CN107056967A - A kind of extracting method of liquaemin - Google Patents
A kind of extracting method of liquaemin Download PDFInfo
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- CN107056967A CN107056967A CN201710379292.8A CN201710379292A CN107056967A CN 107056967 A CN107056967 A CN 107056967A CN 201710379292 A CN201710379292 A CN 201710379292A CN 107056967 A CN107056967 A CN 107056967A
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
- C08B37/0063—Glycosaminoglycans or mucopolysaccharides, e.g. keratan sulfate; Derivatives thereof, e.g. fucoidan
- C08B37/0075—Heparin; Heparan sulfate; Derivatives thereof, e.g. heparosan; Purification or extraction methods thereof
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0003—General processes for their isolation or fractionation, e.g. purification or extraction from biomass
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Abstract
The invention discloses a kind of extracting method of liquaemin.This method comprises the following steps：(1) small intestine is collected；(2) added water into small intestinal mucosa, dispersed with stirring；(3) digest；(4) resin is pre-processed；(5) adsorb；(6) elute, then digest；(7) purify.The inventive method can effectively lift the yield and purity of obtained liquaemin.
Description
Technical field
The invention belongs to beaters' skin extractive technique field, and in particular to a kind of extracting method of liquaemin.
Background technology
Heparin is that one kind is alternately made up of GLUCOSAMINE, D- glucuronic acids, N-Acetyl-D-glucosamine and L- iduronic acids
Acidic mucopolysaccharide, be widely present in mammal intestinal mucosa, heart, liver, lung, blood, pancreas and placenta etc. tissue in, tool
There is the activity in terms of good anticoagulation, reducing blood lipid, rush fibrinolysis, anti-middle film smooth muscle cell proliferation, it is extensive
Treatment for cardiovascular and cerebrovascular disease.
In heparin extraction process, generally by it in the form of sodium salt preparation-liquaemin, be easy to drug effect and activity guarantor
Deposit, used at the same time as pharmaceutical preparation, it is in clinical practice the history with more than 60 years, but at present still can only be from
Extracted in animal part tissue, it is impossible to prepared by artificial synthesized, wherein using small intestinal mucosa as extraction optimum feed stock；But existing enterprise
The liquaemin purity that industry is obtained by small intestinal mucosa extraction is low, and yield is low.
The content of the invention
For above-mentioned deficiency of the prior art, the present invention provides a kind of extracting method of liquaemin, can effectively solve liver
The problem of plain sodium extract yield is low and purity is low.
A kind of extracting method of liquaemin, comprises the following steps：
(1) fresh intestinal mucosa is collected, is saved backup in 0~4 DEG C；
(2) water of 4~5 times of its weight is incorporated as into small intestinal mucosa, 60~65 DEG C are warming up to, and stirred while heating
Mix scattered, be incubated 20~30min, then be cooled to 30~45 DEG C；
(3) compound protein of 0.4~0.8% small intestinal mucosa weight is added into the small intestinal mucosa solution obtained by step (2)
Enzyme, regulation solution ph is 8.5~9, and in digesting 3~4h at 40~45 DEG C, enzymolysis liquid I and filter residue are collected in filtering；Using filter residue as
Substrate, repeats above-mentioned enzymolysis operation, and centrifugation collects enzymolysis liquid II, enzymolysis liquid I and enzymolysis liquid II are merged；
(4) resin is soaked with 20%~25% sodium chloride solution of 2~3 times of weight resins, and stirs 2~3h, then distinguished
Resin is eluted using 10% sodium chloride solution and 5% sodium chloride solution of 0.5~1.5 times of weight resin, every time 1~2h；
(5) sodium chloride solution is added into the enzymolysis liquid after merging, stirring 5~10min of mixing is incubated 10 in 60~70 DEG C
~20min, adds the resin after processing in step (4), at room temperature 4~6h of stirring and adsorbing, and mixing speed is 50~100r/
Filtrate and resin are collected in min, filtering respectively；And the sodium chloride solution of its volume 6%~8% is incorporated as into filtrate, add
Excessive 90% ethanol precipitation, collects and dries precipitate I；Wherein, the envelope-bulk to weight ratio V of enzymolysis liquid and resin:M=12~15；
Sodium chloride solution volume accounts for the 1%~4% of enzymolysis liquid volume；The concentration of sodium chloride solution is 0.5~1mol/L；
(6) sodium chloride solution that 1~2 times of weight resin is added into resin elutes 2~3 times, every time 3~5h, and merging is washed
Liquid is taken off, 0.4% 2~3h of trypsin digestion is added, then isometric 95% ethanol precipitation, collects and simultaneously dries precipitate
Ⅱ；
(7) precipitate I and precipitate II are mixed, are dissolved in water, then be separately added into the peroxidating of 0.1% mixture weight
5~10min, filtering, then addition etc. into filtrate are placed in the tannic acid of hydrogen and 4%~8% mixture weight, stirring mixing
90% ethanol precipitation of volume, collects and dries precipitate, obtain heparin sodium pure product.
Further, the weight of compound protease is the 0.4% of small intestinal mucosa weight in step (3).
Further, compound protease includes trypsase and papain, trypsase and pawpaw egg in step (3)
The weight ratio of white enzyme is 2:1.
Further, sodium chloride concentration is 0.8mol/L in step (5), and addition is 1.5% enzymolysis liquid volume.
Further, resin is the D-254 resins or WD-1701 resins that particle diameter is 150~200 mesh, its grain in step (5)
Footpath is 150~200 mesh.
Further, concentration of sodium chloride solution is 1.6mol/L in step (6).
Further, the weight of tannic acid is the 4% of mixture weight in step (7).
Beneficial effects of the present invention are：
1st, the heparin that compound protease helps to make in small intestinal mucosa extracts more complete, simultaneously as band is negative in solution
Floating preteins meeting of electricity and resin reaction, by resin adsorption, therefore, ensure to extract more completely under the conditions of, it is as few as possible
Addition compound protease, while before resin adsorption, high temperature makes the amount of floating preteins in albumen enzyme denaturation, reduction solution, carries
Rise the adsorption rate of resin.
2nd, particle diameter has larger specific surface area for the resin of 150~200 mesh, has aperture larger on the outside of its solid space
Water-absorbing resin net, in addition, the resin after being soaked using sodium chloride has stronger permeability, can effectively lift the absorption of resin
Efficiency, lifts the yield of liquaemin.
3rd, liquaemin is by after resin adsorption, and concentration has maximum elution effect to liquaemin for 1.6mol/L sodium chloride solution
Rate.
4th, the present invention through digesting twice, Adsorption and desorption is accompanied by and the technique that digests again extracts heparin in small intestinal mucosa
Sodium, by the inventive method, can make the yield of liquaemin reach 100,000,000 U/1000~1200 piece small intestines, potency is up to 160U/mg.
5th, add 1% hydrogen peroxide for mixing precipitate weight to aoxidize precipitate, make product oxidation thorough, lifting
Product potency, meanwhile, it can also avoid the problem of product causes inactivation because of over oxidation.
6th, by the effect of tannic acid, the impurity coagulating sedimentation such as remaining albumen in precipitate solution can be made, to lift system
The purity of standby obtained liquaemin.
Embodiment
The embodiment to the present invention is described below, in order to which those skilled in the art understand this hair
It is bright, it should be apparent that the invention is not restricted to the scope of embodiment, for those skilled in the art,
As long as various change is in the spirit and scope of the present invention that appended claim is limited and is determined, these changes are aobvious and easy
See, all are using the innovation and creation of present inventive concept in the row of protection.
Embodiment 1
A kind of extracting method of liquaemin, comprises the following steps：
(1) fresh intestinal mucosa is collected, is saved backup in 4 DEG C；
(2) water of 4 times of its weight is incorporated as into small intestinal mucosa, 65 DEG C, and dispersed with stirring while heating is warming up to,
30min is incubated, then is cooled to 45 DEG C；
(3) trypsase and the pawpaw of 0.8% small intestinal mucosa weight are added into the small intestinal mucosa solution obtained by step (2)
The mixture of protease, regulation solution ph is 9, in digesting 4h at 45 DEG C, filters, collects enzymolysis liquid I and filter residue；Using filter residue as
Substrate, repeats above-mentioned enzymolysis operation, and centrifugation collects enzymolysis liquid II, enzymolysis liquid I and enzymolysis liquid II are merged；Wherein, trypsase
Weight ratio with papain is 2:1；
(4) resin is soaked for 25% sodium chloride solution of the D-254 weight resins of 180 mesh with 3 times of particle diameters, and stirs 3h,
Again respectively using 10% sodium chloride solution and 5% sodium chloride solution elution resin of 1.5 times of weight resins, each 2h；
(5) it is the 4% of enzymolysis liquid volume that volume is added into the enzymolysis liquid after merging, and concentration is molten for 1mol/L sodium chloride
Liquid, stirring mixing 10min, 20min is incubated in 70 DEG C, and the D-254 resins added after being handled in step (4) are stirred at room temperature
Absorption 6h is mixed, mixing speed is 100r/min, filtrate and D-254 resins are collected in filtering respectively；And its body is incorporated as into filtrate
Product 8%, concentration is 1mol/L sodium chloride solution, adds excessive 90% ethanol precipitation, collects and dries precipitate I；Wherein,
The envelope-bulk to weight ratio V of enzymolysis liquid and D-254 resins:M=12；
(6) 1~2 times of its weight of addition into D-254 resins, the sodium chloride solution elution that concentration is 1.6mol/L 3 times, often
Secondary 3.5h, merges eluent, adds 0.4% trypsin digestion 3h, then isometric 95% ethanol precipitation, collects and do
Dry precipitate II；
(7) precipitate I and precipitate II are mixed, are dissolved in water, then be separately added into the peroxidating of 0.1% mixture weight
The tannic acid of hydrogen and 8% mixture weight, stirs mixing, places 10min, and then filtering adds in equal volume into filtrate
90% ethanol precipitation, collects and dries precipitate, obtain heparin sodium pure product.
Embodiment 2
A kind of extracting method of liquaemin, comprises the following steps：
(1) fresh intestinal mucosa is collected, is saved backup in 4 DEG C；
(2) water of 4 times of its weight is incorporated as into small intestinal mucosa, 60 DEG C, and dispersed with stirring while heating is warming up to,
20min is incubated, then is cooled to 30 DEG C；
(3) trypsase and wood of 0.4~% small intestinal mucosa weight are added into the small intestinal mucosa solution obtained by step (2)
The mixture of melon protease, regulation solution ph is 8.5, in digesting 3h at 4 DEG C, filters, collects enzymolysis liquid I and filter residue；To filter
Slag is substrate, repeats above-mentioned enzymolysis operation, and centrifugation collects enzymolysis liquid II, enzymolysis liquid I and enzymolysis liquid II are merged；Wherein, pancreas egg
The weight ratio of white enzyme and papain is 2:1；
(4) D-254 resins are soaked for 20% sodium chloride solution of the D-254 weight resins of 200 mesh with 2 times of particle diameters, and stirred
2~3h is mixed, then uses 10% sodium chloride solution and 5% sodium chloride solution of 0.5 times of D-254 weight resin to elute respectively, every time
1.5h；
(5) it is the 1% of enzymolysis liquid volume that volume is added into the enzymolysis liquid after merging, and concentration is 0.5mol/L sodium chloride
Solution, stirring mixing 5min, 10min is incubated in 60 DEG C, and the D-254 resins added after being handled in step (4) are stirred at room temperature
Absorption 4h is mixed, mixing speed is 80r/min, filtrate and D-254 resins are collected in filtering respectively；And its body is incorporated as into filtrate
The sodium chloride solution of product 6%, adds excessive 90% ethanol precipitation, collects and dries precipitate I；Wherein, enzymolysis liquid and D-254
The envelope-bulk to weight ratio V of resin:M=13.5；
(6) 1 times of its weight of addition into D-254 resins, the sodium chloride solution elution that concentration is 1.6mol/L 2 times, every time
4h, merges eluent, adds 0.4% trypsin digestion 2h, then isometric 95% ethanol precipitation, collects and dries analysis
Go out thing II；
(7) precipitate I and precipitate II are mixed, are dissolved in water, then be separately added into the peroxidating of 0.1% mixture weight
8min is placed in the tannic acid of hydrogen and 4% mixture weight, stirring mixing, and then filtering adds isometric 90% into filtrate
Ethanol precipitation, collects and dries precipitate, obtain heparin sodium pure product.
Embodiment 3
A kind of extracting method of liquaemin, comprises the following steps：
(1) fresh intestinal mucosa is collected, is saved backup in 4 DEG C；
(2) water of 5 times of its weight is incorporated as into small intestinal mucosa, 65 DEG C, and dispersed with stirring while heating is warming up to,
28min is incubated, then is cooled to 40 DEG C；
(3) trypsase and the pawpaw of 0.4% small intestinal mucosa weight are added into the small intestinal mucosa solution obtained by step (2)
The mixture of protease, regulation solution ph is 8.5, in digesting 3h at 40 DEG C, filters, collects enzymolysis liquid I and filter residue；With filter residue
For substrate, above-mentioned enzymolysis operation is repeated, centrifugation collects enzymolysis liquid II, enzymolysis liquid I and enzymolysis liquid II are merged；Wherein, tryptose
The weight ratio of enzyme and papain is 2:1；
(4) resin is soaked for 25% sodium chloride solution of the D-254 weight resins of 200 mesh with 2 times of particle diameters, and stirs 3h,
Eluted respectively using 10% sodium chloride solution and 5% sodium chloride solution of 1 times of D-254 weight resin again, each 2h；
(5) it is the 4% of enzymolysis liquid volume that volume is added into the enzymolysis liquid after merging, and concentration is molten for 1mol/L sodium chloride
Liquid, stirring mixing 8min, 20min is incubated in 70 DEG C, and the D-254 resins added after being handled in step (4) are stirred at room temperature
6h is adsorbed, mixing speed is 100r/min, filtrate and D-254 resins are collected in filtering respectively；And its volume is incorporated as into filtrate
6% sodium chloride solution, adds excessive 90% ethanol precipitation, collects and dries precipitate I；Wherein, enzymolysis liquid and resin
Envelope-bulk to weight ratio V:M=12~15；
(6) 2 times of weight resins are added into resin, concentration is eluted 2 times for 1.6mol/L sodium chloride solution, each 4h,
Merge eluent, add 0.4% trypsin digestion 2h, then isometric 95% ethanol precipitation, collect and dry precipitation
Thing II；
(7) precipitate I and precipitate II are mixed, are dissolved in water, then be separately added into the peroxidating of 0.1% mixture weight
5min is placed in the tannic acid of hydrogen and 4% mixture weight, stirring mixing, and then filtering adds isometric 90% into filtrate
Ethanol precipitation, collects and dries precipitate, obtain heparin sodium pure product.
Detect example
The yield and purity of the heparin sodium pure product of the gained of embodiment 1~3 are detected under the same conditions, and it the results are shown in Table 1.
The liquaemin yield of table 1 and purity
| Yield (%) | Purity (%) | |
| Embodiment 1 | 94.2 | 97.6 |
| Embodiment 2 | 93.5 | 96.4 |
| Embodiment 3 | 96.8 | 97.3 |
As shown in Table 1, the gained liquaemin yield of embodiment 1~3 is all higher than 90%, and purity is above 95%, especially in fact
Example 3 is applied, yield reaches 96.8%, and purity is 97.3%.
Claims (7)
1. a kind of extracting method of liquaemin, it is characterised in that comprise the following steps：
(1) fresh intestinal mucosa is collected, is saved backup in 0~4 DEG C；
(2) water of 4~5 times of its weight is incorporated as into small intestinal mucosa, 60~65 DEG C, and the stirring point while heating is warming up to
Dissipate, be incubated 20~30min, then be cooled to 30~45 DEG C；
(3) compound protease of 0.4~0.8% small intestinal mucosa weight is added into the small intestinal mucosa solution obtained by step (2), is adjusted
It is 8.5~9 to save solution ph, and in digesting 3~4h at 40~45 DEG C, enzymolysis liquid I and filter residue are collected in filtering；Using filter residue as substrate,
Above-mentioned enzymolysis operation is repeated, centrifugation collects enzymolysis liquid II, enzymolysis liquid I and enzymolysis liquid II are merged；
(4) resin is soaked with 20%~25% sodium chloride solution of 2~3 times of weight resins, and stirs 2~3h, then used respectively
10% sodium chloride solution and 5% sodium chloride solution of 0.5~1.5 times of weight resin elute resin, every time 1~2h；
(5) sodium chloride solution is added into the enzymolysis liquid after merging, stirring 5~10min of mixing, it is incubated 10 in 60~70 DEG C~
20min, adds the resin after processing in step (4), at room temperature 4~6h of stirring and adsorbing, and mixing speed is 50~100r/
Filtrate and resin are collected in min, filtering respectively；And the sodium chloride solution of its volume 6%~8% is incorporated as into filtrate, add
Excessive 90% ethanol precipitation, collects and dries precipitate I；Wherein, the envelope-bulk to weight ratio V of enzymolysis liquid and resin:M=12~15；
Sodium chloride solution volume accounts for the 1%~4% of enzymolysis liquid volume；The concentration of sodium chloride solution is 0.5~1mol/L；
(6) sodium chloride solution that 1~2 times of weight resin is added into resin is eluted 2~3 times, and 3~5h, merges eluent every time,
Add 0.4% 2~3h of trypsin digestion, then isometric 95% ethanol precipitation collects and simultaneously dries precipitate II；
(7) precipitate I and precipitate II are mixed, are dissolved in water, then be separately added into 0.1% mixture weight hydrogen peroxide and
5~10min is placed in the tannic acid of 4%~8% mixture weight, stirring mixing, and then filtering adds isometric into filtrate
90% ethanol precipitation, collect simultaneously dry precipitate, obtain heparin sodium pure product.
2. the extracting method of liquaemin according to claim 1, it is characterised in that compound protease described in step (3)
Weight be small intestinal mucosa weight 0.4%.
3. the extracting method of liquaemin according to claim 1 or 2, it is characterised in that compound protein described in step (3)
Enzyme includes trypsase and papain, and the weight ratio of trypsase and papain is 2:1.
4. the extracting method of liquaemin according to claim 1, it is characterised in that sodium chloride concentration described in step (5)
For 0.8mol/L, sodium chloride solution volume accounts for the 1.5% of enzymolysis liquid volume.
5. the extracting method of liquaemin according to claim 1, it is characterised in that resin described in step (5) is particle diameter
For the D-254 resins or WD-1701 resins of 150~200 mesh.
6. the extracting method of liquaemin according to claim 1, it is characterised in that sodium chloride solution described in step (6)
Concentration is 1.6mol/L.
7. the extracting method of liquaemin according to claim 1, it is characterised in that the weight of tannic acid described in step (7)
Measure as the 4% of mixture weight.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101831009A (en) * | 2010-05-11 | 2010-09-15 | 新疆立实生物科技有限公司 | Process for producing concentrated and purified heparin |
| CN101864002A (en) * | 2010-06-21 | 2010-10-20 | 广元市海天实业有限责任公司 | Method for extracting sodium heparin |
| KR101447123B1 (en) * | 2014-02-27 | 2014-10-06 | 박상협 | Extraction Method of Heparin |
| CN104725531A (en) * | 2013-12-24 | 2015-06-24 | 安徽宝迪肉类食品有限公司 | Production process of effectively extracting heparin sodium |
-
2017
- 2017-05-25 CN CN201710379292.8A patent/CN107056967A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101831009A (en) * | 2010-05-11 | 2010-09-15 | 新疆立实生物科技有限公司 | Process for producing concentrated and purified heparin |
| CN101864002A (en) * | 2010-06-21 | 2010-10-20 | 广元市海天实业有限责任公司 | Method for extracting sodium heparin |
| CN104725531A (en) * | 2013-12-24 | 2015-06-24 | 安徽宝迪肉类食品有限公司 | Production process of effectively extracting heparin sodium |
| KR101447123B1 (en) * | 2014-02-27 | 2014-10-06 | 박상협 | Extraction Method of Heparin |
Non-Patent Citations (1)
| Title |
|---|
| 王国良主编: "《财产保险 核赔实务指南 化学工业篇 上篇》", 30 June 2001, 重庆出版社 * |
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