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CN-121975030-A - Extraction and purification method of clematis polysaccharide and polysaccharide obtained by extraction and purification method

CN121975030ACN 121975030 ACN121975030 ACN 121975030ACN-121975030-A

Abstract

The invention relates to separation, purification and polysaccharide preparation of a clematis polysaccharide, which comprises the steps of S1, alcohol precipitation of the clematis crude polysaccharide in an 80% alcohol solution overnight, dialysis and drying to obtain an 80% alcohol precipitation component of the clematis crude polysaccharide, S2, enzymolysis of the 80% alcohol precipitation component of the clematis crude polysaccharide obtained in S1 at 70 ℃ by using alpha-amylase for 2h, enzyme inactivation, dialysis and drying to obtain a clematis polysaccharide enzymolysis product, and S3, purifying the clematis polysaccharide enzymolysis product by using a DEAE-anion exchange chromatography column to obtain the clematis purified polysaccharide. The purification method of the clematis polysaccharide has the advantages of mild conditions, short purification time, simple overall process, and capability of quickly and efficiently obtaining the purified components of the clematis polysaccharide with high purity, and has practical and popularization values.

Inventors

  • Jia Bingtao
  • LI XIANG
  • WEI YUKUN
  • YANG SHUILAN
  • FENG LEI
  • XIE ANQI
  • WAN HAO
  • WAN YIQUN

Assignees

  • 江西省检验检测认证总院
  • 南昌大学

Dates

Publication Date
20260505
Application Date
20260117

Claims (6)

  1. 1. The extraction and purification method of the polysaccharide of the clematis and the polysaccharide obtained are characterized in that the preparation method is as follows: S1, performing alcohol precipitation on crude polysaccharide of the clematis with 80% ethanol, placing the crude polysaccharide in a refrigerator at a temperature of 4 ℃, standing overnight, centrifuging an alcohol solution of the crude polysaccharide of the clematis (3000-5000 rpm,5-10 min), removing supernatant, re-dissolving the precipitate with water, dialyzing, performing rotary evaporation, and freeze-drying to obtain an alcohol precipitation component of the crude polysaccharide of the clematis; S2, weighing 500 mg of the ethanol precipitation component of the crude polysaccharide of the clematis chinensis, which is obtained in the step S1, dissolving the ethanol precipitation component in a buffer solution of 100 mL PBS, stirring the solution until the ethanol precipitation component is completely dissolved, adding 20-40 mu L of alpha-amylase, carrying out water bath 2-h at 70 ℃, placing the reaction in a refrigerator at-80 ℃, then taking out the solution to dissolve the solution at 25 ℃, centrifuging the solution, taking the supernatant, repeatedly freezing and thawing the supernatant for 2-4 times, dialyzing the obtained supernatant for 48-h, concentrating and freeze-drying the supernatant to obtain an enzymolysis product of the crude polysaccharide of the clematis chinensis; S3, dissolving the crude polysaccharide enzymolysis product of the clematis chinensis obtained in the step S2 in Tris-HCl buffer solution, separating by using a DEAE anion exchange chromatography, continuously eluting an eluent which is a Tris-HCl buffer solution containing 0-0.5 mol/L NaCl, respectively collecting each eluting component in an eluting mode, dialyzing, steaming in a rotary mode, freeze-drying, performing high-performance liquid chromatography detection, and obtaining the 4 th eluting component with single and symmetrical liquid chromatography peak and highest yield which is named as AFP-80-0.4.
  2. 2. The method for extracting and purifying a polysaccharide from Leuconostoc as claimed in claim 1, wherein the enzyme activity of the alpha-amylase in the step S2 is 3000U/mL.
  3. 3. The method for extracting and purifying polysaccharide of Leuconostoc as claimed in claim 1, wherein the PBS solution in step S3 has a pH of 7.
  4. 4. The method for extracting and purifying polysaccharide of Leuconostoc as claimed in claim 1, wherein the PBS solution in step S3 contains 0.02% NaN 3 (w/v%).
  5. 5. The method for extracting and purifying polysaccharide of Leuconostoc and polysaccharide obtained according to claim 1, wherein the loading and eluting flow rate in step S3 is preferably 0.5 mL/min.
  6. 6. The method for extracting and purifying polysaccharide of Leuconostoc as claimed in claim 1, wherein the NaCl solution gradient in step S3 is 0, 0.2, 0.3, 0.4, 0.5 mol/L.

Description

Extraction and purification method of clematis polysaccharide and polysaccharide obtained by extraction and purification method Technical Field The invention belongs to the technical field of extraction of plant polysaccharide, and particularly relates to an extraction and purification method of clematis polysaccharide and the obtained polysaccharide. Background The herba Lobeliae chinensis is a rare plant which is used as both crude drugs and food for many years, and the whole herb of herba Lobeliae chinensis can be used as a medicine, has sweet taste and cool nature, and has the effects of clearing heat and cooling blood, dispelling wind and promoting diuresis, strengthening heart and promoting urination, reinforcing kidney, calming liver and the like. At present, the polysaccharide in the solution can be precipitated by ethanol to extract the polysaccharide, but the method has the defects that firstly, the non-binding protein in the polysaccharide can form coprecipitation with the polysaccharide, and the precipitate is glycoprotein composed of the polysaccharide and the binding protein, so that the polysaccharide extraction rate of the polysaccharide in the polysaccharide is low, the purity of the extracted polysaccharide is low, and the polysaccharide becomes a bottleneck which hinders the practical application of the polysaccharide; the second alcohol precipitation method is easy to precipitate polysaccharide alcohols in different molecular weight ranges, so that the obtained polysaccharide is not pure polysaccharide, but is a combination of polysaccharide with different molecular weights, and therefore, the research and development of the separation and purification process of the polysaccharide of the clematis is of great practical significance for the development and application of the polysaccharide of the clematis. Disclosure of Invention The invention provides an extraction and purification method of clematis polysaccharide and the polysaccharide obtained by the method, which are characterized in that the crude clematis polysaccharide is primarily purified by an alcohol precipitation method, and then the polysaccharide is further purified by an enzymolysis method and an ion exchange column chromatography method, so that the clematis polysaccharide component with higher purity is separated, and the problems of low extraction rate of the clematis polysaccharide and low purity of the clematis polysaccharide in the related technology are solved. The extraction and purification method of the polysaccharide of the clematis and the polysaccharide obtained by the method are as follows: S1, performing alcohol precipitation on crude polysaccharide of the clematis with 80% ethanol, placing the crude polysaccharide in a refrigerator at a temperature of 4 ℃, standing overnight, centrifuging an alcohol solution of the crude polysaccharide of the clematis (3000-5000 rpm,5-10 min), removing supernatant, re-dissolving the precipitate with water, dialyzing, performing rotary evaporation, and freeze-drying to obtain an alcohol precipitation component of the crude polysaccharide of the clematis; S2, weighing 500 mg of the ethanol precipitation component of the crude polysaccharide of the clematis chinensis, which is obtained in the step S1, dissolving the ethanol precipitation component in a buffer solution of 100 mLPBS, stirring the solution until the ethanol precipitation component is completely dissolved, adding 20-40 mu L of alpha-amylase, carrying out water bath 2-h at 70 ℃, placing the reaction in a refrigerator at-80 ℃, then taking out the solution to dissolve the solution at 25 ℃, centrifuging the solution, taking the supernatant, repeatedly freezing and thawing the supernatant for 2-4 times, dialyzing the obtained supernatant for 48-h, concentrating and freeze-drying the supernatant to obtain an enzymolysis product of the crude polysaccharide of the clematis chinensis; S3, dissolving the crude polysaccharide enzymolysis product of the clematis in the step S2 in Tris-HCl buffer solution, separating by DEAE anion exchange chromatography, continuously eluting the eluent which is Tris-HCl buffer solution containing 0-0.5 mol/LNaCl, respectively collecting each eluting component, dialyzing, steaming, freeze-drying, performing high performance liquid chromatography detection, and obtaining the 4 th eluting component with single and symmetrical liquid chromatography peak and highest yield which is named AFP-80-0.4. Further, the enzyme activity of the alpha-amylase in the step S2 is 3000U/mL; Further, the pH of the PBS solution in step S3 is 7; Further, the PBS solution in step S3 contains 0.02% NaN 3 (w/v%); further, the loading and eluting flow rate in the step S3 is preferably 0.5 mL/min; Further, the NaCl solution gradient in step S3 is 0, 0.2, 0.3, 0.4, 0.5 mol/L. The invention has the beneficial effects that: 1. The invention establishes a complete and feasible technical route for separating and purifying the