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

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

A new method for the preparation of polysaccharides from the pericarp of Corvus japonica and its product application

technical field

The invention relates to the technical field of extraction technology of plant polysaccharides, in particular to an extraction method and application of polysaccharides from the pericarp of Jacuna japonica.

Background technique

In recent years, more and more researchers have paid attention to plant polysaccharides because of their good biological activities such as anti-tumor, hypoglycemic, and anti-oxidation, and have developed various medicines and health care products such as ganoderma polysaccharide, lentinan, and ginseng polysaccharide. .

Euscaphis japonica (Thunb.) Dippel is a plant of the genus Euscaphis japonica (Thunb.) Dippel, which is a unique medicinal ornamental tree species in China. In recent years, the planting area in Fujian, Jiangxi and other provinces has continued to expand. Only Fujian Sanming Qingliu, Taining, Nanping Shaowu, Jianyang, Quanzhou Dehua and other places have more than 100 hectares of planting area, rich in plant resources. The research group has been committed to the resource development and utilization of Javenica japonica. It was found in the early stage that the water extract of its pericarp has a high polysaccharide content, and has the effect of inhibiting α -glucosidase, showing hypoglycemic activity. In order to develop the polysaccharides of Corvus japonicus, the research team used the traditional polysaccharide extraction method in the early stage, that is, water extraction-ethanol precipitation-centrifugation-concentration to obtain a crude polysaccharide extract, which is weaker in inhibiting α -glucosidase activity than the positive control drug Aka wave sugar. In order to further improve the biological activity of the polysaccharide extract of S. avenica, it is necessary to remove the ineffective components in the polysaccharide extract as much as possible.

Extraction of plant polysaccharides by water extraction and alcohol precipitation often has a large amount of water-soluble tannins. Polyamide is a kind of high molecular compound formed by the polymerization of amide groups. It contains abundant amide groups. It can form hydrogen bonds with phenols, quinones, nitro compounds, etc. and be adsorbed, and it can be separated from compounds that cannot form hydrogen bonds. . Therefore, our research group used polyamide as the chromatography column filler, so that the water-soluble tannins in the polysaccharides of S. javenica were adsorbed on the polyamide chromatographic column, and the polysaccharides were eluted by water, so that the tannins of S. Crude polysaccharide extraction was further purified. The activity of inhibiting α -glucosidase of the polysaccharide purified by this method is equivalent to that of the positive control drug, and can be further developed and utilized.

Contents of the invention

The present invention aims to provide a new method for preparing polysaccharides from the pericarp of Javenica ravensia. The preparation process adopts water extraction-alcohol precipitation-centrifugation-polyamide column chromatography purification-concentration under reduced pressure to obtain a high-purity polysaccharide extract from the pericarp of Javenica ravensia; The preparation method is simple and easy to operate, and is suitable for large-scale production, and the prepared polysaccharide from the pericarp of Jacuna japonica has significant activity of inhibiting α-glucosidase.

The technical method provided by the present invention is as follows:

Get the jacaranda pericarp, dry it and crush it into 20 meshes for subsequent use;

Aqueous solution extraction: take 1,000 g of jacarina pericarp powder, add 7,500-10,000 mL of aqueous solution, heat and boil for 2 hours, and filter to obtain the extract; add 7,500-10,000 mL of aqueous solution to the filter residue, boil for 2 hours again, and filter to obtain the extract; Combine the two water extracts, concentrate under reduced pressure to 300-500mL to obtain a concentrated extract;

Alcohol precipitation: Add anhydrous methanol or ethanol to the concentrated extract to make the concentration of methanol or ethanol in the solution 85-90%, let stand for 2-3 hours, separate in a centrifuge at 1600-2000 rpm for 15-20 min, discard Remove the supernatant, and the resulting precipitate is high-concentration Arvenia pericarp polysaccharide; considering the toxicity of methanol, ethanol precipitation is preferred;

Polyamide column chromatographic purification: mix high-concentration polysaccharides of jacarina pericarp and equal mass of polyamide; the elution solvent is water; 5-6 column volumes are eluted, and the water eluate is collected.

Concentration of the water eluate: the water eluate is concentrated to dryness under reduced pressure to obtain the polysaccharide from the pericarp of Javenica japonica.

Inhibition of α-glucosidase by polysaccharides from the pericarp of Arvenia japonica in vitro

0.5 U/mL α -glucosidase, dissolved in pH 6.8 phosphate-buffered saline solution; pipette 40 μL, mix with 20 μL of the polysaccharide extract from the pericarp of Arvenia japonica with different concentration gradients, incubate at 37 ℃ for 10 min, then add 5 mmol/ Add 40 μL of L substrate PNPG, mix well, react at 37 °C for 30 min, then add 100 μL of 0.2 mol/L Na ₂ CO ₃ to terminate the reaction. The above experiments were repeated 3 times. The absorbance of the reaction product was measured at 405 nm. An equal volume of solvent PBS was used as a blank control, and acarbose was used as a positive control.

The inhibition rate of the sample to α-glucosidase %=(1 -Ao/As) ×100,

In the formula: Ao is the absorbance of replacing the sample solution with the same amount of PBS; As is the absorbance of adding the sample. The inhibition rate of α -glucosidase by the polysaccharide extract from the pericarp of Corvus avenae at different concentrations is shown in the table below.

Inhibition rate of α-glucosidase inhibited by polysaccharides from the pericarp of Arvenna japonica in vitro

Detailed ways

The present invention can be better understood from the following examples. However, those skilled in the art can easily understand that the specific material ratios, process conditions and results described in the examples are only used to illustrate the present invention, and should not and will not limit the present invention described in the claims.

Implementation Example 1

Dried jacuba pericarp at 50-60°C, crushed through 20 meshes, and set aside; weigh 1000 g of jacuba pericarp powder, add 10000 mL of aqueous solution, heat and boil for 2 hours, and filter to obtain the extract; add 10000 mL of aqueous solution to the filter residue, Boil again for 2 hours, filter to obtain the extract; combine the two water extracts, concentrate under reduced pressure to 400mL; add absolute ethanol to the concentrated extract to make the ethanol concentration of the solution 86%, and let it stand for 2-3 hours , separated in a centrifuge at 2000 rpm for 20 min, discarded the supernatant, and obtained 236 g of a precipitate; dissolved the precipitate in water, mixed the sample with 236 g of 60-100 mesh polyamide, and eluted it by polyamide column chromatography; Remove the solvent with water; elute with 20,000 mL, collect the water eluate, concentrate under reduced pressure to dryness to obtain 185 g of polysaccharides from the pericarp of Javenica javenica, with a yield of 18.5% and a polysaccharide content of 88.3%.

Example 2

Dry the pericarp of Javenica japonica at 50-60°C, crush it through 20 meshes, and set aside; weigh 1000 g of the pericarp of Javenica japonica, add 1000 mL of aqueous solution, heat and boil for 2 hours, and filter to obtain the extract; add 7500 mL of aqueous solution to the filter residue, Boil again for 2 hours, filter to obtain the extract; combine the two water extracts, concentrate under reduced pressure to 400mL; add anhydrous methanol to the concentrated extract to make the methanol concentration of the solution 86%, and let it stand for 2-3 hours , separated in a centrifuge at 2000 rpm for 20 min, discarded the supernatant to obtain a precipitate; dissolved the precipitate in water, mixed with 236 g of 60-100 mesh polyamide, and eluted by polyamide column chromatography; the elution solvent into water; elute with 20,000 mL, collect the water eluate, concentrate under reduced pressure to dryness to obtain 185 g of polysaccharides from the pericarp of Javenica javenica, with a yield of 18.5% and a polysaccharide content of 88.3%.

Example 3

Dried jacuba pericarp at 50-60°C, crushed through 20 meshes, and set aside; weigh 1000 g of jacuba pericarp powder, add 7500 mL of aqueous solution, heat and boil for 2 hours, and filter to obtain the extract; add 7500 mL of aqueous solution to the filter residue, Boil again for 2 hours, filter to obtain the extract; combine the two water extracts, concentrate under reduced pressure to 400mL; add absolute ethanol to the concentrated extract to make the ethanol concentration of the solution 86%, and let it stand for 2-3 hours , separated in a centrifuge at 2000 rpm for 20 min, discarded the supernatant to obtain a precipitate; dissolved the precipitate in water, mixed with 236 g of 60-100 mesh polyamide, and eluted by polyamide column chromatography; the elution solvent into water; elute with 20,000 mL, collect the water eluate, concentrate under reduced pressure to dryness to obtain 185 g of polysaccharides from the pericarp of Javenica javenica, with a yield of 18.5% and a polysaccharide content of 88.3%.

Example 4

According to the experiment of inhibiting α-glucosidase in vitro by polysaccharides from the pericarp of Javenica javenica, it can be applied to inhibit α-glucosidase.

Claims (1)

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.