CN114409818B

CN114409818B - Preparation method and application of physalis pubescens polysaccharide selenide

Info

Publication number: CN114409818B
Application number: CN202210078278.5A
Authority: CN
Inventors: 高品一; 周可; 刘学贵; 李丹琦
Original assignee: Shenyang University of Chemical Technology
Current assignee: Shenyang University of Chemical Technology
Priority date: 2022-01-24
Filing date: 2022-01-24
Publication date: 2022-11-11
Anticipated expiration: 2042-01-24
Also published as: CN114409818A

Abstract

A preparation method and application of physalis pubescens selenka selenolysaccharide relate to a preparation method and application of a natural medicine. The invention adopts the methods of petroleum ether degreasing, ethanol removing micromolecules and deionized water extracting crude polysaccharide to separate and purify crude polysaccharide from natural medicine physalis pubescens fruits to obtain three new polysaccharides: PPFP-1, PPFP-2 and PPFP-3, and three new modified polysaccharides are obtained by selenization: se-PPFP-1, se-PPFP-2 and Se-PPFP-3. The prepared selenized polysaccharide has good antioxidant activity and nerve cell protection activity, is a novel natural selenized modified polysaccharide, and has an application prospect of preventing and treating neurodegenerative diseases. The new polysaccharide has remarkable effect of eliminating DPPH and ABTS free radicals, and can be used for treating H ₂ O ₂ The induced SH-SY5Y cell injury experiment shows obvious neuroprotective effect.

Description

Preparation method and application of physalis pubescens polysaccharide selenide

Technical Field

The invention relates to a preparation method and application of a natural medicine, in particular to a preparation method and application of physalis pubescens polysaccharide selenide.

Background

Physalis pubescens Pubescens L. is a annual or perennial herbaceous plant of Physalis L. of Solanaceae (Solanaceae), is native to south America, is planted in the three provinces of northeast China, inner Mongolia and other places of China, and is also called as calyx seu fructus Physalis, physalis pubescens and the like. The wild cherry fruit is a spherical berry, is yellow when ripe, can be directly eaten after being ripe, and has sour and sweet taste and bright color; the fruit with calyx is used as the downy groundcherry fruit medicine, has sour and mild nature and taste, enters lung channels, and has the effects of clearing away heat and toxic materials, promoting urination and stopping bleeding. The history of the cultivation technique of the hair acid pulp in China is long and recorded long ago. Polysaccharides are widely distributed in various organisms in the nature, are natural high-molecular polymers formed by connecting monosaccharides through alpha or beta glycosidic bonds, are one of basic substances forming life activities, have large molecular weight and complex structure, and become one of the hotspots of the current life science research because of having important pharmacological action, biological activity and wide application fields.

Modern pharmacological research shows that the physalis pubescens has obvious pharmacological activities of resisting tumor, bacteria, oxidation, diuresis, immunity and the like. At present, more than 120 chemical components are separated from physalis pubescens, and mainly comprise steroid physalins, luteolin, flavonoids, sterols, alkaloids, volatile oils, abundant inorganic elements, polysaccharides and the like. Among many active ingredients, the polysaccharide is one of four basic substances forming life, is one of the most important active substances, has various pharmacological actions, has very good treatment and protection effects on diseases such as diabetes, tumor, immunity and the like of a human body, and can improve the immunity of the organism by increasing the volume of macrophages; has cytotoxic effect on tumor cells to exert direct anti-tumor effect; directly acts on islet beta cells, promotes insulin secretion, increases insulin sensitivity and enhances anti-oxidative stress to play a role in reducing blood sugar.

Researches show that the high-level spatial structure, monosaccharide composition, molecular weight and specific groups of the polysaccharide closely influence the biological activity and physicochemical properties. The invention performs antioxidant activity investigation on the polysaccharide of the separated and purified physalis pubescens and the selenized polysaccharide of the physalis pubescens, and the result shows that the polysaccharide has a certain scavenging effect on two free radicals, and the scavenging rate is continuously improved along with the increase of the concentration of a sample. The protection condition of the polysaccharide of the physalis pubescens on H2O 2-induced SH-SY5Y cell damage is detected by an MTT method, and experimental results show that the polysaccharide has certain neuroprotective activity and certain dose dependence.

Disclosure of Invention

The invention aims to provide a preparation method and application of physalis pubescens polysaccharide selenide, and discloses three new natural polysaccharides obtained by separating and extracting physalis pubescens fruits, and three selenized polysaccharides obtained by selenizing and modifying. The polysaccharide and the selenized polysaccharide prepared by the invention have antioxidant activity and nerve cell protection activity, and lay a foundation for clinical research on prevention or treatment of neurodegenerative diseases in the future.

The invention is realized by the following technical scheme:

a preparation method of physalis pubescens selenka polysaccharide comprises Se-PPFP-1, se-PPFP-2 and Se-PPFP-3, and comprises the following preparation steps:

a. firstly, preparing natural polysaccharide by the following steps:

(1) Cleaning fresh Physalis alkekengi fruit, oven drying, grinding into powder, extracting with petroleum ether for defatting, reflux extracting with ethanol for removing small molecules, adding deionized water, heating and refluxing for 2-6 times, filtering, and mixing filtrates;

(2) Using n-butyl alcohol: adding chloroform = (0.5-2): (3-5), v/v reagent into supernatant, stirring for 20-40 min, centrifuging for 5-20 min to remove protein; repeating the process until the intermediate layer no longer produces protein precipitates; subsequently, the supernatant was collected;

(3) Decolorizing with AB-8, D101, D301, D307 macroporous resin to obtain crude polysaccharide;

(4) The crude polysaccharide is subjected to separation, purification, dialysis and freeze drying to obtain three single-component natural polysaccharides with relatively large amount and high purity: PPFP-1, PPFP-2 and PPFP-3;

b. the preparation of the selenized polysaccharide is realized by the following steps:

(1) PPFP-1, PPFP-2 and PPFP-3 are weighed and respectively added into HNO3 (0.4-0.8%, v/v) solution for reaction;

(2) Adding Na2SeO3 and BaCl2, and stirring at 50-90 ℃ for 8-12 h;

(3) After the reaction is stopped, adjusting the pH value to 5-8 by using 0.5-2.5 mol/L NaOH solution, and then adding 0.5-2.5 mol/L sodium sulfate to remove Ba < 2+ >;

centrifuging the supernatant, dialyzing with distilled water, concentrating, and lyophilizing to obtain Se-PPFP-1, se-PPFP-2, and Se-PPFP-3.

According to the preparation method of the physalis pubescens selenylation polysaccharide, the molecular weight range of the natural polysaccharide PPFP-1 is 29-36 KDa, the monosaccharide composition and the molar ratio range are as follows: arabinose: fructose: mannose: glucose = (0.20-0.45): (0.05-0.18): (0.01-0.03): (0.01-0.05): (0.35-0.65), the skeleton structure is as follows:

→2)-α-L-Rhap-(1→，→2,3)-α-L-Rhap-(1→，→3)-α-L-Araf-(1→，→3)-α-L-Araf-，T-β-L-Rhap-(1，→3)-β-D-Glcp-(1→，T-β-D-Glcp-(1→，→6)-β-D-Glcp-(1→，→4)-β-D-Glcp-(1→。

according to the preparation method of the physalis pubescens selenylation polysaccharide, the molecular weight range of the natural polysaccharide PPFP-2 is 10-14 KDa, the monosaccharide composition and the molar ratio range are as follows: arabinose: fructose: mannose: glucose = (0.10-0.18): (0.20-0.45): (0.12-0.24): (0.02-0.10): (0.24-0.34), the skeleton structure is as follows:

→2,3)-α-L-Rhap-(1→，→3)-α-L-Araf-(1→，α-L-Fucp-(2→，α-L-Fucp-(2→，→2)-β-L-Rhap-(1→，T-β-L-Rhap-(1→，→3)-β-D-Glcp-(1→，T-β-D-Glcp-(1→，→6)-β-D-Glcp-(1→，→4)-β-D-Glcp-(1→。

according to the preparation method of the physalis pubescens selenylation polysaccharide, the molecular weight range of the natural polysaccharide 3 PPFP-3 is 220-240 KDa, the monosaccharide composition and the molar ratio range are as follows: arabinose: fructose: mannose: glucose = (0.23-0.33): (0.19-0.27): (0.12-0.20): (0.01-0.07): (0.25-0.34), the skeleton structure is as follows:

→2)-α-L-Araf-(1→，α-L-Fucp-(2→，→3)-β-L-Rhap-(1→，α-L-Araf-(1→，T-β-L-Rhap-(1→，→3)-β-D-Glcp-(1→，→6)-β-D-Glcp-(1→，→4)-β-D-Glcp-(1→。

according to the preparation method of the polysaccharide selenide of physalis pubescens, the polysaccharide selenide Se-PPFP-1, se-PPFP-2 and Se-PPFP-3 are modified through selenylation, and the selenylation site is an O-H group at the C-6 position.

The application of the polysaccharide selenide of physalis pubescens has potential antioxidant activity and nerve cell protection activity, and is applied to potential pharmaceutical preparations for preventing and treating neurodegenerative diseases.

Drawings

FIG. 1 is a 1H-NMR spectrum of PPFP-1;

FIG. 2 is a 13C-NMR spectrum of PPFP-1;

FIG. 3 is an HSQC map of PPFP-1;

FIG. 4 is an HMBC map of PPFP-1;

FIG. 5 is a 1H-NMR spectrum of PPFP-2;

FIG. 6 is a 13C-NMR spectrum of PPFP-2;

FIG. 7 is an HSQC map of PPFP-2;

FIG. 8 is an HMBC map of PPFP-2;

FIG. 9 is a 1H-NMR spectrum of PPFP-3;

FIG. 10 is a 13C-NMR spectrum of PPFP-3;

FIG. 11 is an HSQC map of PPFP-3;

FIG. 12 is a 1H-NMR spectrum of Se-PPFP-1;

FIG. 13 is a 13C-NMR spectrum of Se-PPFP-1;

FIG. 14 is a 1H-NMR spectrum of Se-PPFP-2;

FIG. 15 is a 13C-NMR spectrum of Se-PPFP-2;

FIG. 16 is a 1H-NMR spectrum of Se-PPFP-3;

FIG. 17 is a 13C-NMR spectrum of Se-PPFP-3;

FIG. 18 is a spectrum of the monosaccharide composition of physalis pubescens polysaccharide;

FIG. 19 is a graph of data on the ability of Physalis pubescens native polysaccharide and selenized polysaccharide (PPFP-1, PPFP-2, PPLP3, se-PPFP-1, se-PPFP-2, se-PPFP-3) to scavenge DPPH, ABTS free radicals;

FIG. 20 is a graph of H2O 2-induced SH-SY5Y cell neuroprotective activity of natural polysaccharide of Physalis pubescens and selenized polysaccharide (PPFP-1, PPFP-2, PPLP3, se-PPFP-1, se-PPFP-2, se-PPFP-3).

Detailed Description

The present invention will be described below with reference to specific examples, but the present invention is not limited thereto. The experimental methods described in the following examples are all conventional methods unless otherwise specified; the reagents and biological materials, both analytically or chromatographically pure, are commercially available without specific reference.

The preparation, separation and purification of the physalis pubescens polysaccharide:

cleaning fresh Physalis pubescens fruit, drying, grinding thoroughly, refluxing with petroleum ether and ethanol to remove lipid and small molecular compounds, refluxing with distilled water, condensing and extracting for 2-6 times, filtering, concentrating under reduced pressure, and mixing the extracts. Then adding Sevage reagent (1:3-1, 5, v/v) prepared from n-butanol and chloroform in a volume of 1/2-1/4 to the polysaccharide aqueous solution, stirring for a certain period of time, removing protein, centrifuging, collecting supernatant, and removing protein by Sevage reagent method for 2-5 times until no protein layer is precipitated. Concentrating the supernatant, decolorizing with AB-8 resin column, eluting with distilled water, and concentrating under reduced pressure. Separating the crude polysaccharide with DEAE-52 cellulose column, and eluting with distilled water and NaCl solution of different concentrations. The content thereof was measured by the phenol-sulfuric acid method. Purifying by Sephadex G-200 Sephadex, collecting three components with high sugar content, and concentrating and drying under reduced pressure to obtain three new natural polysaccharides: PPFP-1, PPFP-2 and PPFP-3.

Preparing selenized polysaccharide:

carrying out selenylation reaction on the obtained new natural polysaccharides PPFP-1, PPFP-2 and PPFP-3 with large yield, respectively weighing 40-70 mg sample polysaccharide, adding 4-10 mL of 0.3-0.9% HNO3 solution, and stirring at 50-90 ℃ for 6-14 h. Then adding 30-70 mg of Na2SeO3 and 80-120 mL of BaCl2 to react at 50-90 ℃ for 7-13 h, adjusting the pH to 4-11 by using 0.6-1.4 mol/L NaOH solution after the reaction is stopped, and adding 0.6-1.4 mol/L sodium sulfate to remove Ba2+. Dialyzing the supernatant with distilled water for 65-80 h, concentrating and freezing to obtain Se-PPFP-1, se-PPFP-2 and Se-PPFP-3 powder.

Analyzing the structure of the physalis pubescens polysaccharide:

PPFP-1, PPFP-2 and PPFP-3 samples 10-30 mg were weighed into round bottom flasks 5-15 mL of 10-30 mol/L trifluoroacetic acid was hydrolyzed under reflux in a 100-120 ℃ oil bath 2-6 h, cooled and then spun dry with anhydrous methanol, several times to remove residual trifluoroacetic acid, finally acetonitrile: water =82:18 dissolve the sample and pass through a 0.22 μm organic frit.

The three new physalis pubescens polysaccharides obtained by the method have the molecular weight range of 29-36 KDa in PPFP-1, 10-14 KDa in PPFP-2 and 220-240 KDa in PPFP-3. PPFP-1 is composed of rhamnose, arabinose, fructose, mannose and glucose, and the molar ratio is (0.20-0.45): (0.05-0.18): (0.01-0.03): (0.01-0.05): (0.35-0.65); the PPFP-2 and the PPFP-3 are composed of rhamnose, arabinose, fructose, mannose and glucose, and the molar ratio ranges are respectively as follows: (0.10-0.18): (0.2-0.45): (0.12-0.24): (0.02-0.10): (0.24-0.34) and (0.23-0.33): (0.19-0.27): (0.12-0.20): (0.01-0.07): (0.25-0.34). The molecular weight ranges of the selenizing polysaccharides Se-PPFP-1, se-PPFP-2 and Se-PPFP-3 are 23 KDa-28 KDa, 8-10 KDa and 50-53 KDa respectively, and the selenium content ranges from 423.9-431.9 mug/g, 294.5-302.5 mug/g and 306.0-312.0 mug/g respectively.

The removal effect of the three new physalis pubescens polysaccharide and the three selenium polysaccharide on DPPH and ABTS free radicals is determined through related experiments, the natural physalis pubescens polysaccharide and the selenium polysaccharide both have certain removal effect on the two free radicals and are dose-dependent at the concentration of 0.005 mg/mL-5 mg/mL, and the removal effect sequence of PPFP-1, PPFP-2 and PPFP-3 on DPPH free radicals is PPFP-1 >; wherein, at a high concentration of 5.0mg/mL, the clearance of the Se-PPFP-3 to DPPH free radicals is about 2.3 times or more of that of the PPFP-3. The effect of eliminating ABTS free radicals is in the range of 0.08-5.0 mg/mL, PPFP-1 >. In an experiment for detecting H2O 2-induced SH-SY5Y cell damage by means of an MTT method, polysaccharide compounds with different concentrations of 15.625, 31.25, 62.5 and 125 mu g/mL are used for pretreating cells, and the result shows that the physalis pubescens polysaccharide can remarkably recover cell activity and presents dose dependence, and the potential protection effect of Se-PPFP-2 is 73.781 +/-0.019 (%) at 50 mu g/mL and is superior to that of a positive control group.

The following are specific examples:

example 1

Extraction of polysaccharide from physalis pubescens

The physalis pubescens fruit is cleaned, dried, ground and degreased by 3 times of petroleum ether under the condition of 60 ℃ and refluxing for 4 h. Adding 3 times volume of 80% ethanol, refluxing for 3 h, filtering, washing the residue with ethanol, air drying, refluxing with distilled water, condensing, extracting for 4 times, filtering, concentrating under reduced pressure, and oven drying. Sevage reagent (1, 4,v/v) prepared from n-butanol and chloroform was added to the polysaccharide solution, the protein was removed with vigorous stirring, centrifuged, and the supernatant was collected and the protein was removed 3 times by the Sevage method until no protein layer precipitated. Concentrating the obtained supernatant, decolorizing with AB-8 resin column, dissolving and sampling with 5 g crude polysaccharide each time, eluting with deionized water as mobile phase, and concentrating to obtain crude polysaccharide of herba Oxalidis Corniculatae.

Separating crude polysaccharide with DEAE-52 cellulose column, setting the elution flow rate range to 0.8 mL/min, collecting time interval of each tube to 10 min, eluting with distilled water and NaCl solution of different concentrations, detecting polysaccharide solution in the test tube with phenol-sulfuric acid method, measuring its absorbance at 490 nm, collecting, combining eluates with symmetrical gradient peak shapes, concentrating and dialyzing. And eluting the obtained polysaccharide components by using a Sephadex G-200 column, further purifying and collecting three components with high sugar content, and decompressing, concentrating, dialyzing and drying to obtain polysaccharides PPFP-1, PPFP-2 and PPFP-3.

A nuclear magnetic spectrum (600 MHz, D2O) as shown, from 1H-NMR in combination with 13C-NMR and HSQC, the PPFP-1 spectrum indicates the presence of → 2) - α -L-Rhap- (1 → fragment at a displacement value of 5.27/97.7 ppm, the presence of → 2,3) - α -L-Rhap- (1 → fragment at 5.26/97.3 ppm, the presence of → 3) - α -L-ara- (1 → fragment at 5.17/108.3 ppm, the presence of → 3) - α -L-ara-fragment at 5.14/109.3 ppm, the presence of T- β -L-Rhap- (1 fragment at 4.88/97.6 ppm, the presence of → 3) - β -D-Glcp- (1 → fragment at 4.83/99.5 ppm, the presence of T- β -D-Glcp- (1 → fragment at 4.58/103.6 ppm, the presence of T- β -D-Glcp- (1 → fragment at 4.42/5 ppm, the presence of T- β -Glcp → fragment at 4.42 → 4.3 ppm; PPFP-2 indicates the presence of → 2,3) - α -L-Rhap- (1 → fragment at a spectral chemical shift value of 5.27/97.5 ppm, → 3) - α -L-Araf- (1 → fragment at 5.17/108.0 ppm, α -L-Fucp- (2 → fragment at 5.16/98.3 ppm, α -L-Fucp- (2 → fragment) at 5.14/109.2 ppm, the presence → 2) - β -L-Rhap- (1 → fragment at 5.02/98.9 ppm, the presence T- β -L-Rhap- (1 → fragment at 4.92/99.6 ppm, the presence → 3) - β -D-Glcp- (1 → fragment at 4.88/97.7 ppm, the presence T- β -D-Glcp- (1 → fragment at 4.52/102.7 ppm, the presence → 6) - β -D-Glcp- (1 → fragment at 4.41/102.8 ppm, the presence → 4) - β -D-Glcp- (1 → fragment at 4.35/103.3 ppm, the presence → 4) - β -D-Glcp- (1 → fragment at 5.68/106.8 ppm, the presence → 2) - α -L-Araf- (1 → fragment at 5.15/98.5 ppm, the presence → 2-L-Glcp- (2 → fragment at 4.99/99.99 ppm, the presence → fragment at 4.42-1 → fragment at 4.42 ppm, the presence → fragment at 5.15/4.15/98.99 → 4.99 ppm, the presence → fragment at 4.1 → fragment at 4.42 ppm, the presence → fragment at 3.42-1 → fragment at 4.42 ppm, the presence → fragment at 4.9 ppm, the presence → 4.42-1 → fragment at 3.42-7.42-1 → fragment.

Example 2

Synthesis of physalis pubescens selenium polysaccharide

Adding 10 ml of 0.6% HNO3 into 70 mg sodium selenite to fully dissolve the sodium selenite, adding a 50 mg sample to continuously dissolve the sodium selenite, reacting the sodium selenite with 10 h under 70 oC, cooling to room temperature after the solution is completed, adjusting the pH of the solution to 7 by using a NaOH solution, adding 1 mol/L sodium sulfate to remove Ba2+, dialyzing by using distilled water for 72 h to dialyze small molecular compounds such as sodium selenite and the like, stopping dialysis and freeze-drying to obtain selenized polysaccharides Se-PPFP-1, se-PPFP-2 and Se-PPFP-3. From 13C-NMR, it was found that: a new signal peak at δ 62.4 ppm, indicating C-6 was replaced by a selenium atom.

Example 3

Analysis of monosaccharide composition of Physalis pubescens polysaccharide

Taking a proper amount of PPFP-1, PPFP-2 and PPFP-3 samples, dissolving the samples by trifluoroacetic acid with a certain concentration, carrying out oil bath reaction on the samples for 6 h under the condition of 120 oC, cooling the samples to room temperature after hydrolysis, carrying out reduced pressure spin drying, and carrying out a small amount of residual trifluoroacetic acid for many times until no acid smell exists. Then with 600 μ L acetonitrile: water =82:18 dissolve the sample and pass through a 0.22 μm organic frit.

Chromatographic conditions are as follows: LC 3000 high performance liquid chromatograph;

and (3) chromatographic column: sepax HP-Amino (4.6X 250 mm);

mobile phase: acetonitrile: water =82:18;

column temperature: 20-40 oC;

flow rate: 0.4 mL/min;

sample injection amount: 60. mu.L;

the chromatogram is shown in FIG. 18, PPFP-1 is composed of rhamnose, arabinose, fructose, mannose and glucose, and the molar ratio is 0.39:0.12:0.02:0.03:0.44; the PPFP-2 and the PPFP-3 consist of rhamnose, arabinose, fructose, mannose and glucose, and the molar ratio of monosaccharides is respectively 0.13:0.34:0.18:0.06:0.29 and 0.28:0.23:0.16:0.04:0.29.

example 4

Determination of molecular weight of polysaccharide of physalis pubescens and polysaccharide selenide

Weighing a certain amount of glucan standard with molecular weight of T4 (4000 Da), T10 (10000 Da), T20 (20000 Da), T40 (40000 Da), T200 (200000 Da) and T500 (500000 Da) and PPFP-1, PPFP-2, PPFP-3, se-PPFP-1, se-PPFP-2 and Se-PPFP-3. Adding 1 mL Wahaha purified water, dissolving, mixing well to obtain standard solution, and filtering with 0.22 μm filter membrane.

Chromatographic conditions are as follows: LC-20AR high performance liquid chromatograph;

a detector: RID-20A shows a differential refractive detector;

a chromatographic column: sugar column (i.d. =4.6 mm, L =250 mm);

mobile phase: 0.01-0.04 mol/L KH2PO4;

column temperature: 20-40 oC;

flow rate: 0.6 mL/min;

sample injection amount: 60. mu L;

obtaining a standard curve according to the molecular weight logarithm and the peak-off time of the standard glucan, wherein the molecular weights of PPFP-1, PPFP-2 and PPFP-3 are respectively 33 KDa,12 KDa and 229 KDa according to the peak-off time of the polysaccharide; the molecular weights of Se-PPFP-1, se-PPFP-2 and Se-PPFP-3 are 26 KDa, 9 KDa and 51 KDa respectively.

Example 5

Determination of selenium content in selenized polysaccharide

Weighing a certain amount of sample in a triangular flask, adding 6-12 mL nitric acid and 2-6 mL perchloric acid, placing the mixture above 12-20 h in the dark at normal temperature, heating and digesting the mixture on a heating sleeve at 160-220 oC, adding 6-12 mL hydrochloric acid (0.5-1.0 mol/mL) when the solution is colorless or yellowish, continuously heating the mixture until the solution is about 1 mL, cooling the mixture, and simultaneously performing a blank test. Diluting concentrated hydrochloric acid to 4-8 mol/L with distilled water, adding the digested solution into a colorimetric tube, adding 2-8 mol/L hydrochloric acid, placing in a 70-100 oC constant temperature tank, heating for 1-3 h, taking out, cooling, diluting with distilled water, and detecting with an atomic fluorescence spectrometer. The results show that the selenium content of Se-PPFP-1, se-PPFP-2 and Se-PPFP-3 is 431.9. Mu.g/g, 299.6. Mu.g/g and 309.0. Mu.g/g, respectively.

Example 6

DPPH radical scavenging ability

Preparing the samples into a series of aqueous solutions with different concentrations, taking 100 mu L of samples with different concentrations, adding 100 mu L of 0.2 mmol/L DPPH stock solution (prepared from absolute ethyl alcohol and DPPH powder), reacting for 30 min in a dark place, measuring the absorbance value under 517 nm, and using Vc as a positive control. The formula for the scavenging ability is R% = [ A0- (A1-A2) ]/A0 × 100%. A0 is 100. Mu.L of 0.2 mmol/L DPPH stock solution, 100. Mu.L of pure water, as a blank group; a1 is 100 μ L of ethanol and samples of different concentrations of 100 μ L, which is a control group; a2 is a 100. Mu.L DPPH stock with 100. Mu.L samples at different concentrations, for the sample set. The capacity and the concentration of the physalis pubescens polysaccharide for removing DPPH free radicals are dose-dependent, when the concentration reaches 5 mg/mL, the effect of removing DPPH free radicals is PPFP-1>se-PPFP-1, PPFP-2>, se-PPFP-2, se-PPFP-3>, PPFP-3, wherein the removal rate of the Se-PPFP-3 to the DPPH free radicals is about 2.3 times or more than that of the PPFP-3 when the concentration is 5.0 mg/mL.

Example 7

ABTS free radical scavenging ability

Preparing a series of aqueous solutions with different concentrations from the sample, taking 10 mu L of the sample with different concentrations, adding 190 mu L of ABTS stock solution, reacting for 10 min in a dark place, placing the mixture into an enzyme-linked immunosorbent assay, and detecting the absorbance (A1) at the wavelength of 734 nm. Vc was used as a positive control. Blank (A0) was 100. Mu.L of ABTS and 100. Mu.L of purified water. 100. mu.L of different concentrations of sample and 100. Mu.L of ABTS, as a sample background group (A2). The formula for the scavenging ability is R% = [ A0- (A1-A2) ]/A0 × 100%.

As can be seen in FIG. 19, the ability of the physalis pubescens polysaccharide to scavenge ABTS free radicals is dose-dependent on concentration. PPFP-1 >.

Example 8

H2O 2-induced SH-SY5Y cell neuroprotective activity of physalis pubescens polysaccharide

After conventional culture of SH-SY5Y cells for passaging, the cells were seeded on a 96-well plate and cultured statically in an incubator with 5% CO2 at 37 oC for 24 h until the cells were attached to the wall. Samples with the concentrations of 25, 50 and 100 mu mol/L are respectively added into a 96-well plate, placed in an incubator to be cultured for 12H, then 100 mu mol/L H2O2 is added for reaction, and MTT detects the activity of cells. No cells were inoculated into 96 wells as a blank control, and no H2O2 or drug treatment was used, and the other experimental steps were identical to those of the administered group.

Survival (%) = [ a490 (administered group) -a490 (blank) ]/[ a490 (negative control) -a490 (blank) ] × 100%

In an experiment for detecting H2O2 induced SH-SY5Y cell damage by physalis pubescens polysaccharide through an MTT method, results show that compared with a blank group, cells are pretreated by polysaccharide compounds with different concentrations, cell activity is remarkably recovered in a dose-dependent mode, and potential protection effect of Se-PPFP-2 (73.781 +/-0.019 (%)) is more obvious than that of a positive control at 50 mu mol/L.

Claims

1. A preparation method of physalis pubescens selenka polysaccharide is characterized in that the physalis pubescens selenka polysaccharide comprises Se-PPFP-1, se-PPFP-2 and Se-PPFP-3, and the preparation method comprises the following steps:

a. firstly, preparing natural polysaccharide by the following steps:

(4) The crude polysaccharide is subjected to separation, purification, dialysis and freeze drying to obtain three kinds of single-component natural polysaccharides with relatively large amount and high purity: PPFP-1, PPFP-2 and PPFP-3;

(1) PPFP-1, PPFP-2 and PPFP-3 are weighed and respectively added into 0.4-0.8% (v/v) HNO ₃ Reacting in a solution;

(2) Then adding Na ₂ SeO ₃ And BaCl ₂ Stirring the mixture at 50-90 ℃ for 8-12 h;

(3) After the reaction is stopped, the pH is adjusted to 5 to 8 by 0.5 to 2.5 mol/L NaOH solution, and then 0.5 to 2.5 mol/L sodium sulfate is added to remove Ba ²⁺ ；

Centrifuging the supernatant, dialyzing with distilled water, concentrating, and lyophilizing to obtain Se-PPFP-1, se-PPFP-2, and Se-PPFP-3;

the molecular weight range of the natural polysaccharide PPFP-1 is 29-36 KDa, the composition and the molar ratio range of monosaccharides are as follows: arabinose: fructose: mannose: glucose = (0.20-0.45): (0.05-0.18): (0.01-0.03): (0.01-0.05): (0.35-0.65), the skeleton structure is as follows:

→2)-α-L-Rhap-(1→，→2,3)-α-L-Rhap-(1→，→3)-α-L-Araf-(1→，→3)-α-L-Araf-，T-β-L-Rhap-(1，→3)-β-D-Glcp-(1→，T-β-D-Glcp-(1→，→6)-β-D-Glcp-(1→，→4)-β-D-Glcp-(1→；

the molecular weight range of the natural polysaccharide PPFP-2 is 10-14 KDa, the monosaccharide composition and the molar ratio range are as follows: arabinose: fructose: mannose: glucose = (0.10-0.18): (0.20-0.45): (0.12-0.24): (0.02-0.10): (0.24-0.34), the skeleton structure is as follows:

→2,3)-α-L-Rhap-(1→，→3)-α-L-Araf-(1→，α-L-Fucp-(2→，α-L-Fucp-(2→，→2)-β-L-Rhap-(1→，T-β-L-Rhap-(1→，→3)-β-D-Glcp-(1→，T-β-D-Glcp-(1→，→6)-β-D-Glcp-(1→，→4)-β-D-Glcp-(1→；

the molecular weight range of the natural polysaccharide 3 PPFP-3 is 220-240 KDa, the monosaccharide composition and the molar ratio range are as follows: arabinose: fructose: mannose: glucose = (0.23-0.33): (0.19-0.27): (0.12-0.20): (0.01-0.07): (0.25-0.34), the skeleton structure is as follows:

2. the preparation method of the selenized polysaccharide of physalis pubescens as claimed in claim 1, wherein the selenized polysaccharides Se-PPFP-1, se-PPFP-2 and Se-PPFP-3 are modified by selenization, and the selenization site is an O-H group at C-6 position.

3. The application of the polysaccharide selenide of physalis pubescens prepared by the method of claim 1, wherein the polysaccharide selenide has potential antioxidant activity and nerve cell protection activity, and is applied to the preparation of potential pharmaceutical preparations for preventing and treating neurodegenerative diseases.