CN110845632B

CN110845632B - Novel preparation method of euscaphis konishii hayata pericarp polysaccharide and product application thereof

Info

Publication number: CN110845632B
Application number: CN201911062581.0A
Authority: CN
Inventors: 倪林; 邹双全; 陈景新; 徐会有; 黄维; 邹小兴
Original assignee: Fujian Tianwu Forestry Development Co ltd; Fujian Agriculture and Forestry University
Current assignee: Fujian Tianwu Forestry Development Co ltd; Fujian Agriculture and Forestry University
Priority date: 2019-11-03
Filing date: 2019-11-03
Publication date: 2023-02-10
Anticipated expiration: 2039-11-03
Also published as: CN110845632A

Abstract

A novel preparation method of euscaphis konishii hayata pericarp polysaccharide comprises crushing euscaphis konishii hayata pericarp, extracting with water, precipitating with alcohol, centrifuging, purifying by polyamide resin column chromatography, and concentrating under reduced pressure to dry to obtain euscaphis konishii hayata pericarp polysaccharide. On the basis of preparing polysaccharide by traditional water extraction and alcohol precipitation, the crude polysaccharide is further purified by using a polyamide column chromatography, so that the content of euscaphis konishii hayata polysaccharide is over 85 percent, and in vitro tests show that the product can replace acarbose to inhibit the acarbose equivalentlyα-the activity of a glucosidase enzyme.

Description

Novel preparation method of euscaphis konishii hayata pericarp polysaccharide and product application thereof

Technical Field

The invention relates to the technical field of extraction processes of plant polysaccharides, in particular to an extraction method and application of euscaphis konishii hayata pericarp polysaccharide.

Background

In recent years, plant polysaccharides have attracted more and more researchers' attention because of their biological activities such as anti-tumor, blood sugar lowering, and anti-oxidation, and have developed various drugs and health products such as ganoderan, lentinan, and ginseng polysaccharide.

Euscaphis japonicaEuscaphis japonica(Thunb.) Dippel, a plant of the genus Euscaphis of the family Staphyleaceae, is a unique medicinal ornamental tree species in our country. In recent years, the planting area of Fujian provinces, jiangxi provinces and the like is continuously enlarged, the planting area of the Fujian Sanming Qingliu, taining, the Nanping Shaowu province, the Jianyang province, the Quanzhou Germany province and the like is more than 100 hectares, and the plant resources are rich. The subject group always focuses on the resource development and utilization of euscaphis konishii hayata, and the early stage discovers that the polysaccharide content of the water extract of the fruit peel of the euscaphis konishii hayata is high and the water extract has the effect of inhibitingα-the action of glycosidases, showing hypoglycemic activity. In order to develop euscaphis konishii hayata polysaccharide, the prior stage of the subject group adopts the traditional polysaccharide extraction method, namely water extraction-ethanol precipitation-centrifugation-concentration to obtain crude polysaccharide extraction, and the crude polysaccharide extraction inhibits the inhibitionαThe activity of glucosidase is weaker than that of the positive control drug acarbose. In order to further improve the biological activity of the euscaphis konishii hayata polysaccharide extract, ineffective components in the polysaccharide extract must be removed as much as possible.

The water extraction and alcohol precipitation method for extracting the plant polysaccharide usually has a large amount of water-soluble tannin components. Polyamides are high molecular compounds obtained by amide group polymerization, contain abundant amide groups, and can be adsorbed by forming hydrogen bonds with phenols, quinones, nitro compounds, etc., and separated from compounds incapable of forming hydrogen bonds. Therefore, the subject group utilized polyamide as the column packing to adsorb the water-soluble tannin components in euscaphis konishii hayata polysaccharide in the polyamide column, and the polysaccharide components were eluted with water to further purify the crude extract of euscaphis konishii hayata polysaccharide. The euscaphis japonica polysaccharide purified by the method can inhibitαThe activity of the glucosidase is equivalent to that of a positive control drug, and the glucosidase can be further developed and utilized.

Disclosure of Invention

The invention aims to provide a novel preparation method of euscaphis konishii hayata pericarp polysaccharide, which adopts the preparation process of water extraction, alcohol precipitation, centrifugation, polyamide column chromatography purification and reduced pressure concentration to obtain a high-purity euscaphis konishii hayata pericarp polysaccharide extract; the preparation method is simple, easy to operate and suitable for large-scale production, and the prepared euscaphis konishii hayata pericarp polysaccharide has obvious alpha-glucosidase inhibition activity.

The technical method provided by the invention comprises the following steps:

taking euscaphis konishii hayata pericarp, drying, crushing and sieving by a 20-mesh sieve for later use;

aqueous solution extraction: taking euscaphis konishii hayata pericarp powder 1000 g, adding water solution 7500-10000 mL, heating and boiling for 2 hours, and filtering to obtain extract; adding 7500-10000 mL water solution into the residue, boiling for 2 hr, and filtering to obtain extractive solution; mixing the two water extracting solutions, and concentrating under reduced pressure to 300-500mL to obtain a concentrated extracting solution;

alcohol precipitation: adding anhydrous methanol or ethanol into the concentrated extractive solution to make the concentration of methanol or ethanol 85-90%, standing for 2-3 hr, centrifuging at 1600-2000 rpm for 15-20 min, and discarding supernatant to obtain precipitate as high-concentration Euscaphis konishii pericarp polysaccharide; ethanol precipitation is preferred in view of the toxicity of methanol;

and (3) polyamide column chromatography purification: mixing high-concentration euscaphis konishii hayata pericarp polysaccharide with polyamide of equal mass; the elution solvent is water; eluting 5-6 column volumes, and collecting water eluate.

Concentrating the water eluent: concentrating the water eluate under reduced pressure to dry to obtain Euscaphis japonica pericarp polysaccharide.

Experiment for inhibiting alpha-glucosidase in vitro by euscaphis japonica pericarp polysaccharide

0.5 U/mL α-glucosidase dissolved in phosphate buffered saline pH 6.8; sucking 40 μ L, mixing with 20 μ L of Corona sinensis pericarp polysaccharide extract with different concentration gradients, incubating at 37 deg.C for 10 min, adding 5 mmol/L substrate PNPG40 μ L, mixing, reacting at 37 deg.C for 30 min, adding 0.2 mol/L Na ₂ CO ₃ 100. mu.L of the reaction was stopped. The above experiment was repeated 3 times. The reaction product was measured for absorbance at 405 nm. An equal volume of solvent PBS was used as a blank control and acarbose was used as a positive control.

The sample has% inhibition rate on alpha-glucosidase (= (1-Ao/As) × 100,

in the formula: ao is the absorbance of the sample solution replaced by an equal amount of PBS; as is the absorbance of the added sample. Palmaria tomentosa peel polysaccharide extract pair with different concentrationsαThe inhibition of glucosidase is shown in the table below.

In vitro inhibition rate table for alpha-glucosidase inhibition by euscaphis japonica pericarp polysaccharide

Detailed Description

The invention will be better understood from the following examples. However, it is easily understood by those skilled in the art that the specific material ratios, process conditions and results thereof described in the examples are only for illustrating the present invention and should not be construed as limiting the invention described in detail in the claims.

Working example 1

Drying Euscaphis japonica pericarp at 50-60 deg.C, pulverizing, and sieving with 20 mesh sieve; weighing euscaphis konishii hayata pericarp powder 1000 g, adding water solution 10000 mL, heating and boiling for 2 hours, and filtering to obtain an extracting solution; adding 10000 mL water solution into the filter residue, boiling for 2 hours again, and filtering to obtain an extracting solution; mixing the two water extracting solutions, and concentrating under reduced pressure to 400mL; adding anhydrous ethanol into the concentrated extractive solution to make ethanol concentration of the solution be 86%, standing for 2-3 hr, centrifuging at 2000 rpm for 20 min, and discarding supernatant to obtain precipitate 236 g; dissolving the precipitate with water, mixing with 236 g of 60-100 mesh polyamide, stirring, and eluting by polyamide column chromatography; the elution solvent is water; eluting 20000 mL, collecting water eluate, concentrating under reduced pressure to dry to obtain 185g of euscaphis japonica pericarp polysaccharide with yield of 18.5% and polysaccharide content of 88.3%.

Example 2

Drying Euscaphis japonica pericarp at 50-60 deg.C, pulverizing, and sieving with 20 mesh sieve; weighing euscaphis konishii peel powder 1000 g, adding water solution 1000 mL, heating and boiling for 2 hours, and filtering to obtain an extracting solution; adding 7500 mL water solution into the residue, boiling for 2 hr, and filtering to obtain extractive solution; mixing the two water extracting solutions, and concentrating under reduced pressure to 400mL; adding anhydrous methanol into the concentrated extractive solution to make the concentration of methanol be 86%, standing for 2-3 hr, centrifuging at 2000 rpm for 20 min, and discarding supernatant to obtain precipitate; dissolving the precipitate with water, mixing with 236 g of 60-100 mesh polyamide, stirring, and eluting by polyamide column chromatography; the elution solvent is water; eluting 20000 mL, collecting water eluate, concentrating under reduced pressure to dry to obtain 185g of euscaphis japonica pericarp polysaccharide with yield of 18.5% and polysaccharide content of 88.3%.

Example 3

Drying Euscaphis japonica pericarp at 50-60 deg.C, pulverizing, and sieving with 20 mesh sieve; weighing euscaphis konishii hayata pericarp powder 1000 g, adding water solution 7500 mL, heating and boiling for 2 hours, and filtering to obtain extract; adding 7500 mL water solution into the residue, boiling for 2 hr, and filtering to obtain extractive solution; mixing the two water extracting solutions, and concentrating under reduced pressure to 400mL; adding anhydrous ethanol into the concentrated extractive solution to make ethanol concentration of the solution be 86%, standing for 2-3 hr, centrifuging at 2000 rpm for 20 min, and discarding supernatant to obtain precipitate; dissolving the precipitate with water, mixing with 236 g of 60-100 mesh polyamide, stirring, and eluting by polyamide column chromatography; the elution solvent is water; eluting 20000 mL, collecting water eluate, concentrating under reduced pressure to dry to obtain 185g of euscaphis japonica pericarp polysaccharide with yield of 18.5% and polysaccharide content of 88.3%.

Example 4

According to the experiment of inhibiting alpha-glucosidase in vitro by the euscaphis konishii hayata pericarp polysaccharide, the euscaphis konishii hayata polysaccharide can be applied to alpha-glucosidase inhibition.

Claims

1. An application of a euscaphis konishii hayata pericarp polysaccharide product is characterized in that a high-purity euscaphis konishii hayata pericarp polysaccharide extract obtained by water extraction, alcohol precipitation, centrifugation, polyamide column chromatography purification and reduced pressure concentration is used for preparing a medicine for inhibiting the activity of alpha-glucosidase; the method comprises the following steps and parameters:

(1) Taking euscaphis konishii hayata pericarp, drying, crushing and sieving by a 20-mesh sieve for later use;

(2) Aqueous solution extraction: taking Euscaphis japonica pericarp powder 1000 g, adding water solution 7500-10000 mL, heating and boiling for 2 hours, and filtering to obtain extract; adding 7500-10000 mL water solution into the residue, boiling for 2 hr, and filtering to obtain extractive solution; mixing the two water extracting solutions, and concentrating under reduced pressure to 300-500mL to obtain a concentrated extracting solution;

(3) Alcohol precipitation: adding anhydrous methanol or ethanol into the concentrated extractive solution to make the alcohol concentration of the solution 85-90%, standing for 2-3 hr, centrifuging at 1600-2000 rpm for 15-20 min, discarding supernatant to obtain precipitate as high-concentration fructus Euscaphis Japonica pericarp polysaccharide;

(4) And (3) polyamide column chromatography purification: mixing high-concentration euscaphis konishii hayata pericarp polysaccharide with polyamide of equal mass; the elution solvent is water; eluting for 5-6 column volumes, and collecting water eluate;

(5) Concentrating the water eluent: concentrating the water eluate under reduced pressure to dry to obtain Euscaphis japonica pericarp polysaccharide.