Method for separating hyaluronate tetrasaccharide from hyaluronate hexasaccharide

A hyaluronic acid tetrasaccharide and separation method technology, applied in the field of bioengineering, can solve problems such as unsuitable separation, and achieve the effects of low detection limit, good recovery rate and high purity

Active Publication Date: 2015-05-13
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, these methods are not suitable for the isolation of hyaluronic acid hydrolyzed products from leech-derived hyaluronidase

Method used

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  • Method for separating hyaluronate tetrasaccharide from hyaluronate hexasaccharide
  • Method for separating hyaluronate tetrasaccharide from hyaluronate hexasaccharide
  • Method for separating hyaluronate tetrasaccharide from hyaluronate hexasaccharide

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0017] Embodiment 1 is the preparation of hyaluronic acid oligosaccharides with glucuronic acid as the end

[0018] Add 1g of hyaluronic acid to a 38°C enzyme reactor containing 98ml of deionized water. After incubating at 38°C for 15 minutes, add 2ml of leech hyaluronidase solution with an enzyme activity of 800,000 U / ml, and react for 32 hours. The bath was heated for 10 min. After centrifugation at 8000 rpm for 15 min, the precipitate was discarded and the supernatant was taken. Such as figure 1 As shown, the supernatant is a mixture containing disaccharides, tetrasaccharides and hexasaccharides of hyaluronic acid. The sample with a retention time of 8.636 is hyaluronic acid disaccharide, the sample with a retention time of 10.884 is hyaluronic acid tetraose, and the sample with a retention time of 13.608 is hyaluronic acid hexasaccharide.

Embodiment 2

[0019] Example 2 Separation and purity detection of hyaluronic acid tetrasaccharide and hexasaccharide

[0020] Prepare solution A (balance solution): pH 8, 10mM Tris-HCl buffer solution, solution B (elution solution): 0.125M NaCl buffer solution prepared from solution A, and carry out hyaluronic acid tetrasaccharide and hexasaccharide by the following conditions separation.

[0021] (1) Equilibrate the HiPrep 16 / 10Q FF ion exchange column (height diameter 3:1) with 5 column volumes of solution A at a speed of 5ml / min.

[0022] (2) Load the sample (the mixture containing hyaluronic acid tetrasaccharide and hexasaccharide prepared in Example 1) at a flow rate of 1 ml / min until the sample breakthrough point is 5%.

[0023] (3) Wash away the sample with solution A, elute for 5 column volumes, and flow rate is 5ml / min.

[0024] (4) Carry out linear elution with liquid A and liquid B, so that the NaCl concentration in the eluent is 0.000M-0.125M, the elution volume is 15 column v...

Embodiment 3

[0028] Embodiment 3 hyaluronic acid tetrasaccharide and hexasaccharide recovery rate

[0029] Take 0.1000 g of the tetrasaccharides and hexasaccharides separated in Example 2, mix them and dissolve them in 500 μL, follow the operation in Example 2, the sample volume is 500 μL, separate and obtain desalted hyaluronic acid oligosaccharides, and measure after lyophilization The weight of oligosaccharides, including 0.9385g of tetrasaccharides and 0.9061g of hexasaccharides. The recovery rate of tetrasaccharide was 93.85%, and the recovery rate of hexasaccharide was 90.61%.

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Abstract

The invention discloses a method for separating hyaluronate tetrasaccharide from hyaluronate hexasaccharide, and belongs to the technical field of bioengineering. The method comprises the following steps: adopting Q Sepharose fast flow as an ion exchanger, Tris-HCl with the pH value of 8-9 as an equilibrium liquid, and 0.125 M NaCl with the pH value of 8, performing linear gradient elution to separate hyaluronate tetrasaccharide from hyaluronate hexasaccharide, and desalting to obtain pure products of hyaluronate tetrasaccharide and hyaluronate hexasaccharide.

Description

technical field [0001] The invention relates to a method for separating tetrasaccharides and hexasaccharides of hyaluronic acid, in particular to a method for separating tetrasaccharides and hexasaccharides of hyaluronic acid with glucuronic acid as the terminal obtained by hydrolyzing hyaluronic acid with leech hyaluronidase , belonging to the field of bioengineering technology. Background technique [0002] Hyaluronic acid (HA for short), commonly known as hyaluronic acid, is a repeating disaccharide unit composed of D-glucuronic acid and N-acetylglucosamine, through β-(1→4) and β-(1→ 3) Macromolecular acidic viscous polysaccharides formed by alternating glycosidic bonds were first extracted from bovine glass globe eyes by Meyer et al. in 1934, and are widely used in medicine, cosmetics, food and other fields. Studies have shown that hyaluronic acid can interact with the cell surface receptor CD44, thereby affecting the drug resistance of cancer cells. The hyaluronic aci...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C08B37/08C07H7/033C07H1/06
Inventor 王淼吕梦娴郝文幸
Owner JIANGNAN UNIV
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