CN103645150A - Detection method for enzymatic hydrolysate in extraction process of sodium hyaluronate by cockscombs - Google Patents
Detection method for enzymatic hydrolysate in extraction process of sodium hyaluronate by cockscombs Download PDFInfo
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- CN103645150A CN103645150A CN201310673738.XA CN201310673738A CN103645150A CN 103645150 A CN103645150 A CN 103645150A CN 201310673738 A CN201310673738 A CN 201310673738A CN 103645150 A CN103645150 A CN 103645150A
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- sodium hyaluronate
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Abstract
The invention relates to a detection method for the content of sodium hyaluronate in cockscomb enzymatic hydrolysate in the process of preparing the sodium hyaluronate by a cockscomb extraction method. According to the detection method, a treated test sample solution is detected by using an ultraviolet and visible spectrophotometer; the content of the sodium hyaluronate in the detected cockscomb enzymatic hydrolysate is calculated by preparing a gradient standard solution, making a standard curve and establishing a regression equation. The detection method is accurate and reliable in result, simple in steps, low in detection cost and easy to popularize; the content of the sodium hyaluronate in the cockscomb enzymatic hydrolysate is detected and an optimal enzymolysis condition can be regulated, so that the quality of downstream products is guaranteed.
Description
Technical field
The present invention relates to a kind of detection method of sodium hyaluronate, be specifically related to cockscomb and extract the detection method in legal system sodium hyaluronate process, relate in particular to the detection method of enzymolysis liquid in a kind of sodium hyaluronate cockscomb leaching process.
Background technology
Sodium hyaluronate is a kind of macromolecule polysaccharide, for aspects such as surgery arthropathy, organization restoration, ophthalmologic operation, auxiliary diagnosises.Its production technology is mainly divided into two large classes, take extraction method and the bacterial fermentation method that animal tissue is raw material.In cockscomb extraction method (animal tissue), produce in the process of sodium hyaluronate, the mensuration by sodium hyaluronate in cockscomb enzymolysis liquid, can regulate and control best enzymatic hydrolysis condition, for process modification provides Data support more accurately, thus the quality of assurance downstream finished product.
Summary of the invention
Technical matters to be solved by this invention is to provide the detection method of enzymolysis liquid in a kind of sodium hyaluronate cockscomb leaching process, to detect the content of the sodium hyaluronate in enzymolysis liquid.
In order to realize object of the present invention, the technical scheme adopting is as follows:
A detection method for enzymolysis liquid in sodium hyaluronate cockscomb leaching process, the method comprises the following steps:
(1) prepare reference substance solution: get D-Glucose aldehydic acid reference substance 60mg, accurately weighed, put in 100ml measuring bottle, be dissolved in water and be diluted to scale, shake up; Precision measures 5ml, puts in 50ml volumetric flask, is diluted with water to scale, shakes up and makes the reference substance solution that contains D-Glucose aldehydic acid 60 μ g in every 1ml;
(2) prepare need testing solution: take cockscomb enzymolysis liquid 5.2g in 50ml centrifuge tube, add 10ml ethanol, put into centrifuge, with rotating speed 4000rmp, after centrifugal 20 minutes, take out centrifuge tube; Outwell the ethanol supernatant in centrifuge tube, retain the white depositions in centrifuge tube; Then add a small amount of water that white depositions in centrifuge tube is all transferred in 250ml volumetric flask and dissolves and spend the night and in centrifuge tube, add suitable quantity of water dissolution precipitation residual; All pour aqueous solution in centrifuge tube in volumetric flask next day, shakes up, and is settled to scale;
(3) make reference substance typical curve solution: precision measures reference substance solution 0,0,0.2,0.4,0.6,0.8,1.0ml, put respectively in 25ml tool plug test tube, add water to respectively successively 1.0ml, mix, cooling in ice bath, and slowly drip 0.025mol/L borax sulfuric acid solution 5.0ml under constantly stirring, close plug, boiling water bath heating 10 minutes, middle jolting is once, cooling rapidly, the ethanol solution 0.2ml that adds 0.125% carbazole, shakes up, and heats 15 minutes in boiling water bath, middle jolting once, is cooled to room temperature; 0 pipe is blank solution.
(4) precision measures need testing solution 0.5ml and water 0.5ml, puts in same 25ml tool plug test tube, mixes, cooling in ice bath, and slowly drip 0.025mol/L borax sulfuric acid solution 5.0ml, close plug under constantly stirring, boiling water bath heating 10 minutes, middle jolting once, cooling rapidly, adds the ethanol solution 0.2ml of 0.125% carbazole, shake up, in boiling water bath, heat 15 minutes, middle jolting once, is cooled to room temperature;
(5) with ultraviolet-visible pectrophotometer to reference substance typical curve solution and need testing solution, under 530nm wavelength, measure, by the concentration of regression equation calculation sodium hyaluronate, its formula is:
M in formula: be the amount (μ g/ml) of glucuronic acid in test sample pipe;
W
supply: be test sample weight (g).
The method of enzymolysis liquid sodium hyaluronate content in detection sodium hyaluronate cockscomb extraction method of the present invention, speed is fast, and degree of accuracy is high, can be used to cockscomb extraction method and produce in the process of sodium hyaluronate, regulates and controls best enzymatic hydrolysis condition.
Embodiment
Mode below by embodiment further illustrates the present invention, but the present invention is not limited in described embodiment.
Embodiment 1:
Cockscomb extraction method is produced a detection method for cockscomb enzymolysis liquid in sodium hyaluronate process, and the method comprises the following steps:
(1) prepare reference substance solution: get D-Glucose aldehydic acid reference substance 60mg, accurately weighed, put in 100ml measuring bottle, be dissolved in water and be diluted to scale, shake up; Precision measures 5ml, puts in 50ml volumetric flask, is diluted with water to scale, shakes up.In every 1ml, contain glucuronic acid 60 μ g.
(2) prepare need testing solution: take in the about 5.2g of cockscomb enzymolysis liquid and 50ml centrifuge tube, add 10ml ethanol, put into centrifuge, with rotating speed 4000rmp, after centrifugal 20 minutes, take out centrifuge tube.Outwell the ethanol supernatant in centrifuge tube, attention can not make white depositions in pipe run off.Add a small amount of water that white depositions in centrifuge tube is all transferred in 250ml volumetric flask to dissolve and spend the night (can in centrifuge tube, add suitable quantity of water dissolution precipitation residual).All pour aqueous solution in centrifuge tube in volumetric flask next day, shakes up, and is settled to scale.
(3) make reference substance typical curve solution: precision measures reference substance solution 0,0,0.2,0.4,0.6,0.8,1.0ml, put respectively in 25ml tool plug test tube, add water to respectively successively 1.0ml, mix, cooling in ice bath, and slowly drip 0.025mol/L borax sulfuric acid solution 5.0ml under constantly stirring, close plug, 10 minutes (middle jolting once) of boiling water bath heating, cooling rapidly, add the ethanol solution 0.2ml of 0.125% carbazole, shake up, in boiling water bath, heat 15 minutes (middle jolting once), be cooled to room temperature.0 pipe is blank solution.
(4) precision measures need testing solution 0.5ml and water 0.5ml, put in same 25ml tool plug test tube, mix, cooling in ice bath, and slowly drip 0.025mol/L borax sulfuric acid solution 5.0ml under constantly stirring, close plug, 10 minutes (middle jolting is once) of boiling water bath heating, cooling rapidly, the ethanol solution 0.2ml that adds 0.125% carbazole, shake up, in boiling water bath, heat 15 minutes (middle jolting once), be cooled to room temperature.
(5) with ultraviolet-visible pectrophotometer to reference substance typical curve solution and need testing solution, under 530nm wavelength, measure, by the concentration of regression equation calculation sodium hyaluronate, its formula is:
M in formula: be the amount (μ g/ml) of glucuronic acid in test sample pipe;
W
supply: be test sample weight (g).
According to said method, a certain batch of cockscomb newly entering carried out to enzymolysis, 1kg cockscomb is used the enzyme that the enzyme activity of 5g is 60W to carry out enzymolysis 1h, and sodium hyaluronate content in enzymolysis liquid is detected, and its test result is in Table 1.
Table 1
| Sampling batch | Sodium hyaluronate content (g/L) |
| A | 1.94 |
| B | 1.93 |
| C | 1.95 |
| D | 1.93 |
| E | 1.95 |
Average: 1.94 RSD%:0.1
Embodiment 2:
According to the method described in embodiment 1, a certain batch of cockscomb newly entering carried out to enzymolysis, 1kg cockscomb is used the enzyme that the enzyme activity of 7g is 60W to carry out enzymolysis 1h, and sodium hyaluronate content in enzymolysis liquid is detected, and its test result is in Table 2.
Table 2
| Sampling batch | Sodium hyaluronate content (g/L) |
| A | 2.55 |
| B | 2.56 |
| C | 2.60 |
| D | 2.63 |
| E | 2.61 |
Average: 2.59 RSD%:0.1
Embodiment 3:
According to the method for embodiment 1, a certain batch of cockscomb newly entering carried out to enzymolysis, 1kg cockscomb is used the enzyme that the enzyme activity of 9g is 60W to carry out enzymolysis 1h, and sodium hyaluronate content in enzymolysis liquid is detected, and its test result is in Table 3.
Table 3
| Sampling batch | Sodium hyaluronate content (g/L) |
| A | 3.13 |
| B | 3.18 |
| C | 3.14 |
| D | 3.16 |
| E | 3.15 |
Average: 3.15 RSD%:0.1
According to above-mentioned analysis of experimental data, in the present invention, cockscomb extraction method is produced to sodium hyaluronate, by regulating and controlling best enzymatic hydrolysis condition, for process modification provides Data support more accurately.
Claims (1)
1. a detection method for enzymolysis liquid in sodium hyaluronate cockscomb leaching process, is characterized in that, the method comprises the following steps:
(1) prepare reference substance solution: get D-Glucose aldehydic acid reference substance 60mg, accurately weighed, put in 100ml measuring bottle, be dissolved in water and be diluted to scale, shake up; Precision measures 5ml, puts in 50ml volumetric flask, is diluted with water to scale, shakes up and makes the reference substance solution that contains D-Glucose aldehydic acid 60 μ g in every 1ml;
(2) prepare need testing solution: take cockscomb enzymolysis liquid 5.2g in 50ml centrifuge tube, add 10ml ethanol, put into centrifuge, with rotating speed 4000rmp, after centrifugal 20 minutes, take out centrifuge tube; Outwell the ethanol supernatant in centrifuge tube, retain the white depositions in centrifuge tube; Then add a small amount of water that white depositions in centrifuge tube is all transferred in 250ml volumetric flask and dissolves and spend the night and in centrifuge tube, add suitable quantity of water dissolution precipitation residual; All pour aqueous solution in centrifuge tube in volumetric flask next day, shakes up, and is settled to scale;
(3) make reference substance typical curve solution: precision measures reference substance solution 0,0,0.2,0.4,0.6,0.8,1.0ml, put respectively in 25ml tool plug test tube, add water to respectively successively 1.0ml, mix, cooling in ice bath, and slowly drip 0.025mol/L borax sulfuric acid solution 5.0ml under constantly stirring, close plug, boiling water bath heating 10 minutes, middle jolting is once, cooling rapidly, the ethanol solution 0.2ml that adds 0.125% carbazole, shakes up, and heats 15 minutes in boiling water bath, middle jolting once, is cooled to room temperature; 0 pipe is blank solution.
(4) precision measures need testing solution 0.5ml and water 0.5ml, puts in same 25ml tool plug test tube, mixes, cooling in ice bath, and slowly drip 0.025mol/L borax sulfuric acid solution 5.0ml, close plug under constantly stirring, boiling water bath heating 10 minutes, middle jolting once, cooling rapidly, adds the ethanol solution 0.2ml of 0.125% carbazole, shake up, in boiling water bath, heat 15 minutes, middle jolting once, is cooled to room temperature;
(5) with ultraviolet-visible pectrophotometer to reference substance typical curve solution and need testing solution, under 530nm wavelength, measure, by the concentration of regression equation calculation sodium hyaluronate, its formula is:
M in formula: be the amount (μ g/ml) of glucuronic acid in test sample pipe;
W
supply: be test sample weight (g).
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106053369A (en) * | 2016-07-13 | 2016-10-26 | 浙江景嘉医疗科技有限公司 | Method for detecting content of free sodium hyaluronate in medical cross-linking sodium hyaluronate gel |
| CN108303386A (en) * | 2017-12-15 | 2018-07-20 | 浙江景嘉医疗科技有限公司 | A kind of detection method of the external enzymatic hydrolyzation of medical cross-linking sodium hyaluronate gel |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102134287A (en) * | 2010-01-22 | 2011-07-27 | 上海昊海生物科技股份有限公司 | Preparation method for pipelined extraction of sodium hyaluronate through cockscombs |
| CN102492755A (en) * | 2011-11-30 | 2012-06-13 | 上海景峰制药有限公司 | Sodium hyaluronate enzymolysis method |
-
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- 2013-12-10 CN CN201310673738.XA patent/CN103645150A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102134287A (en) * | 2010-01-22 | 2011-07-27 | 上海昊海生物科技股份有限公司 | Preparation method for pipelined extraction of sodium hyaluronate through cockscombs |
| CN102492755A (en) * | 2011-11-30 | 2012-06-13 | 上海景峰制药有限公司 | Sodium hyaluronate enzymolysis method |
Non-Patent Citations (3)
| Title |
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| 凌沛学: "《玻璃酸钠及其在外科中的应用》", 31 January 2003, 中国医药科技出版社 * |
| 杨雅莉等: "药用真菌戴氏虫草中透明质酸的定性定量分析", 《药物分析杂志》 * |
| 高继国等: "《普通生物化学教程实验指导》", 31 August 2009, 化学工业出版社 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106053369A (en) * | 2016-07-13 | 2016-10-26 | 浙江景嘉医疗科技有限公司 | Method for detecting content of free sodium hyaluronate in medical cross-linking sodium hyaluronate gel |
| CN108303386A (en) * | 2017-12-15 | 2018-07-20 | 浙江景嘉医疗科技有限公司 | A kind of detection method of the external enzymatic hydrolyzation of medical cross-linking sodium hyaluronate gel |
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Application publication date: 20140319 |