hours; Then etc. enzymolysis solution was warming up to 90 DEG C in 40 minutes, static 30 minutes, stirs, when being cooled to 55 DEG C, PH to 12.0 is adjusted, static 30 hours, layering, siphon supernatant with 6mol sodium hydroxide solution, filter, stay supernatant liquor to be precipitated, lower sediment puts centrifuge, stays upper strata centrifugate after centrifugal; Treat that supernatant liquor and centrifugate temperature are down to 30 DEG C, add 30g/L sodium-chlor, stirring and dissolving, adjust PH to 12.0 with hydrochloric acid, stir while adding concentration is 80% ethanolic soln, precipitate 12 hours; Take off a layer throw out purified water to dissolve, with sodium hydroxide solution, solution is adjusted PH to 12.0, then add 5% hydrogen peroxide oxidation 12 hours; Solution is added 30g/L sodium-chlor, after stirring and dissolving, with hydrochloric acid soln, solution is adjusted PH to 7.0, add ethanolic soln, precipitation 10-12 hour; Take off a layer throw out purified water to dissolve, adjust PH to 12.0 with sodium hydroxide solution, then add volume 5% hydrogen peroxide, be oxidized 12 hours; Solution is added 30g/L sodium-chlor, adjust PH to 12.0 with hydrochloric acid soln, stir while add the ethanolic soln that concentration is 97%, precipitate 12 hours; Take off a layer throw out purified water to dissolve, adjust PH to 12.0 with sodium hydroxide solution, then add 5% hydrogen peroxide, be oxidized 12 hours; Solution is added 30g/L sodium-chlor, adjust PH to 7.0 with hydrochloric acid soln, be then refrigerated to-5 DEG C, stir while add acetone, 30 hours are incubated in 0 DEG C, abandoning supernatant, chondroitin polysulfate is taken away by acetone, by purified water by precipitate dissolves, add sodium-chlor, adjust PH to 7.0 with hydrochloric acid soln, stir while add ethanol, precipitate 12 hours; After being dissolved by purified water by throw out, with micron membrane filter board and frame machine micro-filtration, with alcohol settling, then add 30g/L sodium chloride solution, quiescent setting 12 hours, taking precipitate, with dissolve with ethanol, leave standstill 30 hours; Discard upper liquid, stainless steel core rod is put in the product, drain with vacuum pump is logical; Product is placed in vacuum drying oven, dries 80 hours, obtains product.After testing, it tires as 189U/mg to obtain product, and the rate of recovery is 89% chondroitin sulfate be 0.002mg/mL dermatan sulfate is 0.003mg/mL, and indices all meets Chinese Pharmacopoeia requirement.
Claims (7)
1. prepare a technique for high-purity sulfuric acid chrondroitin, its step is as follows:
Crude product chondroitin sulfate is dissolved in tap water, stirs 5-10 hour, it is all dissolved;
By lysate in step (1), adjust PH to 7.0-8.0 with hydrochloric acid soln, add stomach en-when being then warming up between 50-55 DEG C, then add pancreatin, insulation 2-4 hour;
Enzymolysis solution in step (2) was warming up to 85-90 DEG C in 30 ~ 40 minutes, then etc. static 10-30 minute, stirs, when being cooled to 50-55 DEG C, PH to 10.0-12.0 is adjusted with sodium hydroxide solution, static 20-30 hour, layering, siphon supernatant, filter, stay supernatant liquor to be precipitated, lower sediment puts centrifuge, stays upper strata centrifugate after centrifugal;
Treat that in step (3), supernatant liquor and centrifugate temperature are down to 20-30 DEG C, add sodium-chlor, stirring and dissolving, adjust PH to 10.0-12.0 with hydrochloric acid, stir while add ethanolic soln, precipitation 10-12 hour;
Get lower sediment thing purified water in step (4) to dissolve, with sodium hydroxide solution, solution is adjusted PH to 10.0-12.0, then add hydrogen peroxide oxidation 10-12 hour;
Solution in step (5) is added sodium-chlor, after stirring and dissolving, with hydrochloric acid soln, solution is adjusted PH to 6.0-7.0, add ethanolic soln, precipitation 10-12 hour;
Get lower sediment thing purified water in step (6) to dissolve, adjust PH to 10.0-12.0 with sodium hydroxide solution, then add volume 3-5% hydrogen peroxide, oxidation 10-12 hour;
Solution in step (7) is added sodium-chlor, adjusts PH to 10.0-12.0 with hydrochloric acid soln, stir while add the ethanolic soln that concentration is 95 ~ 97%, precipitation 10-12 hour;
Get lower sediment thing purified water in step (8) to dissolve, adjust PH to 10.0-12.0 with sodium hydroxide solution, then add hydrogen peroxide, oxidation 10-12 hour;
Solution in step (9) is added sodium-chlor, adjust PH to 6.0-7.0 with hydrochloric acid soln, be then refrigerated to-10 DEG C--5 DEG C, stir while add acetone, 24-30 hour is incubated in-5 DEG C-0 DEG C, abandoning supernatant, chondroitin polysulfate is taken away by acetone, by purified water by precipitate dissolves, add sodium-chlor, adjust PH to 6.0-7.0 with hydrochloric acid soln, stir while add ethanol, precipitation 10-12 hour;
After being dissolved by purified water by throw out in step (10), with micron membrane filter board and frame machine micro-filtration, with alcohol settling, then add sodium chloride solution, quiescent setting 10-12 hour, taking precipitate, with dissolve with ethanol, leave standstill 24-30 hour;
Discard upper liquid, stainless steel core rod is put in the product, drain with vacuum pump is logical;
Product in step (11) is placed in vacuum drying oven, dries 70-80 hour, obtain product.
2. technique according to claim 1, is characterized in that: the pepsin concn in step (1) is 5-10g/ hundred million unit, and the concentration of pancreatin is 10-20g/ hundred million unit.
3. technique according to claim 1, is characterized in that: in step (2), the concentration of sodium hydroxide is 2-6mol/L.
4. technique according to claim 1, is characterized in that: in step (3), step (6), step (8), step (10) and step (11), the concentration of sodium-chlor is 20-30g/L.
5. technique according to claim 1, is characterized in that: in step (4), the concentration of ethanolic soln is 75% ~ 80%.
6. technique according to claim 1, is characterized in that: the volume fraction of the hydrogen peroxide in step (5) and step (9) is 3-5%.
7. technique according to claim 1, is characterized in that: in step (10), the concentration of acetone is 40-45%.
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| CN201410813443.2A CN104479046A (en) | 2014-12-24 | 2014-12-24 | Method for increasing yield of chondroitin sulfate |
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101575385A (en) * | 2008-05-09 | 2009-11-11 | 青岛九龙生物医药有限公司 | Method for separating chondroitin polysulfate from heparin sodium by extraction method |
| CN103641934A (en) * | 2013-11-21 | 2014-03-19 | 青岛佰众化工技术有限公司 | Chondroitin sulfate preparation method |
| CN103694373A (en) * | 2013-11-23 | 2014-04-02 | 青岛九龙生物医药有限公司 | Method for removing DS impurity in refined heparin sodium |
| CN103804524A (en) * | 2013-11-24 | 2014-05-21 | 青岛九龙生物医药有限公司 | Method for improving yield of heparin sodium |
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- 2014-12-24 CN CN201410813443.2A patent/CN104479046A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101575385A (en) * | 2008-05-09 | 2009-11-11 | 青岛九龙生物医药有限公司 | Method for separating chondroitin polysulfate from heparin sodium by extraction method |
| CN103641934A (en) * | 2013-11-21 | 2014-03-19 | 青岛佰众化工技术有限公司 | Chondroitin sulfate preparation method |
| CN103694373A (en) * | 2013-11-23 | 2014-04-02 | 青岛九龙生物医药有限公司 | Method for removing DS impurity in refined heparin sodium |
| CN103804524A (en) * | 2013-11-24 | 2014-05-21 | 青岛九龙生物医药有限公司 | Method for improving yield of heparin sodium |
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Application publication date: 20150401 |