CN103044564A - Preparation method for holothuria leucospilota glycosaminoglycans - Google Patents
Preparation method for holothuria leucospilota glycosaminoglycans Download PDFInfo
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- CN103044564A CN103044564A CN2012105862000A CN201210586200A CN103044564A CN 103044564 A CN103044564 A CN 103044564A CN 2012105862000 A CN2012105862000 A CN 2012105862000A CN 201210586200 A CN201210586200 A CN 201210586200A CN 103044564 A CN103044564 A CN 103044564A
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Abstract
The invention belongs to the field of extraction of natural active materials by a biotechnology, and particularly relates to a preparation method for holothuria leucospilota glycosaminoglycans. The preparation method comprises the following steps: step 1, pretreating the holothuria leucospilota; step 2, performing alkaline hydrolysis on the body wall of the holothuria leucospilota; step 3, using bienzyme to perform enzymolysis on the body wall of the holothuria leucospilota; step 4, after the enzymolysis, using trichloroacetic acid to remove the protein in the bienzyme enzymatic hydrolysate; step 5, using ethanol to precipitate the holothuria leucospilota glycosaminoglycans; step 6, drying the holothuria leucospilota glycosaminoglycan raw sugar; step 7, using the cellulose ion exchange column chromatography and the gel column sequentially to purify the holothuria leucospilota glycosaminoglycans; and step 8, drying the holothuria leucospilota glycosaminoglycan refined sugar. According to the invention, the relation of the yield and the degree of purity of the holothuria leucospilota glycosaminoglycans can be balanced, and the degree of purity of the holothuria leucospilota glycosaminoglycans is improved.
Description
Technical field
The invention belongs to the biotechnology natural active matter and extract the field, be specifically related to a kind of preparation method of holothuria leucospilota glycosaminoglycan.
Background technology
Along with the raising of aging population degree, cardiovascular and cerebrovascular diseases has become the biggest threat of human health.It is reported that cardiovascular and cerebrovascular diseases is one of three large major causes of death of the elderly, at present because the cardiovascular and cerebrovascular diseases died accounts for 34% of total death toll, in survival, have approximately patient about 3/4 have in various degree work capacity and the forfeiture of self care ability.And ishemic stroke is modal a kind of disease in the cardiovascular and cerebrovascular, accounts for 43 ~ 65% of cerebro-vascular diseases, and this disease incidence is high, case fatality rate is high, disability rate is high, recurrence rate is high, serious human survival and the quality of life of endangering.Apoplexy world population mark sickness rate is 239.4/10 ten thousand, and wherein ishemic stroke accounts for 53.6%.Along with aging population and human life style's variation, its sickness rate also has the trend of increase year after year.Cardiovascular and cerebrovascular diseases has become human dead first-class reason, and drug development and the exploitation of therefore actively developing this disease of prevention are extremely urgent.
The ocean is a huge natural product treasure-house, and abundant marine organisms and marine active substance provide a huge marine pharmaceutical resource storehouse for us.Therefore the special marine eco-environment and the Close relation between the biologic chain are significant to the Application and Development of marine polysaccharide so that the marine organisms secondary metabolite has the biological activity with terrestrial organism difference chemical structure and differential high efficient.From the marine active polysaccharide, seek the active drug with blood coagulation resisting function, caused the close attention of domestic and international the world of medicine.
Holothuria leucospilota glycosaminoglycan (Holothuria leucospilota GlycosaminoGlycans, HG) is once called as acidic mucopolysaccharide or mucopolysaccharide.Holothuria leucospilota glycosaminoglycan has anti-freezing, fibrinolytic, reduction blood viscosity, regulates the blood fat isoreactivity, acts on the links of ishemic stroke (cerebral infarction), thereby reaches the purpose for the treatment of ishemic stroke.Glycosaminoglycan is main pharmacodynamics composition relevant with curing apoplexy in the sea cucumber; But the holothuria leucospilota glycosaminoglycan that extracts from hojothuria leucospilota at present is crude product (quality purity only is 50% ~ 60%), can not be directly used in and make injection.
Summary of the invention
For solving above-mentioned problems of the prior art, the object of the present invention is to provide a kind of preparation method of holothuria leucospilota glycosaminoglycan, holothuria leucospilota glycosaminoglycan is used cellulose ion-exchange column chromatography method and gel column purifying successively, obtain the higher holothuria leucospilota glycosaminoglycan of purity.
Technical scheme of the present invention is: a kind of preparation method of holothuria leucospilota glycosaminoglycan comprises the following steps: step 1: the hojothuria leucospilota pre-treatment; Step 2: alkaline hydrolysis hojothuria leucospilota body wall; Step 3: two enzyme enzymolysis hojothuria leucospilota body walls; Step 4: after enzymolysis is complete, with the protein in the two enzyme enzymolysis solutions of trichoroacetic acid(TCA) removal; Step 5: precipitate holothuria leucospilota glycosaminoglycan with ethanol; Step 6: dry holothuria leucospilota glycosaminoglycan raw sugar; Step 7: use successively cellulose ion-exchange column chromatography method and gel column purifying holothuria leucospilota glycosaminoglycan; Step 8: dry holothuria leucospilota glycosaminoglycan refined sugar.
Optimize, the concrete steps of gel column purifying are in the described step 7: with the distilled water soaked overnight of 40g dextrane gel with 3 times of volumes, degassed rear dress post is with the damping fluid balance of 2 times of column volumes; 30mg glycosaminoglycan raw sugar with 10mL damping fluid dissolving upper props under 40～60 ℃, is used the distilled water wash-out, and flow velocity adopts 10mL/h, collects with the 3mL/ pipe; Respectively manage the content of total osamine glycan with A Li Xinlan colorimetric determination, collect the sample liquid that the elution peak part is arranged, behind the dialysis desalting, rotatory evaporator is concentrated.
Optimize, described damping fluid adopts the NaAC/HAC of 0.5mol/L, and its pH is 6.0～6.5.
The g-100 of sephadex described in the present invention is dextrane gel, and the present invention does not have particular requirement to reagent, material and the instrument that adopts, and it is the commercially available prod, can buy by multiple commercial channel.
Beneficial effect of the present invention is: set up a kind of method that effectively prepares highly purified holothuria leucospilota glycosaminoglycan, when alkaline process extracts, in conjunction with trypsinase and pepsic pair of collagenase treatment, namely at first adopt alkaline purification hojothuria leucospilota tissue, use again trypsinase and pepsin hydrolysis, gather the release of glycosaminoglycan to strengthen hojothuria leucospilota body wall tissue.For improving the purity of glycosaminoglycan, the present invention uses cellulose ion-exchange column chromatography method and gel column purifying purifying holothuria leucospilota glycosaminoglycan raw sugar successively.Method of the present invention can the balance holothuria leucospilota glycosaminoglycan yield and the relation of purity, improved the purity of holothuria leucospilota glycosaminoglycan.
Embodiment
The present invention will be further described below in conjunction with specific embodiment.
Embodiment 1
The method of extracting glycosaminoglycan from hojothuria leucospilota of present embodiment comprises the following steps: step 1: the hojothuria leucospilota pre-treatment; Step 2: alkaline hydrolysis hojothuria leucospilota body wall; Step 3: two enzyme enzymolysis hojothuria leucospilota body walls; Step 4: after enzymolysis is complete, with the protein in the two enzyme enzymolysis solutions of trichoroacetic acid(TCA) removal; Step 5: precipitate holothuria leucospilota glycosaminoglycan with ethanol; Step 6: dry holothuria leucospilota glycosaminoglycan raw sugar; Step 7: use successively cellulose ion-exchange column chromatography method and gel column purifying holothuria leucospilota glycosaminoglycan; Step 8: dry holothuria leucospilota glycosaminoglycan refined sugar.The concrete grammar of each step is:
Step 1: hojothuria leucospilota pre-treatment; Use rapidly the distilled water flushing hojothuria leucospilota, removes the foreign matter that the hojothuria leucospilota body surface adheres to, with microscler pocket knife from belly only anus dissect forward aquatic foods and join, reject immediately internal organ, with distilled water flush away dirt, after the weighing hojothuria leucospilota body wall is placed clean large beaker rapidly, shred the hojothuria leucospilota body wall;
Step 2: alkaline hydrolysis hojothuria leucospilota body wall: add the distilled water of 2 times of weight in the hojothuria leucospilota body wall, then add the NaOH of 2mol/L, the limit edged stirs, and making NaOH solution final concentration is 0.5mol/L, 4 ℃ of digested overnight;
Step 3: two enzyme enzymolysis of hojothuria leucospilota body wall:
Trypsin digestion: reconcile pH value to 8.2 with Glacial acetic acid, add the trypsinase of hojothuria leucospilota body wall quality 0.45%, 52 ℃ of water-bath 5h fully stir in the water-bath process, and drip the NaOH solution of 5 mmol/L, keep pH 8.2; Then enzymolysis solution is cooled to rapidly below 30 ℃ termination reaction behind the maintenance 30min;
Stomach en-enzymolysis: reconcile between the pH value to 2 with 6mol/L HCl, add again the stomach en-of hojothuria leucospilota body wall quality 0.40%, in 50 ℃ water-bath, continue enzymolysis 4h, fully stir in the water-bath process; Enzymolysis is got in enzymolysis solution 5mL and the glass test tube before stopping, and the trichoroacetic acid(TCA) solution of dropping 5% detects, and when solution is little muddiness, enzymolysis solution is cooled to rapidly below 30 ℃, keeps the 30min enzymolysis reaction; Then collect supernatant with the centrifugal 15min of 5000r/min;
Step 4: after enzymolysis is complete, process two enzyme enzymolysis supernatant with trichoroacetic acid(TCA), the addition of trichoroacetic acid(TCA) is 10% of enzymolysis solution, progressively adds, and the limit edged stirs, and enzymolysis solution is become till the muddiness; Solution is kept at leaves standstill 12h in the refrigerating chamber, then collect supernatant with the centrifugal 25min of 5000r/min;
Step 5: ethanol precipitation holothuria leucospilota glycosaminoglycan: the NaOH solution that drips 5mmol/L is reconciled the pH value to 6.8 of supernatant liquor, the centrifugal 10min of 7000r/min collects supernatant and adds dehydrated alcohol, and the limit edged stirs, making the ethanol final concentration is 55%, 4 ℃ of precipitation of spending the night;
Step 6: dry holothuria leucospilota glycosaminoglycan raw sugar: the centrifugal 10min of 7000r/min, collecting precipitation, the washing with alcohol with 95% 3 times is washed respectively with dehydrated alcohol and acetone successively and is dewatered 1 time; Ethanol and the acetone of precipitating species are volatilized naturally, further dry with low temperature vacuum drier during the throw out partial desiccation, obtain rough holothuria leucospilota glycosaminoglycan;
Step 7: at first with 50g diethylaminoethyl cellulose damping fluid soaked overnight, degassed rear dress post is with the damping fluid balance of 2 times of column volumes; With 10mL damping fluid dissolving upper props under 40 ℃, with each 150mL gradient elution of NaCl of damping fluid and 1.5mol/L, flow velocity employing 8mL/h is with the collection of 3mL/ pipe with 30mg glycosaminoglycan raw sugar; Respectively manage the content of total osamine glycan with A Li Xinlan colorimetric determination, collect the sample liquid that the elution peak part is arranged, behind the dialysis desalting, rotatory evaporator is concentrated.
Then with the distilled water soaked overnight of 40g sephadex g-100 gel with 3 times of volumes, degassed rear dress post is with the damping fluid balance of 2 times of column volumes; The above-mentioned enriched material of 30mg with 10mL damping fluid dissolving upper props under 40 ℃, is used the distilled water wash-out, and flow velocity adopts 10mL/h, collects with the 3mL/ pipe; Respectively manage the content of total osamine glycan with A Li Xinlan colorimetric determination, collect the sample liquid that the elution peak part is arranged, behind the dialysis desalting, rotatory evaporator is concentrated, obtains the holothuria leucospilota glycosaminoglycan refined sugar.Described damping fluid adopts the NaAC/HAC of 0.5mol/L, and its pH is 6.0.
Step 8: dry holothuria leucospilota glycosaminoglycan refined sugar:
The holothuria leucospilota glycosaminoglycan refined sugar is used respectively 80%, 95%, 100% washing with alcohol 1 time, wash respectively with dehydrated alcohol and acetone successively again and dewater 1 time; Then the ethanol for the treatment of and acetone volatilize naturally, and during the refined sugar partial desiccation, further dry with low temperature vacuum drier, the unformed powder of the white dried that obtains is refining polysaccharide.
Embodiment 2
The method of extracting glycosaminoglycan from hojothuria leucospilota of present embodiment comprises the following steps: step 1: the hojothuria leucospilota pre-treatment; Step 2: alkaline hydrolysis hojothuria leucospilota body wall; Step 3: two enzyme enzymolysis hojothuria leucospilota body walls; Step 4: after enzymolysis is complete, with the protein in the two enzyme enzymolysis solutions of trichoroacetic acid(TCA) removal; Step 5: precipitate holothuria leucospilota glycosaminoglycan with ethanol; Step 6: dry holothuria leucospilota glycosaminoglycan raw sugar; Step 7: use successively cellulose ion-exchange column chromatography method and gel column purifying holothuria leucospilota glycosaminoglycan; Step 8: dry holothuria leucospilota glycosaminoglycan refined sugar.The concrete grammar of each step is:
Step 1: hojothuria leucospilota pre-treatment; Use rapidly the distilled water flushing hojothuria leucospilota, removes the foreign matter that the hojothuria leucospilota body surface adheres to, with microscler pocket knife from belly only anus dissect forward aquatic foods and join, reject immediately internal organ, with distilled water flush away dirt, after the weighing hojothuria leucospilota body wall is placed clean large beaker rapidly, shred the hojothuria leucospilota body wall;
Step 2: alkaline hydrolysis hojothuria leucospilota body wall: add the distilled water of 2 times of weight in the hojothuria leucospilota body wall, then add the NaOH of 2mol/L, the limit edged stirs, and making NaOH solution final concentration is 0.5mol/L, 4 ℃ of digested overnight;
Step 3: two enzyme enzymolysis of hojothuria leucospilota body wall:
Trypsin digestion: reconcile pH value to 9.0 with Glacial acetic acid, add the trypsinase of hojothuria leucospilota body wall quality 0.45%, 52 ℃ of water-bath 5h fully stir in the water-bath process, and drip the NaOH solution of 5 mmol/L, keep pH 9.0; Then enzymolysis solution is cooled to rapidly below 30 ℃ termination reaction behind the maintenance 30min;
Stomach en-enzymolysis: reconcile between the pH value to 2.5 with 6mol/L HCl, add again the stomach en-of hojothuria leucospilota body wall quality 0.40%, in 50 ℃ water-bath, continue enzymolysis 4h, fully stir in the water-bath process; Enzymolysis is got in enzymolysis solution 5mL and the glass test tube before stopping, and the trichoroacetic acid(TCA) solution of dropping 5% detects, and when solution is little muddiness, enzymolysis solution is cooled to rapidly below 30 ℃, keeps the 30min enzymolysis reaction; Then collect supernatant with the centrifugal 15min of 5000r/min;
Step 4: after enzymolysis is complete, process two enzyme enzymolysis supernatant with trichoroacetic acid(TCA), the addition of trichoroacetic acid(TCA) is 10% of enzymolysis solution, progressively adds, and the limit edged stirs, and enzymolysis solution is become till the muddiness; Solution is kept at leaves standstill 12h in the refrigerating chamber, then collect supernatant with the centrifugal 25min of 5000r/min;
Step 5: ethanol precipitation holothuria leucospilota glycosaminoglycan: the NaOH solution that drips 5mmol/L is reconciled the pH value to 7.2 of supernatant liquor, the centrifugal 10min of 7000r/min collects supernatant and adds dehydrated alcohol, and the limit edged stirs, making the ethanol final concentration is 55%, 4 ℃ of precipitation of spending the night;
Step 6: dry holothuria leucospilota glycosaminoglycan raw sugar: the centrifugal 10min of 7000r/min, collecting precipitation, the washing with alcohol with 95% 3 times is washed respectively with dehydrated alcohol and acetone successively and is dewatered 1 time; Ethanol and the acetone of precipitating species are volatilized naturally, further dry with low temperature vacuum drier during the throw out partial desiccation, obtain rough holothuria leucospilota glycosaminoglycan;
Step 7: at first with 50g diethylaminoethyl cellulose damping fluid soaked overnight, degassed rear dress post is with the damping fluid balance of 2 times of column volumes; With 10mL damping fluid dissolving upper props under 60 ℃, with each 150mL gradient elution of NaCl of damping fluid and 1.5mol/L, flow velocity employing 8mL/h is with the collection of 3mL/ pipe with 30mg glycosaminoglycan raw sugar; Respectively manage the content of total osamine glycan with A Li Xinlan colorimetric determination, collect the sample liquid that the elution peak part is arranged, behind the dialysis desalting, rotatory evaporator is concentrated.
Then with the distilled water soaked overnight of 40g sephadex g-100 gel with 3 times of volumes, degassed rear dress post is with the damping fluid balance of 2 times of column volumes; The above-mentioned enriched material of 30mg with 10mL damping fluid dissolving upper props under 60 ℃, is used the distilled water wash-out, and flow velocity adopts 10mL/h, collects with the 3mL/ pipe; Respectively manage the content of total osamine glycan with A Li Xinlan colorimetric determination, collect the sample liquid that the elution peak part is arranged, behind the dialysis desalting, rotatory evaporator is concentrated, obtains the holothuria leucospilota glycosaminoglycan refined sugar.Described damping fluid adopts the NaAC/HAC of 0.5mol/L, and its pH is 6.5.
Step 8: dry holothuria leucospilota glycosaminoglycan refined sugar:
The holothuria leucospilota glycosaminoglycan refined sugar is used respectively 80%, 95%, 100% washing with alcohol 1 time, wash respectively with dehydrated alcohol and acetone successively again and dewater 1 time; Then treat that ethanol and acetone volatilize naturally, during the refined sugar partial desiccation, further dry with low temperature vacuum drier, the unformed powder of the white dried that obtains is refining polysaccharide.
The method of extracting glycosaminoglycan from hojothuria leucospilota of implementing in the embodiment of the invention all can balance holothuria leucospilota glycosaminoglycan yield and the relation of purity, the purity of raising holothuria leucospilota glycosaminoglycan.
Claims (3)
1. the preparation method of a holothuria leucospilota glycosaminoglycan is characterized in that: comprise the following steps: step 1: the hojothuria leucospilota pre-treatment; Step 2: alkaline hydrolysis hojothuria leucospilota body wall; Step 3: two enzyme enzymolysis hojothuria leucospilota body walls; Step 4: after enzymolysis is complete, with the protein in the two enzyme enzymolysis solutions of trichoroacetic acid(TCA) removal; Step 5: precipitate holothuria leucospilota glycosaminoglycan with ethanol; Step 6: dry holothuria leucospilota glycosaminoglycan raw sugar; Step 7: use successively cellulose ion-exchange column chromatography method and gel column purifying holothuria leucospilota glycosaminoglycan; Step 8: dry holothuria leucospilota glycosaminoglycan refined sugar.
2. the preparation method of a kind of holothuria leucospilota glycosaminoglycan according to claim 1, it is characterized in that: the concrete steps of gel column purifying are in the described step 7: with the distilled water soaked overnight of 40g dextrane gel with 3 times of volumes, degassed rear dress post is with the damping fluid balance of 2 times of column volumes; 30mg glycosaminoglycan raw sugar with 10mL damping fluid dissolving upper props under 40～60 ℃, is used the distilled water wash-out, and flow velocity adopts 10mL/h, collects with the 3mL/ pipe; Respectively manage the content of total osamine glycan with A Li Xinlan colorimetric determination, collect the sample liquid that the elution peak part is arranged, behind the dialysis desalting, rotatory evaporator is concentrated.
3. the preparation method of a kind of holothuria leucospilota glycosaminoglycan according to claim 2 is characterized in that: described damping fluid adopts the NaAC/HAC of 0.5mol/L, and its pH is 6.0～6.5.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101724086A (en) * | 2009-11-25 | 2010-06-09 | 深圳海王药业有限公司 | Oligomerization pineapple ginseng glycosaminoglycan and preparation method thereof |
| CN101787084A (en) * | 2010-02-12 | 2010-07-28 | 青岛市市立医院 | Method for preparing stichopus japonicus selenka glycosaminoglycans |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101724086A (en) * | 2009-11-25 | 2010-06-09 | 深圳海王药业有限公司 | Oligomerization pineapple ginseng glycosaminoglycan and preparation method thereof |
| CN101787084A (en) * | 2010-02-12 | 2010-07-28 | 青岛市市立医院 | Method for preparing stichopus japonicus selenka glycosaminoglycans |
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Application publication date: 20130417 |