CN107522796A - A kind of method that hyaluronic acid is extracted in the eye from tuna - Google Patents
A kind of method that hyaluronic acid is extracted in the eye from tuna Download PDFInfo
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- CN107522796A CN107522796A CN201710768321.XA CN201710768321A CN107522796A CN 107522796 A CN107522796 A CN 107522796A CN 201710768321 A CN201710768321 A CN 201710768321A CN 107522796 A CN107522796 A CN 107522796A
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- hyaluronic acid
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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
- C08B37/0072—Hyaluronic acid, i.e. HA or hyaluronan; Derivatives thereof, e.g. crosslinked hyaluronic acid (hylan) or hyaluronates
-
- 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
Abstract
The invention discloses a kind of method that hyaluronic acid is extracted in eye from tuna, methods described is：Tuna eye powder is added in deionized water and extracts complete, centrifugation, acquisition supernatant；Trypsase is added into supernatant, 60~180min is reacted under conditions of being 7.0~8.5 in 20~45 DEG C, pH, after reaction completely, obtains hyaluronic acid crude product；Hyaluronic acid crude product is configured to the hyaluronic acid solution of mass concentration 1 5% with deionized water, final concentration 0.1~0.3mol/L sodium chloride is added, mother liquor is made；The hexadecylpyridinium chloride aqueous solution of mass concentration 1~10% is added into mother liquor, stirring reaction is complete at 30 DEG C, obtains hyaluronic acid；The hyaluronan molecule amount that tuna is extracted is smaller, is 0.470MDa, and process route is simple and easy, and gained hyaluronic acid purity is higher, and loss of hyaluronic acid is smaller in technological process.
Description
(1) technical field
The present invention relates to a kind of tuna eye source production its extraction process of medical hyaluronic acid and purifying products method.
(2) background technology
Hyaluronic acid (hyaluronic acid, HA), is a kind of anionic acid mucopolysaccharide, by β-(1 → 3)-D-N second
Acylamino- glucose repeats to be formed by connecting with β-(1 → 4)-D-Glucose aldehydic acid dissacharide units.Hyaluronic acid has unique guarantor
Moist and bioaffinity, pass through the physical actions such as its viscoelasticity (macromolecular network structure) and high degree of hydration in body
Physics function and its important function is played with the physiologic functions of HA receptor actings, at present both functions extensively should
For cosmetics, pharmaceuticals industry.The production technology of hyaluronic acid mainly has biological tissue extracted method and Streptococcal fermentation method at present.
Research shows that HA is contained in nearly all animal tissue, but considers cost of material, HA contents, is easy to the factors such as acquirement degree,
The main production raw material of the hyaluronic acid of biology extraction at present is rooster comb, Human Umbilical Cord and animal vitreum.With cockscomb and the mankind
The biological pollution risk such as avian influenza virus, hepatitis B, AIDS virus be present in the hyaluronic acid that the terrestrial animals such as umbilical cord are extracted.
And the hyaluronic acid in tuna eye source has higher biological safety because of species variation.Have at present and many tuna is discarded
Internal organ, the research of fish-bone comprehensive utilization, but tunny head is a large amount of caused by it can not utilize to be wasted and pollutes.Therefore with tuna
Eye reduces the extraction cost of hyaluronic acid, can be the low value-added tuna production in China as raw material extraction hyaluronic acid
Industry provides more selections.
The demand of hyaluronic acid at home and abroad in the market is very big, but is limited cause by the prices of raw materials and fermentation technique
Make holding at high price for hyaluronic acid, and then limit its application in medicine, cosmetics and health food etc..Therefore
How to obtain cheap, safe hyaluronic acid turns into new research direction.The present invention uses Enzymatic Extraction tuna eye hyalomitome
Acid, and quaternary ammonium salt complexometry Purification of hyaluronic acid is used, provide new approach for the production of hyaluronic acid.
(3) content of the invention
The present invention relates to a kind of its extraction of tuna eye source production medical hyaluronic acid and purification process, carried using enzyme process
Flake hyaluronic acid is taken, and uses quaternary ammonium salt complexometry Purification of hyaluronic acid product.This method produces hyaluronic acid product tool
There is the advantages that cheap, safe, technology path is simple, easy to operation, solve existing HA production technology costs height, it is originally rare,
The problems such as complex operation.
The technical solution adopted by the present invention is：
The present invention provides a kind of method that hyaluronic acid is extracted in eye from tuna, and methods described is：(1) removed cleaned
Miscellaneous dried tuna eye powder is added in deionized water, and extraction is stirred at room temperature completely, centrifugation, obtains supernatant；(2) to step
Suddenly add trypsase in the supernatant of (1), 20~45 DEG C (preferably 40 DEG C), pH be 7.0~8.5 under conditions of reaction 60~
180min, after reaction completely, by (preferably 90 DEG C of heating water bath inactivations) centrifugation after reaction solution heat inactivation, supernatant is taken to add body
Product ratio 4:1 chloroform and n-butanol carries out extract and separate, takes upper liquid to add the ethanol water of volumetric concentration 95% stirring precipitation
After centrifuge, take precipitation drying, obtain hyaluronic acid crude product；The trypsase addition adds tuna eye powder with step (1)
The amount at end is calculated as 200~1000U/g (preferably 200-500U/g)；(3) step (2) hyaluronic acid crude product is prepared with deionized water
Into mass concentration 1-5% (preferably 1-2%) hyaluronic acid solution, final concentration 0.1~0.3mol/L sodium chloride is added, mother is made
Liquid；Mass concentration 1~10% (preferably 1-5%) hexadecylpyridinium chloride aqueous solution is added into mother liquor, is stirred at 30 DEG C anti-
Should be complete, stand and separate out precipitation, remove supernatant, 0.4~1.0mol/L NaCl aqueous solution is added into precipitation, in 30 DEG C of stirring solutions
From 1~2h, and 0.45 μm of membrane filtration is used, filtrate uses the ethanol water of volumetric concentration 95% stirring precipitation, gained precipitation
40-50 DEG C of vacuum drying after rinsing 2~3 times repeatedly using absolute ethyl alcohol, obtain hyaluronic acid；The hexadecylpyridinium chloride
Aqueous solution volumetric usage is calculated as 4~8mL/g with hyaluronic acid crude product quality.
Further, the method for step (1) the cleaning removal of impurities is：Frozen fresh flake is taken, after normal temperature unfreezing, except going
Shell and structure of fish muscle, optic nerve, collecting black flake vitreum, (in operation, liquid easily flows out in vitreum, should try one's best
Recovery)；3 times of vol acetones are added after black glass body is shredded, 6h is stirred at room temperature, 10000rpm is centrifuged under the conditions of subsequent 4 DEG C,
Taking precipitate is dried in vacuo 6~10h at 30~45 DEG C, needs not timing to take out upset product, crushes after sediment drying, exist again
30~45 DEG C of 6~10h of vacuum drying, gained powder is tuna eye powder.
Further, step (1) extraction time is 1-10h (preferably 4-6h), and the volumetric usage of the deionized water is with golden rifle
Flake powder weight is calculated as 6~50mL/g (preferably 20-35mL/g).
Further, step (2) the trypsase addition is calculated as 200 with the amount of step (1) addition tuna eye powder
~500U/g.
Further, step (3) the hyaluronic acid solution mass concentration is 1.0~2.0%.
Further, step (3) the hexadecylpyridinium chloride aqueous solution volumetric usage is in terms of hyaluronic acid crude product quality
For 4~6mL/g.
Tuna eye feature of the present invention is as follows：Flake exophthalmos, corneal transparency, surface mucus is limpid, without obvious
Corrupt smell.This is fresh flake, if flake is corrupt, extracts hyaluronic acid volume of production, significant decrease of molecular weight.
Enzymatic Extraction flake hyaluronic acid of the present invention uses trypsase, is purchased from raw work bioengineering (Shanghai) share
Co., Ltd, enzyme activity 2000U/g.
It is hexadecylpyridinium chloride (cetylpyridinium chloride the present invention relates to the quaternary ammonium salt used
Monohydrate, CPC), it is purchased from BBI Life Scienses.
It is as follows with existing hyaluronic acid manufacture method compared, outstanding advantages of the invention：
(1) tuna eye has huge supply in China as raw material, and this invention can also solve substantial amounts of fishery and discard
Thing pollutes；(2) hyaluronic acid in tuna eye source has higher biological safety；(3) hyaluronic acid that flake is extracted
Molecular weight is smaller, is 0.470MDa, has some superiority in small molecule class cosmetics.(4) this process route is simple and easy, gained
Hyaluronic acid purity is higher, and loss of hyaluronic acid is smaller in technological process.It is rare to solve raw material using discarded tuna eye,
Terrestrial animal organizes pathogen contamination problem that may be present, reduces production cost.
(4) illustrate
Fig. 1 tuna eye hyaluronan extraction purification circuits.
Fig. 2 glucuronic acid standard curves.
The regression equation of Fig. 3 hyaluronic acid samples concentration and viscosity.
Fig. 4 hyaluronic acids standard items and flake hyaluronic acid infared spectrum.
(5) embodiment
With reference to specific embodiment, the present invention is described further, and as described below is only being preferable to carry out for the present invention
Mode：Room temperature refers to 25-30 DEG C described in the embodiment of the present invention.
Embodiment 1：Tuna eye pretreatment prepares flake powder
Frozen fresh flake 4314g is taken, after normal temperature unfreezing, shell and structure of fish muscle, optic nerve etc. is removed, retains black
Flake vitreum, its weight are 1431g.In operation, liquid easily flows out in vitreum, and should try one's best recovery.By black glass
Glass body adds 3 times of vol acetones after shredding, and 6h is stirred at room temperature.10000rpm is centrifuged under the conditions of subsequent 4 DEG C, and taking precipitate is at 45 DEG C
Vacuum drying oven dries 10h, needs not timing to take out upset product.Dried again in 45 DEG C of vacuum drying ovens after sediment drying through crushing
10h, gained powder are flake powder 210g, can be used as extraction hyaluronic acid raw material, can also be placed in -20 DEG C long-term preserve.
Embodiment 2：Tuna eye pretreatment prepares flake powder
Frozen fresh flake 2932g is taken, after normal temperature unfreezing, shell and structure of fish muscle, optic nerve etc. is removed, retains black
Flake vitreum, its weight are 941g.In operation, liquid easily flows out in vitreum, and should try one's best recovery.By black glass
Body adds 3 times of vol acetones after shredding, and 4h is stirred at room temperature.10000rpm is centrifuged under the conditions of subsequent 4 DEG C, and taking precipitate is true at 30 DEG C
Empty oven drying 6h, not timing is needed to take out upset product.6h is dried in 30 DEG C of vacuum drying ovens again through crushing after sediment drying,
Gained powder is flake powder 154g, can be used as extraction hyaluronic acid raw material, can also be placed in -20 DEG C long-term preserve.
Embodiment 3：Enzymatic Extraction flake hyaluronic acid
It is raw material by flake powder obtained by the method for embodiment 1, enzymatic isolation method extraction hyaluronic acid technique is as follows
(1) 25g flake powder is weighed, and adds 500mL deionized waters, 6h is stirred at room temperature.
(2) (1) reaction solution is centrifuged, takes supernatant to add 500U trypsase/g flake powder, at 40 DEG C, the conditions of pH 8.5
Lower reaction 120min.
(3) after (2) reaction solution is inactivated in 90 DEG C of water-baths, cooling.10000rpm is centrifuged under the conditions of subsequent 4 DEG C, is obtained
Obtain supernatant.
(4) supernatant 500mL in (3) is added into separatory funnel, adds 200mL chloroforms, acutely stirred after 50mL n-butanols
20min is mixed, stratification (be divided into, neutralize lower three layers) in separatory funnel, releases lower floor's chloroform and intermediate layer protein, is repeated
Layering 3 times.
(5) ethanol water of volumetric concentration 95% of 3 times of volumes is added in (4) at the middle and upper levels clear liquid, is centrifuged after stirring 4h,
It is powdered to Baise that 45 DEG C of vacuum drying oven dryings will be deposited in, as hyaluronic acid crude product 0.117g, quality containing hyaluronic acid point
Number is 83.1%, and albumen quality fraction is 2.1%.
The hyaluronic acid contents that tuna eye of the present invention is extracted by raw material use Bitter-Muir sulfuric acid carbazole methods
Detection, albumen are detected using Coomassie Brilliant Blue, and the method for detection is：Detected using Bitter-Muir sulfuric acid carbazole methods transparent
Matter acid, its principle are：Hyaluronic acid is hydrolyzed into glucuronic acid (GA) in boiling water bath by the concentrated sulfuric acid, and GA is presented with carbazole reaction
Reddish violet, there is an obtained the maximum absorption at 530nm, and absorbance is directly proportional to GA content.Specific detection method is as follows：
The configuration of 0.125% ethanol carbazole solution：Precision weighs 0.125g carbazoles, and it is molten to add 100mL absolute ethyl alcohol ultrasounds
Solution, is placed in 4.0 DEG C and is kept in dark place, term of validity 15d.
The configuration of 0.025mol/L sodium tetraborate sulfuric acid solutions：Precision weighs 4.77g sodium tetraborates (Na2BO7·10H2O),
It is added in 500mL top pure grade sulfuric acid, not timing shakes repeatedly, until being completely dissolved, room temperature is kept in dark place, and the term of validity is
180d。
The configuration of glucuronic acid standard items：Precision weighs 0.100g glucuronic acid standard items, is dried in 105.0 DEG C of vacuum
Dried to constant weight in case, with distilled water constant volume in 100mL volumetric flasks.5.0mL fully is taken after dissolving, is placed in 100mL volumetric flasks
With distilled water constant volume, glucuronic acid concentration is 50 μ g/mL, and 4.0 DEG C preserve.
The formulation of glucuronic acid standard curve：Glucuronic acid standard liquid is prepared by table 1..
The preparation of the glucuronic acid standard liquid of table 1
Each test tube of titer series is placed in cooling more than 2h in mixture of ice and water, 5mL is slowly added into each test tube is adherent
Sodium tetraborate sulfuric acid solution, side edged concussion, keeps test tube lower end to be immersed in ice-water bath, each test tube is placed in into boiling water bath after mixing
In boil 15min, cooled down after taking-up in ice-water bath, then add and 0.2mL ethanol carbazole solution and fully shake into each test tube,
15min is boiled in boiling water bath again, is cooled to room temperature.Using No. 0 test tube as control, each test tube suction is detected at ultraviolet 530nm
Luminosity.Using glucuronic acid content as abscissa, absorbance is ordinate, does linear regression, and standard curve is shown in Fig. 2.
Embodiment 4：Enzymatic Extraction flake hyaluronic acid
It is raw material by flake powder obtained by the method for embodiment 2, enzymatic isolation method extraction hyaluronic acid technique is as follows
(1) 15g flake powder is weighed, and adds 500mL deionized waters, 4h is stirred at room temperature.
(2) (1) reaction solution is centrifuged, takes supernatant to add 200U trypsase/g flake powder, at 40 DEG C, the conditions of pH 8.0
Lower reaction 120min.
(3) after (2) reaction solution is inactivated in 90 DEG C of water-baths, cooling.10000rpm is centrifuged under the conditions of subsequent 4 DEG C, is taken
Supernatant.
(4) it supernatant 500ml will be added in (3) in separatory funnel, and add 200mL chloroforms, after 50mL n-butanols acutely
20min is stirred, is stood in separatory funnel and is divided into upper, middle and lower-ranking, released lower floor's chloroform and intermediate layer protein, be repeated 3 times.
(5) ethanol water of volumetric concentration 95% of 3 times of volumes is added in (4) at the middle and upper levels clear liquid, after 4h is stirred at room temperature
Centrifugation, it is powdered to Baise to be deposited in 45 DEG C of vacuum drying oven dryings, as hyaluronic acid crude product 0.071g, hyaluronic acid quality
Fraction is 81.0%, and albumen quality fraction is 2.7%.
Embodiment 5：The method of hexadecylpyridinium chloride method Purification of hyaluronic acid
Being configured to mass concentration with deionized water as raw material using the hyaluronic acid crude product 5.0g of the enzymatic isolation method of embodiment 3 extraction is
1% hyaluronic acid solution 500mL, then with CPC complex reaction Purification of hyaluronic acid, comprise the following steps that：
(1) 5.84g sodium chloride is added in the hyaluronic acid solution 500mL of mass concentration 1.0% makes its concentration be
0.2mol/L is as reaction mother liquor.
(2) after adding the 30mL mass concentration 1.0%CPC aqueous solution to step (1), being sufficiently stirred under 30 DEG C of water-baths makes it
Reaction completely, 1h, siphon supernatant are stood after precipitating completely.
(3) wall of cup will be adhered in (2), the complex compound sediment of bottom of a cup is existed using 0.4mol/L NaCl aqueous solution 500mL
30 DEG C of stirring dissociation 2h, and use 0.45 μm of membrane filtration.
(4) filtrate is stirred into precipitation using the ethanol water of volumetric concentration 95% of 3 times of volumes, gained precipitation uses anhydrous
45 DEG C of vacuum dryings after ethanol rinses 2~3 times repeatedly, produce hyaluronic acid product 4.56g, the HA rate of recovery 91.3%, HA mass
Fraction 90.1%, albumen quality fraction are 0.1%, and hyaluronan molecule amount is 0.470MDa, and final products pass through infared spectrum
Identification is compared, and product purity is higher, and infared spectrum is shown in Fig. 4.
Tuna eye of the present invention is viscosimetry by the assay method for the hyaluronan molecule amount that raw material extracts, and is had
The advantages that easy to operate, detection time is short, and molecular weight detection scope is wide, concrete operations are as follows：Precision weighs HA standard items 0.1g,
100mL is settled to the 0.2mol/L NaCl aqueous solution.Take the solution 10,15,20 respectively again and 25mL be configured to 0,0.2,
0.3rd, 0.4,0.5 and 0.6g/L HA series standard solution.It is molten using digital display viscosimeter detection sample viscosity η at 25.0 DEG C
The viscosity η of agent0, by formula η sp=(η-η0)/η0Specific viscosity η sp are calculated, are horizontal stroke with the concentration (mg/100mL) of HA standard items
Coordinate, the ratio for increasing specific concentration and concentration make regression analysis for ordinate, calculate coefficient correlation and regression equation, HA standard items
Regression equation see Fig. 3.Intercept of the curve of regression equation fitting on the longitudinal axis is inherent viscosity [η], and HA is calculated by formula
The mean molecule quantity (MDa) of standard items.
[η]=3.6x10-4M0.78(M is mean molecule quantity)
Embodiment 6：The method of hexadecylpyridinium chloride method Purification of hyaluronic acid
It is dense that the hyaluronic acid crude product 10.0g extracted using enzymatic isolation method in embodiment 3 as raw material with deionized water is configured to quality
The hyaluronic acid solution 500mL of degree 2%, then with CPC complex reaction Purification of hyaluronic acid, is comprised the following steps that：
(1) 2.92g sodium chloride is added in the hyaluronic acid solution 500mL of mass concentration 2.0% makes its concentration be
0.1mol/L is as reaction mother liquor.
(2) after adding the 40mL mass concentration 5.0%CPC aqueous solution to step (1), being sufficiently stirred under 40 DEG C of water-baths makes it
Reaction completely, 2h, siphon supernatant are stood after precipitating completely.
(3) wall of cup will be adhered in (2), the complex compound sediment of bottom of a cup is existed using 0.6mol/L NaCl aqueous solution 500mL
40 DEG C of stirring dissociation 2h, and use 0.45 μm of membrane filtration.
(4) filtrate is stirred into precipitation using the ethanol water of volumetric concentration 95% of 3 times of volumes, gained precipitation uses anhydrous
45 DEG C of vacuum dryings after ethanol rinses 2-3 times repeatedly, produce hyaluronic acid 8.42g, the HA rate of recovery 84.1%, HA mass fractions
82.6%, albumen quality fraction is 0.2%.
Above-described embodiment is the preferable implementation of the present invention, and in addition, the present invention can be realized with other manner,
Do not depart from the premise of present inventive concept it is any it is obvious replace, within protection scope of the present invention.
Claims (6)
1. the method for hyaluronic acid is extracted in a kind of eye from tuna, it is characterised in that methods described is：(1) by cleaned removal of impurities
Dried tuna eye powder is added in deionized water, and extraction is stirred at room temperature completely, centrifugation, obtains supernatant；(2) to step
(1) trypsase is added in supernatant, reacts 60~180min, reaction under conditions of being 7.0~8.5 in 20~45 DEG C, pH
It after completely, will be centrifuged after reaction solution heat inactivation, take supernatant to add volume ratio 4:1 chloroform and n-butanol carries out extraction point
From taking upper liquid to be centrifuged after adding volumetric concentration 95% ethanol water stirring precipitation, take precipitation drying, it is thick to obtain hyaluronic acid
Product；The amount that the trypsase addition adds tuna eye powder with step (1) is calculated as 200~1000U/g；(3) by step
(2) hyaluronic acid crude product is configured to mass concentration 1-5% hyaluronic acid solutions with deionized water, add final concentration 0.1~
0.3mol/L sodium chloride, is made mother liquor；The addition hexadecylpyridinium chloride aqueous solution of mass concentration 1~10% into mother liquor, 30
Stirring reaction is complete at DEG C, stands and separates out precipitation, removes supernatant, and 0.4~1.0mol/L NaCl aqueous solution is added into precipitation,
30 DEG C of stirring dissociation 1-2h, and 0.45 μm of membrane filtration is used, filtrate stirs precipitation using the ethanol water of volumetric concentration 95%,
40-50 DEG C of vacuum drying after gained precipitation rinses 2~3 times repeatedly using absolute ethyl alcohol, obtains hyaluronic acid；The cetyl
Pyridinium chloride aqueous solution volumetric usage is calculated as 4~8ml/g with hyaluronic acid crude product quality.
2. the method for hyaluronic acid is extracted from tuna eye as claimed in claim 1, it is characterised in that step (1) described cleaning
The method of removal of impurities is：Frozen fresh flake is taken, after normal temperature unfreezing, shell and structure of fish muscle, optic nerve is removed, collects black fish
Vitreum；3 times of vol acetones are added after black glass body is shredded, are stirred at room temperature 6h, under the conditions of subsequent 4 DEG C 10000rpm from
The heart, taking precipitate are dried in vacuo 6-10 hours at 30-45 DEG C, need not timing to take out upset product, are crushed after sediment drying, then
Secondary to be dried in vacuo 6-10 hours at 30-45 DEG C, gained powder is tuna eye powder.
3. the method for hyaluronic acid is extracted from tuna eye as claimed in claim 1, it is characterised in that step (1) extraction time
For 1-10h, the volumetric usage of the deionized water is calculated as 6~50ml/g with tuna eye powder weight.
4. the method for hyaluronic acid is extracted from tuna eye as claimed in claim 1, it is characterised in that step (2) is described
The amount that trypsase addition adds tuna eye powder with step (1) is calculated as 200~500U/g.
5. the method for hyaluronic acid is extracted from tuna eye as claimed in claim 1, it is characterised in that step (3) is described transparent
Matter acid solution mass concentration is 1.0~2.0%.
6. the method for hyaluronic acid is extracted from tuna eye as claimed in claim 1, it is characterised in that step (3) described 16
Alkyl chlorination pyridine solution volumetric usage is calculated as 4~6ml/g with hyaluronic acid crude product quality.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1563109A (en) * | 2004-04-13 | 2005-01-12 | 阮春学 | Method for preparing hyaluronic acid |
| CN105131150A (en) * | 2015-08-19 | 2015-12-09 | 浙江工业大学 | Extraction method for hyaluronic acid in tuna eye |
| CN105504091A (en) * | 2013-07-30 | 2016-04-20 | 青岛九龙生物医药有限公司 | Method for extracting hyaluronic acid from pigskin |
-
2017
- 2017-08-31 CN CN201710768321.XA patent/CN107522796A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1563109A (en) * | 2004-04-13 | 2005-01-12 | 阮春学 | Method for preparing hyaluronic acid |
| CN105504091A (en) * | 2013-07-30 | 2016-04-20 | 青岛九龙生物医药有限公司 | Method for extracting hyaluronic acid from pigskin |
| CN105131150A (en) * | 2015-08-19 | 2015-12-09 | 浙江工业大学 | Extraction method for hyaluronic acid in tuna eye |
Non-Patent Citations (1)
| Title |
|---|
| 姚日生等: "《药用高分子材料 第二版》", 30 April 2008, 化学工业出版社 * |
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Application publication date: 20171229 |