CN111647100B - Preparation method of high molecular weight zinc hyaluronate - Google Patents

Preparation method of high molecular weight zinc hyaluronate Download PDF

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CN111647100B
CN111647100B CN202010673892.7A CN202010673892A CN111647100B CN 111647100 B CN111647100 B CN 111647100B CN 202010673892 A CN202010673892 A CN 202010673892A CN 111647100 B CN111647100 B CN 111647100B
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CN111647100A (en
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郭学平
杨桂兰
肖慧
栾贻宏
刘爱华
石艳丽
耿凤
阚洪玲
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Bloomage Biotech Co Ltd
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Shandong Bloomage Hyinc Biopharm Co Ltd
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    • 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
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Abstract

The invention provides a preparation method of high molecular weight zinc hyaluronate, which comprises the following steps: adding sodium hyaluronate into the replacement solution for replacement to obtain zinc hyaluronate precipitate; washing the precipitate with a washing solution; and dehydrating the washed precipitate by using a dehydrating solution, and drying in vacuum to obtain high molecular weight hyaluronic acid zinc powder. The preparation method of the invention explores the preparation conditions such as pH of the exchange solution, the times of exchange, pH of washing, the times of washing, pH of dehydration solution, the times of dehydration and the like to obtain the optimal preparation conditions. The molecular weight of the zinc hyaluronate obtained by the preparation method can reach more than 100 ten thousand Da, the light transmittance is more than 99.5%, the zinc content is more than 7.0%, and the problems of low molecular weight and low clarity are successfully solved.

Description

Preparation method of high molecular weight zinc hyaluronate
Technical Field
The invention belongs to the field of biological medicines, relates to a preparation method of high molecular weight zinc hyaluronate, and particularly relates to a method for preparing high molecular weight zinc hyaluronate.
Background
Hyaluronic acid (hyaluronic acid) is also called hyaluronic acid, HA for short, hyaluronic acid is called in the fields of cosmetics and foods, hyaluronic acid or hyaluronic acid is called in the field of medicines, sodium hyaluronate is commonly used, carboxyl binding ions of hyaluronic acid are sodium ions, sodium hyaluronate or sodium hyaluronate are also called, and zinc hyaluronate (HA-Zn for short) HA-Zn have the functions of resisting ulcer of digestive tract, treating bacterial infection and fungal infection and inhibiting helicobacter pylori besides the lubrication, moisture retention and viscoelasticity of HA-Na when the carboxyl binding ions of hyaluronic acid are zinc ions. Therefore, the compound has good application prospect in the field of medicine.
Patent CN98804672.5 describes that zinc hyaluronate has a remarkable advantage in treating peptic ulcer, gastric ulcer, duodenal ulcer and other digestive tract ulcers. Because of the viscosity, a protective film can be formed on the surface of the wound, the wound tissue can be repaired, and the wound healing can be promoted. The film agent can be used for treating oral ulcer, and can be applied to the wound surface of the oral cavity to achieve the functions of absorbing ulcer tissue secretion and repairing wound surface tissue.
Patent CN97197886.7 microbial composition with antimicrobial activity: use of zinc or cobalt hyaluronate associate (complex) for the treatment of bacterial and fungal infections and helicobacter pylori induced gastric and duodenal ulcers.
Patent CN200510117601.1, a preparation method of zinc hyaluronate, discloses a method for preparing zinc hyaluronate by conversion in ethanol, but there is no limitation on molecular weight and clarity.
HA-Zn is prepared by the following steps: preparing ethanol replacement solution containing zinc salt, adding HA-Na powder and Zn under stirring2+Replacement of Na on the carboxyl group of HA+. However, hyaluronic acid is easily degraded under acidic conditions, and the molecular weight of HA-Zn is reduced during drying. As is well known, the higher the molecular weight of HA, the better the moisture retention performance, the better the film forming property and the barrier effect, the zinc hyaluronate belongs to a high molecular compound, and the molecular weight can be reduced to different degrees in the storage process, so the HA-Zn M prepared at presentrThe preparation of HA-Zn with the molecular weight of more than 50 ten thousand Da and more than 100 ten thousand Da is a great technical problem, in addition, the light transmittance of the sample prepared by the prior art (the light transmittance is an index for representing the clarity of the sample, and the higher the light transmittance is, the better the clarity is) is often lower than 95%, and the opalescence can be seriously seen by naked eyes.
Disclosure of Invention
In view of the above problems of the prior art, the present invention provides a method for preparing high molecular weight zinc hyaluronate.
Specifically, the present invention relates to the following aspects:
a preparation method of high molecular weight zinc hyaluronate is characterized by comprising the following steps:
adding sodium hyaluronate into the replacement solution for replacement to obtain zinc hyaluronate precipitate;
washing the precipitate with a washing solution;
and dehydrating the washed precipitate by using a dehydrating solution, and drying in vacuum to obtain high molecular weight hyaluronic acid zinc powder.
The method of claim 1, wherein the sodium hyaluronate has a molecular weight greater than or equal to 200 million Da.
The process according to claim 1, wherein the substitution solution has an ethanol concentration of 70 to 85% (v/v), a zinc ion concentration of 0.15 to 1.0% (w/v), and a pH of 5.8 to 6.5.
The production method according to item 3, wherein the number of times of substitution is 2 to 6 times, preferably 3 to 5 times.
The method according to claim 1, wherein the concentration of ethanol in the washing solution is 75% to 85% (v/v), and the pH of the washing solution is 5.5 to 6.5, preferably 5.5 to 6.0.
The production method according to item 5, wherein the number of washing is 3 to 6.
The method according to claim 1, wherein the dehydrated liquid has an ethanol concentration of 90 to 95% (v/v), and the pH of the dehydrated liquid is 5.5 to 6.5, preferably 5.8 to 6.5.
The production method according to item 1, wherein the number of times of dehydration is 3 or more.
The process according to claim 1, wherein the obtained high molecular weight zinc hyaluronate has a molecular weight of more than 100 ten thousand Da.
The method according to item 1, wherein the obtained high molecular weight zinc hyaluronate has a light transmittance of more than 99.5%.
The method according to claim 1, wherein the zinc content of the obtained high molecular weight zinc hyaluronate is 7.0 to 8.0%.
The preparation method of the invention explores the preparation conditions such as pH of the exchange solution, the times of exchange, pH of washing, the times of washing, pH of dehydration solution, the times of dehydration and the like to obtain the optimal preparation conditions. The molecular weight of the zinc hyaluronate obtained by the preparation method can reach more than 100 ten thousand Da, the light transmittance is more than 99.5%, the zinc content is more than 7.0%, and the problems of low molecular weight and low clarity are successfully solved.
Detailed Description
The present invention is further illustrated by the following examples, which are intended to be purely exemplary of the invention and are not intended to be limiting.
Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Although methods and materials similar or equivalent to those described herein can be used in experimental or practical applications, the materials and methods are described below. In case of conflict, the present specification, including definitions, will control, and the materials, methods, and examples are illustrative only and not intended to be limiting. The present invention is further illustrated by the following examples, which are not intended to limit the scope of the invention.
The invention provides a preparation method of high molecular weight zinc hyaluronate, which comprises the following steps: adding powdery sodium hyaluronate into the replacement liquid for replacement to obtain powdery zinc hyaluronate;
washing the precipitate with a washing solution;
and dehydrating the washed precipitate by using a dehydrating solution, and drying in vacuum to obtain high molecular weight hyaluronic acid zinc powder.
In a specific embodiment, the molecular weight of the sodium hyaluronate is greater than or equal to 200 ten thousand Da, for example 200 ten thousand Da, 250 ten thousand Da, 300 ten thousand Da, 400 ten thousand Da, etc.
In a specific embodiment, the ethanol concentration in the substitution solution is 70% to 85% (v/v), and may be, for example, 70%, 72%, 75%, 78%, 80%, 82%, 85%. The zinc ion concentration is 0.15% to 1.0% (w/v), and may be, for example, 0.15%, 0.24%, 0.30%, 0.40%, 0.50%, 0.60%, 0.72%, or 1.0%. The zinc ion can be prepared by adding zinc salt into the replacement liquid, wherein the zinc salt is zinc chloride, zinc sulfate, zinc acetate, zinc nitrate and the like. The pH of the replacement liquid is 5.8 to 6.5, and may be, for example, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, or 6.5. The pH of the substitution solution can be adjusted by using an acid which does not affect the substitution reaction, for example, glacial acetic acid. The number of substitution is 2 to 6, and for example, 2, 3, 4, 5, 6, preferably 3 to 5.
In a specific embodiment, the ethanol concentration in the wash solution is 75% to 85% (v/v), for example 75%, 77%, 78%, 79%, 80%, 82%, 85%. The pH of the washing solution is 5.5 to 6.5, and may be, for example, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, preferably 5.5 to 6.0. The pH of the washing solution can be adjusted by using an acid which does not affect the washing, such as glacial acetic acid. The number of washing is 3 to 6, and for example, the number of washing may be 3, 4, 5, or 6.
In one embodiment, the concentration of ethanol in the dehydrated liquid is 90% to 95% (v/v), and may be, for example, 90%, 91%, 92%, 93%, 94%, 95%. The pH of the dehydrated liquid is 5.5 to 6.5, and may be, for example, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, preferably 5.8 to 6.5. The pH of the dehydrated liquid may be adjusted by using an acid which does not affect washing, such as glacial acetic acid. The number of dehydration is 3 or more, and for example, may be 3, 4, 5, 6, 7, 8, etc.
In a specific embodiment, the molecular weight of the obtained high molecular weight zinc hyaluronate is greater than 100 ten thousand Da, for example, 100 ten thousand Da, 110 ten thousand Da, 120 ten thousand Da, and the like.
In a specific embodiment, the resulting high molecular weight zinc hyaluronate has a light transmittance of greater than 99.5%, such as 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, and the like.
In a specific embodiment, the obtained high molecular weight zinc hyaluronate has a zinc content of 7.0% to 8.0%, and may be 7.0%, 7.1%, 7.2%, 7.3%, 7.4%, 7.5%, 7.6%, 7.7%, 7.8%, 7.9%, 8.0%, and the like.
In the present invention, the method for measuring the molecular weight of the obtained high molecular weight zinc hyaluronate is an intrinsic viscosity method. The specific measurement method is as follows.
Preparing a test solution: precisely weighing the sample about 100mg(W1) (Note: the sample weighing is uncertain, when the intrinsic viscosity is lower than 3.0dL/g or the molecular weight is lower than 10 ten thousand, the sample weighing is increased) to a 100ml volumetric flask, 0.2mol/L sodium chloride solution is added for constant volume, and the solution is shaken up; weighing the solution to a certain amount (W)2) (Note: the sample amount was not determined and adjusted depending on the measured flow-out time of the sample solution) to a 100ml volumetric flask, 0.2mo1/L sodium chloride solution was added to the flask to a constant volume, and the mixture was shaken up to give a sample solution.
Time of solvent flow out (t)0) The determination of (1): collecting appropriate amount of filtered 0.2mol/L sodium chloride solution, discarding the primary filtrate, collecting the subsequent filtrate (not less than 7ml), injecting into clean and dry Ubbelohde viscometer, vertically fixing the viscometer in constant temperature water bath (25 deg.C and 0.1 deg.C), standing for 15 min, accurately recording the outflow time, repeatedly measuring at least two times, and averaging to obtain the outflow time (t) of blank liquid0)。
Test solution flowing time (t)1) The determination of (1): taking appropriate amount of test solution, measuring according to the method for measuring solvent outflow time, repeating the measurement at least twice, the difference between the two measured values should not exceed 0.2 s, and averaging to obtain the outflow time (t) of the test solution1)。
t1/t0The concentration of the sample solution should be 1.3-1.5, otherwise, the concentration of the sample solution should be adjusted and then measured.
The test solution concentration C (g/dL) was calculated using the following formula:
Figure BDA0002583349710000051
ρ25not greater than 1.000g/ml (density of test solution at 25 ℃ C.)
The intrinsic viscosity was calculated using the following formula:
Figure BDA0002583349710000052
calculating the average molecular weight of zinc hyaluronate:
intrinsic viscosity [ eta ]](dL/g)＝3.6×10-4M0.78(M: average molecular weight)
In the present invention, the method for measuring the light transmittance of the obtained high molecular weight zinc hyaluronate is a light absorption method. The specific measurement method is as follows.
A sample (0.50 g) was taken into a conical flask containing 100ml of water, and left overnight in a refrigerator, after dissolution, the sample solution was poured into a 1ml cuvette, blanked with water, and the light transmittance of the solution was measured at a wavelength of 550 nm.
In the present invention, the method for measuring the zinc content of the obtained high molecular weight zinc hyaluronate is a flame atomic absorption method. The specific measurement method is as follows.
Preparation of a standard curve:
1ml of zinc single element solution standard substance (1000 mu g/ml) is precisely weighed into a volumetric flask of 100ml, and the volume is determined by 1% nitric acid to obtain standard stock solution (10 mu g/m 1). Respectively measuring 0ml, 0.2 ml, 0.4 ml, 0.8 ml and 1.6ml of standard stock solutions in 5 clean 10ml volumetric flasks, and fixing the volume by using 1% nitric acid. Obtaining zinc standard solutions with zinc concentrations of 0, 0.2, 0.4, 0.8 and 1.6 mu g/m1 respectively.
Test solution: precisely weighing a sample 50mg to 100ml volumetric flask, adding 1ml nitric acid, heating in a water bath until complete bath decomposition, adding water to a constant volume, taking 1ml to 50ml volumetric flask of the solution, and adding 1% nitric acid to the constant volume to obtain the product.
And (3) determination: after the blank solution was set to zero, an appropriate amount of each solution was taken and measured by flame atomic absorption spectrometry, the detection wavelength was 213.9nm, and the absorbance was measured. The concentration of zinc in the test solution (X, μ g/m1) was linearly regressed by the absorbance (Y) of the test solution to draw a standard curve, and the concentration of zinc in the test solution was calculated by the following formula.
Figure BDA0002583349710000061
In the formula, Ci-the concentration of zinc in the test solution,. mu.g/m 1;
w is the sample weighing and the mg of the sample;
h% -loss on drying of the test article.
According to the preparation method of the high-molecular zinc hyaluronate, the preparation parameters of 70-85% (v/v) of ethanol concentration in a replacement liquid, 0.15-1.0% (w/v) of zinc ion concentration, 5.8-6.5 of pH, 2-6 times of replacement, 75-85% (v/v) of ethanol concentration in a washing liquid, 5.5-6.5 of pH, 3-6 times of washing, 90-95% (v/v) of ethanol concentration in a dehydration liquid, 5.5-6.5 of pH, more than 3 times of dehydration and the like are controlled, so that the molecular weight of the obtained zinc hyaluronate can reach more than 100 Da, the light transmittance is more than 99.5%, and the zinc content is more than 7.0%, and the problems of low molecular weight and low clarity are successfully solved.
Example 1
(1) Preparing displacement liquid, washing liquid and dehydrating liquid. Wherein the replacement solution was adjusted to pH 6.0 with glacial acetic acid while controlling the ethanol concentration at 70% (v/v) and the zinc ion concentration at 0.48% (w/v). The ethanol concentration in the washing solution was 85% (v/v), and the pH of the washing solution was adjusted to 5.5 using glacial acetic acid. The ethanol concentration in the dehydrated liquid was 90% (v/v), and the pH of the dehydrated liquid was adjusted to 5.8 using glacial acetic acid.
(2) Adding sodium hyaluronate with molecular weight of 200 kilodaltons into the ethanol displacement solution while stirring, continuously stirring (displacing) for more than 2 hours, standing, separating supernatant, and displacing for 2 times to obtain zinc hyaluronate precipitate.
(3) The precipitate was washed 6 times with the above washing solution.
(4) The washed zinc hyaluronate precipitate was dehydrated 4 times with a dehydrating solution.
(5) And finally, vacuum drying until the drying weight loss is less than 10 percent to obtain the hyaluronic acid zinc powder.
Examples 2 to 5
Examples 2 to 5 are different from example 1 in the concentration of zinc ions, the concentration of ethanol, and the corresponding pH in the prepared substitution liquid, washing liquid, and dehydrating liquid, and the number of times of substitution, washing, and dehydration are different, and other operation steps are the same. The conditions used in the specific preparation method are shown in table 1.
Comparative example 1
Comparative example 1 differs from example 1 in that the ethanol concentration in the substitution solution was 68% (v/v), and other reaction conditions were the same.
Comparative example 2
Comparative example 2 differs from example 1 in that the ethanol concentration in the substitution solution was 95% (v/v), and other reaction conditions were the same.
Comparative example 3
Comparative example 3 differs from example 1 in that the zinc ion concentration in the substitution solution was 2.4% (w/v), and the other reaction conditions were the same.
Comparative example 4
Comparative example 4 is different from example 1 in that the pH of the substitution solution was 5.0 and other reaction conditions were the same.
Comparative example 5
Comparative example 5 differs from example 1 in that the pH of the substitution solution was 7.0, and other reaction conditions were the same.
Comparative example 6
Comparative example 6 differs from example 1 in that the number of replacements is 1 and other reaction conditions are the same.
Comparative example 7
Comparative example 7 differs from example 1 in that the ethanol concentration in the washing solution was 72% (v/v), and other reaction conditions were the same.
Comparative example 8
Comparative example 8 differs from example 1 in that the pH of the washing solution was 5.0 and other reaction conditions were the same.
Comparative example 9
Comparative example 9 differs from example 1 in that the pH of the washing solution was 7.0 and other reaction conditions were the same.
Comparative example 10
Comparative example 10 is different from example 1 in that the number of washing times is 1 and other reaction conditions are the same.
Comparative example 11
Comparative example 11 is different from example 1 in that the pH of the dehydrated liquid was 5.0 and other reaction conditions were the same.
Comparative example 12
Comparative example 12 is different from example 1 in that the pH of the dehydrated liquid was 7.0 and other reaction conditions were the same.
TABLE 1 conditions in the preparation methods of examples and comparative examples
Figure BDA0002583349710000081
The zinc hyaluronate obtained from the above examples 1 to 5 and comparative examples 1 to 12 was tested for molecular weight, zinc content and transparency. Wherein the method for measuring the molecular weight is an intrinsic viscosity method. The method for measuring the zinc content is a flame atomic absorption method. The method for measuring the light transmittance is a light absorption method.
The test results obtained are shown in table 2.
Table 2 test results of zinc hyaluronate obtained in examples and comparative examples
Figure BDA0002583349710000082
Figure BDA0002583349710000091
Examples 1 to 5 were carried out according to the established preparation parameters, and according to the reaction conditions of table 1 and the test results of table 2, in the preparation of the high molecular zinc hyaluronate of the present invention, by adjusting the preparation conditions such as the pH of the displacement solution, the number of displacements, the pH of washing, the number of washing, the pH of the dehydration solution, and the number of dehydration, the molecular weight of the obtained zinc hyaluronate can reach more than 100 ten thousand Da, the light transmittance is greater than 99.5%, and the zinc content is greater than 7.0%, thereby successfully solving the problems of low molecular weight and low clarity.
Comparative examples 1 and 7 in comparison with examples 1 to 5 and other comparative examples, it is considered that the sample is dissolved due to the decrease in the ethanol concentration and the increase in the water content ratio, and the experiment cannot be performed normally.
In comparative example 2, 95% ethanol is adopted to prepare the replacement solution, so that the replacement cannot be normally carried out, and the zinc content does not meet the standard requirement.
In comparative example 3, the higher concentration of zinc ions was used for the substitution, and since the washing with zinc ions was incomplete during the washing process, the transmittance was low although the concentration of zinc ions reached the requirement of more than 7.0%.
The comparative examples 4, 5, 8, 9 and 11 have lower molecular weight than the examples. It is considered that the comparative examples 4, 5, 8, 9, and 11 were subjected to different degrees of pH change during the preparation process, and the pH change increased instability of the product, resulted in a great degradation of the molecular weight of the product, and a decrease in the molecular weight of the final product.
Comparative example 6 has a reduced number of replacements and a low replacement ratio, resulting in a zinc ion content of less than 7.0%.
Comparative example 10 the number of washes was reduced compared to the example, and the resulting product had a relatively low transmittance and an unsatisfactory clarity of the product solution.
Comparative example 12 is a solution using a higher pH for dehydration, resulting in incomplete washing with zinc ions and a final product with substandard zinc content but substandard light transmittance.

Claims (9)

1. A preparation method of high molecular weight zinc hyaluronate is characterized by comprising the following steps:
adding sodium hyaluronate into the replacement solution for replacement to obtain zinc hyaluronate precipitate;
washing the precipitate with a washing solution;
dehydrating the washed precipitate with a dehydrating solution, and drying in vacuum to obtain high molecular weight zinc hyaluronate powder;
wherein the concentration v/v of ethanol in the replacement liquid is 70-85%, the concentration w/v of zinc ions is 0.15-1.0%, the pH of the replacement liquid is 5.8-6.5, and the replacement times are 2-6 times;
the concentration v/v of ethanol in the washing liquid is 75-85%, the pH value of the washing liquid is 5.5-6.5, and the washing times are 3-6;
the pH value of the dehydration solution is 5.5-6.5.
2. The method of claim 1, wherein the sodium hyaluronate has a molecular weight greater than or equal to 200 million Da.
3. The method according to claim 2, wherein the number of times of substitution is 3 to 5 times.
4. The method according to claim 1, wherein the pH of the washing solution is 5.5 to 6.0.
5. The method according to claim 1, wherein the concentration v/v of ethanol in the dehydrating solution is 90% to 95%.
6. The method according to claim 1, wherein the pH of the dehydrating solution is 5.8 to 6.5.
7. The method according to claim 1, wherein the number of times of dehydration is 3 or more.
8. The method of claim 1, wherein the resulting high molecular weight zinc hyaluronate has a molecular weight greater than 100 million Da.
9. The method of claim 1, wherein the obtained zinc hyaluronate has a light transmittance of more than 99.5% and a zinc content of 7.0% to 8.0%.
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