CN115838441A

CN115838441A - Preparation method of nitraria tangutorum bobr polysaccharide BDP-I (B)

Info

Publication number: CN115838441A
Application number: CN202211434924.3A
Authority: CN
Inventors: 韩丽娟; 邓永蓉; 孙胜男; 岳庆明
Original assignee: Qinghai University
Current assignee: Qinghai University
Priority date: 2022-11-16
Filing date: 2022-11-16
Publication date: 2023-03-24

Abstract

The invention researches the biological activity of BDP-I (B), provides a new revelation for the research of the active basic substance of yellow thorn and also provides a potential product with good efficacy. Therefore, the invention provides a method for separating and preparing polysaccharide compound BDP-I (B) from yellow thorns.

Description

Preparation method of xanthium polysaccharide BDP-I (B)

Technical Field

The invention relates to the field of natural plants and natural medicines.

Background

Yellow thorn, the scientific name berberis (Berberis dasystachyam.), is one of the medicinal and edible berry resources with the characteristics of Qinghai-Tibet plateau and is also a wild berry resource with great development prospect ^[1] . Qinghai province is an important distribution area of Berberis plants, and has the advantages of various types, wide distribution range and abundant resource quantity ^[2] The yellow thorn, the sea buckthorn and the Tanggute white thorn are broadly called three thorns " ^[3] . The yellow thorn fruit is natural and nontoxic, and has a plurality of active ingredients, including phytosterol, organic acid, polysaccharide, polyphenol and the like. So far, fruit juice, fruit powder and health food are processed in the market ^[4-7] . Researches show that the yellow thorn has good function of reducing blood sugar, and the yellow thorn fruit powder is natural and safe, has wider application range, and is more suitable to be used as food, health care products or medicines for treating and preventing diabetes.

For years, most of the researches on yellow thorns at home and abroadFocusing on the alkaloid component berberine, berberine is considered to be the main active component ^[8] However, the study finds that the polysaccharide of the yellow thorn is also an active substance with important medical value ^[9] . After Han Lijuan ^[10] The optimal extraction conditions for extracting the crude polysaccharide of the berberis aristata by a dynamic microwave-assisted method are obtained by utilizing response surface optimization by the people, and the in vitro antioxidant activity of the polysaccharide of the berberis aristata is measured to show that the polysaccharide of the berberis aristata can be used as a natural antioxidant. Meng Zhaojun ^[11] The research of the people finds that the polysaccharide of the yellow thorn fruit has good function of reducing blood sugar and can increase the insulin quantity. However, the active ingredients in the berries of yellow thorn are limited to the preliminary study of the extraction and biological activity of the crude polysaccharides.

Therefore, the separation and purification, structural analysis and structure-activity relationship between structure and function of the polysaccharide from Xanthopanax senticosus have to be further studied, and the antioxidant and hypoglycemic activities and mechanisms of the polysaccharide must be further elucidated from the molecular biological level.

Disclosure of Invention

The invention separates and identifies a brand-new polysaccharide compound BDP-I (B) from yellow thorns by a specific separation and purification means. Meanwhile, the invention also researches the biological activity of BDP-I (B), provides a new inspiration for the research of the active basic substance of the yellow thorn and also provides a potential product with good efficacy.

Based on the discovery, the invention provides a preparation method of nitraria tangutorum bobr polysaccharide BDP-I with good biological activity,

the polysaccharide has peak molecular weight of 38.5-40.0 kDa, weight average molecular weight of 46.5-48.5kDa, and number average molecular weight of 32.0-33.0 kDa;

the method comprises the following operation steps:

(1) Subjecting the aqueous solution of crude polysaccharide of Xanthorrhoea to weak base anion exchange cellulose column chromatography, eluting with water and 0.2M NaCl aqueous solution sequentially, collecting 0.2M NaCl aqueous solution eluate, and dialyzing in 3500Da dialysis bag;

(2) And (3) subjecting the dialyzed polysaccharide to sephadex column chromatography, eluting with water, purifying by a polysaccharide gel purification system in combination with a differential detector, collecting the eluate corresponding to the second-order absorption peak, and drying to obtain BDP-I (B).

Wherein, the weak base anion exchange cellulose column chromatography can be selected from diethylaminoethyl cellulose-52 column.

Wherein said sephadex is selected from sephadex G-200.

The crude polysaccharide of the yellow thorn can be prepared by the prior art, can also be purchased from products sold in markets, and can also be prepared by the following method:

degreasing fructus Rosae Davuricae with petroleum ether, extracting with 85% ethanol, extracting the precipitate with hot water, concentrating the water extract, removing protein, decolorizing, dialyzing with 5000Da dialysis bag, adding ethanol into the dialysate until the alcohol content reaches 75-85%, precipitating with ethanol, and collecting the precipitate to obtain crude polysaccharide of fructus Rosae Davuricae.

The polysaccharide has an infrared spectrum of at least 3397cm ^-1 、2927cm ^-1 、1740cm ^-1 、1612cm ^-1 、1421cm ^-1 、1105cm ^-1 、1020cm ^-1 Has characteristic peaks.

The research of the invention finds that the monosaccharide composing the polysaccharide has a molar ratio of arabinose, galactose, glucose and galacturonic acid of 10.8:9.9:8.9:70.4, wherein the contents of the arabinose, the galactose, the glucose and the galacturonic acid in percentage by mol are 10.8%, 9.9%, 8.9% and 70.4% in sequence.

The inventor researches and discovers that the polysaccharide compound can reduce the damage of oxidation to islet cells, particularly islet beta cells.

The results show that: h ₂ O ₂ Can cause oxidative damage to RIN-m5F cells, and the degree of damage increases with time and concentration of action. Compared with a control group, the injection solution is 250 mu mol/L H ₂ O ₂ Decreased cell viability after treatment (. About.P)<0.05 Increased ROS levels: ( ^# P<0.05 ); and H ₂ O ₂ Model group comparison, channel0.0625-0.5 mg/mL BDP and 0.0625-0.5 mg/mL xanthane polysaccharide BDP-I increase in cell survival rate after stem prediction ([ P ])<0.05 ROS, MDA levels are in a downward trend. In conclusion, the polysaccharide BDP-I of the nitraria tangutorum can improve the survival rate of cells within a certain dosage range, and can improve the survival rate of cells against H ₂ O ₂ The induced oxidative damage has a protective effect, and is beneficial to protecting islet cells from oxidative stress damage, so that diabetes or diabetic complications and the like caused by the oxidative stress damage are prevented or treated.

Diabetes is a complex metabolic disorder disease, seriously harms the health of people, and has no medicine for completely curing the diabetes at present. A plurality of natural plant polysaccharides are proved to have the efficacy of reducing blood sugar ^[12] The research on the polysaccharide hypoglycemic molecular mechanism mainly aims at promoting insulin secretion, inhibiting islet cell apoptosis, reducing insulin resistance, improving antioxidant stress capacity, regulating related signal path and regulating intestinal flora ^[13,14] And the like. Related research shows that glucotoxicity and lipotoxicity can also promote the occurrence of pancreatic beta-cell oxidative stress, increase the apoptosis of beta-cells and insulin resistance ^[15] This therefore also contributes to oxidative stress being one of the risk factors for the development of diabetes. Oxidative stress refers to an unbalanced state in which excessive oxygen radicals (active oxygen) coexist with antioxidant substances in the body. Because normal pancreatic islets are organs with weak oxidation resistance, the normal pancreatic islets are easy to become targets of oxidative attack ¹⁶ . Hydrogen peroxide (H) ₂ O ₂ ) Is an important active oxygen substance, is easy to generate homolytic cleavage to form an active oxygen free radical with extremely strong activity, namely hydroxyl free radical (. OH), is convenient and easy to obtain, has stable property, and becomes an important tool for researching the oxidative damage of cells ^[17] 。

Under the oxidative stress state of the organism, the activity of antioxidant enzymes in the body is gradually reduced, and ROS generated in the body is not cleared in time, so that the ROS are excessively accumulated. Excessive ROS can cause membrane lipid peroxidation, denaturation of intracellular enzymes, and DNA fragmentation, ultimately leading to apoptosis of islet beta cells. Zheng Yansong and the like ^[18] Uses low-concentration hydrogen peroxide (100 mu mol/L) to successfully establish a myocardial cell oxidative damage model,Ankur Maheshwari ^[19] by H ₂ O ₂ In vitro induction of testis sperm cell apoptosis model to study its action path. Warleta et al ^[20] Research shows that squalene can also prevent H to a certain extent ₂ O ₂ Resulting in damage to human breast epithelial cells. Insulinomas, the most common type of endocrine tumors of the pancreas, produce or release large amounts of insulin without depending on changes in blood glucose, can be used as an in vitro model of islet beta cells and widely used clinically in the study of the mechanism of treating diabetes ^[21] 。

The polysaccharide BDP-I (B) is separated and prepared from the yellow thorn for the first time and H with different concentrations ₂ O ₂ Inducing RIN-m5F cells, establishing an oxidative damage model, intervening the model cells by using BDP-I (B) with different concentrations, detecting the cell survival rate, and exploring H pair of BDP-I (B) by using the active oxygen (ROS) level and the Malondialdehyde (MDA) content as indexes ₂ O ₂ The protection effect of inducing the oxidative damage of the islet beta cells has important significance for researching the activity of the polysaccharide of the yellow spine, and provides more sufficient basis for the antioxidant function of the polysaccharide of the yellow spine. In order to determine the material basis and action mechanism of the stichopus japonicus polysaccharide for intervening the islet beta cell apoptosis, the invention carries out structural characterization on the separated and purified polysaccharide and researches the stichopus japonicus polysaccharide on H ₂ O ₂ High-sugar and high-fat induced RIN-m5F islet beta cell apoptosis intervention effect, discussing protection H of polysaccharide of yellow spine by molecular biological level ₂ O ₂ And the action mechanism of RIN-m5F islet beta cells with high sugar and high fat damage is expected to provide theoretical basis for the development and application of the yellow thorn berries and the research and development of yellow thorn polysaccharide health-care food.

The present invention provides a process for the preparation of the above polysaccharide compounds, which process is effective in obtaining the polysaccharide product, and which process also provides insights and possibilities for the production of the polysaccharide product based on its good biological activity.

Drawings

FIG. 1 chromatography elution curve of polysaccharide DEAE anion column

FIG. 2 Sephadex G-200 column chromatography elution curve of polysaccharide from yellow thorn

FIG. 3 HPGPC elution Profile of Nitraria polysaccharide

FIG. 4 HPLC profiles of the purified polysaccharide BDP-I (B) and monosaccharide standards (B) from Xanthoceras sibirica, note: fucose Fuc, galactosamine hydrochloride GalN, rhamnose Rha, arabinose Ara, glucosamine GlcN hydrochloride, galactose Gal, glucose Glc, N-acetyl-D glucosamine GlcNAc, xylose Xyl, mannose Man, fructose Fru, ribose Rib, galacturonic acid GalA, guluronic acid GulA, glucuronic acid GlcA, manA

FIG. 5 Infrared Spectrum of the purified Nitraria polysaccharide BDP-I (B)

FIG. 6 GC-MS Total ion flow diagrams of PMAAs of Nitraria polysaccharide BDP-I (B)

FIG. 7 BDP-I (B) ¹ H-NMR (a) and ¹³ C-NMR spectrum (b)

FIG. 8 Dept135 map

FIG. 9 HSQC (a), HMBC (b), HH-COSY (c) and NOESY (d) maps

FIG. 10H ₂ O ₂ Effect on the survival of RIN-m5F cells, note: (A) H ₂ O ₂ Effect of induction for 3h on cell survival; (B) H ₂ O ₂ Effect of Induction for 6H on cell viability (C) H ₂ O ₂ Induction of 12h effect on cell survival; (D) H ₂ O ₂ Induction of 24h effect on cell survival; * P<0.05 showed significance compared to the control group

FIG. 11H ₂ O ₂ Effect on RIN-m5F cells ROS, note: * P<0.05 indicated significance, compared to the control group;

FIG. 12 effect of different concentrations of BDP-I (B) on cell viability, note: * P <0.05 indicates significance, compared to control

FIG. 13 protective Effect of BDP-I (B) on H2O 2-induced RIN-m5F cells, note: in comparison with the model (H2O 2 group), ^* P<0.05 indicates significance; compared with the cell control group, ^# P<0.05 means significance

FIG. 14 BDP-I (B) vs. H ₂ O ₂ Morphological effects of induced RIN-m5F cells, a-NC: a normal control group; b-PC: positive control group (300. Mu. Mol/L. Alpha. -LA); c-MC: model set (25)0μmol/L H ₂ O ₂ ) (ii) a d-BDP-I (B) low dose group (0.0625 mg/mL); dose group in e-BDP-I (B) (0.125 mg/mL); high dose of f-BDP-I (B) (0.25 mg/mL)

FIG. 15 Effect of BDP-I (B) on ROS levels in RIN-m5F cells, note: NC: a normal control group; PC: positive control group (300. Mu. Mol/L. Alpha. -LA); MC: model group (250. Mu. Mol/L H) ₂ O ₂ )

FIG. 16 Effect of BDP-I (B) on SOD enzyme activity in RIN-m5F cells, note: NC: a normal control group; PC: positive control group (300. Mu. Mol/L. Alpha. -LA); MC: model group (250. Mu. Mol/L H) ₂ O ₂ )

FIG. 17 Effect of BDP-I (B) on CAT enzyme Activity in RIN-m5F cells, note: NC: a normal control group; PC: positive control group (300. Mu. Mol/L. Alpha. -LA); MC: model group (250. Mu. Mol/L H) ₂ O ₂ )

FIG. 18 Effect of BDP-I (B) on MDA content in RIN-m5F cells, note: NC: a normal control group; PC: positive control group (300. Mu. Mol/L. Alpha. -LA); MC: model group (250. Mu. Mol/L H) ₂ O ₂ )

Detailed Description

Example 1 separation, purification and structural characterization of Berberis davidiana Berberis polysaccharide BDP-I (B)

1. Experimental methods

1.1 Process for extracting polysaccharide from Nitraria tangutorum bobr

Dried fruit of yellow thorn → crushing and sieving → degreasing with petroleum ether → 85% ethanol for removing monosaccharide → ultrasonic hot water extraction (repeating for 3 times) → concentration to 1/4 → sevage method for removing protein → D101 macroporous resin for decoloring → 95% ethanol for precipitation → freeze drying → crude polysaccharide BDP of yellow thorn

(1) Degreasing, namely mixing the smashed yellow thorn powder with petroleum ether (60-90 ℃) at 1:5, heating in a water bath at 40 ℃, stirring for 2.5h, and filtering to remove filtrate.

(2) And (3) monosaccharide removal, namely adding ethanol into the degreased raw material until the alcohol content is 80%, heating and stirring the degreased raw material in a water bath at 50 ℃ for 2 hours, and filtering the mixture to remove the filtrate.

(3) Ultrasonic hot water extraction, ultrasonic extracting (70 ℃) of degreased and desugared yellow thorn powder and distilled water for 30min according to the material-liquid ratio of 1.

(4) Removing protein by adding chloroform-n-butanol (4:1) reagent with a volume ratio of 1/5 of polysaccharide solution volume, shaking for 15min, centrifuging (4500 r/min,10 min), collecting upper layer polysaccharide solution, discarding lower organic solvent layer and middle protein emulsion layer, and repeating the above steps until no protein emulsion is produced in the middle layer. And (3) carrying out reduced pressure evaporation on the polysaccharide solution subjected to protein removal to remove the residual sevage solvent.

(5) Decolorizing by adding 1/10D 101 macroporous resin into polysaccharide solution, shaking at 37 deg.C for 48 hr, filtering to remove resin, and repeating for three times.

(6) Evaporating the decolorized polysaccharide solution under reduced pressure to appropriate volume, and dialyzing the polysaccharide solution in a dialysis bag with cut-off of 5000Da for 48h.

(6) Precipitating with ethanol, adding 4 times volume of 80% ethanol into dialyzed polysaccharide solution, stirring, standing at 4 deg.C for 24h, and centrifuging at 4500r/min for 15min to obtain polysaccharide precipitate.

(7) And (3) freeze drying, namely pre-freezing the polysaccharide obtained by centrifugation and drying the polysaccharide in a freeze dryer to obtain crude polysaccharide powder.

1.2 polysaccharide yield calculation:

polysaccharide yield (%) = mass of freeze-dried crude polysaccharide of yellow thorn/mass of yellow thorn fruit powder x 100

1.3 separation and purification of crude polysaccharide of Nitraria tangutorum bobr

(1) DEAE-52 anion exchange chromatography

In order to obtain homogeneous polysaccharide with similar molecular weight and same polarity, the crude polysaccharide obtained by water extraction and alcohol precipitation is separated and purified.

The method comprises the following steps: 1g of crude polysaccharide was dissolved in distilled water, heated, vortexed, centrifuged at 12000rpm, and the supernatant was sampled. Eluting with deionized water 3 times, sequentially eluting with 0.2M NaCl, 0.5M NaCl and 1.0M NaCl at flow rate of 15mL/min, and collecting 10mL eluate per tube. Tracking and detecting the sugar content by adopting a phenol-sulfuric acid method, measuring the absorbance value of the separation tube eluent at 490nm by using an enzyme labeling instrument, and drawing an elution curve.

Respectively combining and collecting the same components according to peak shapes, concentrating at 45 deg.C under reduced pressure, dialyzing the concentrated solution in 3500Da dialysis bag for 48h, freeze drying, and sealing for storage. The obtained components are respectively named as: BDP-W, BDP-I, BDP-II, BDP-III. (see FIG. 1)

(2) Sephadex G-200 gel column chromatography

The sephadex G-200 is used for separating substances with the molecular weight of 5000-600000, and the separated polysaccharide BDP-I is further purified and subjected to purity identification by sephadex G-200 column chromatography. Weighing 100mg of aqueous phase polysaccharide after polar separation, dissolving with 3mL of distilled water, centrifuging at 12000rpm for 10min, taking supernatant, eluting with water, purifying by a polysaccharide gel purification system and collecting by on-line detection combined with a differential detector (RI-502 SHODEX), collecting symmetrical peaks and drawing an elution curve (see figure 2). Evaporating and concentrating the combined components at 45 deg.C under reduced pressure, lyophilizing to obtain polysaccharide separated and purified by gel column, and performing next molecular weight determination.

The polysaccharide gel purification System of the invention is a full-automatic gel purification System (GPC Autopurifier System, BRT-GS), and Borui sugar biotechnology, inc. of Yangzhou, yangzhou.

1.4 molecular weight determination of polysaccharide from Nitraria tangutorum

And performing purity identification and molecular weight determination by high performance liquid gel permeation chromatography.

Drawing a glucan standard curve: the standards were precision weighed to make a 5mg/ml solution, centrifuged at 12000rpm for 10min, the supernatant was filtered through a 0.22 μm microporous membrane, and the sample was transferred to a 1.8ml injection vial. The regression equation was fitted with retention time (x, min) of each standard as the horizontal axis and the logarithm of dextran standard molecular weight (y, log MW) as the vertical axis.

Determination of polysaccharide molecular weight: precisely weighing the sample to prepare a 5mg/ml solution, centrifuging at 12000rpm for 10min, filtering the supernatant with a 0.22 μm microporous filter membrane, transferring the sample into a 1.8ml sample injection vial, and performing high performance liquid chromatography. And recording the peak appearance time of each peak in the hovenia dulcis polysaccharide liquid chromatogram, and calculating the molecular weight distribution of the hovenia dulcis polysaccharide by contrasting a glucan standard curve.

Chromatographic conditions are as follows: LC-10A high performance liquid chromatograph, BRT105-104-102 series gel column (8X 300 mm), difference detector RI-10A. Detection conditions are as follows: 0.05MNaCl solution is used as a mobile phase; the flow rate is 0.6ml/min, and the column temperature is 40 ℃; the amount of the sample was 20. Mu.l.

1.5 Xanthoceratis polysaccharide monosaccharide composition determination

Preparation and calculation method of standard solution: taking 16 monosaccharide standard products (fucose, rhamnose, arabinose, galactose, glucose, xylose, mannose, fructose, ribose, galacturonic acid, glucuronic acid, galactosamine hydrochloride, glucosamine hydrochloride, N-acetyl-D glucosamine, guluronic acid and mannuronic acid) to prepare about 10mg/ml standard solution.

Precisely preparing the monosaccharide Standard solutions into gradient concentration Standard products of 0.1, 0.5, 1,5, 10, 20 and 50mg/L as Standard1-7. According to the absolute quantitative method, the mass of different monosaccharides is determined, and the molar ratio is calculated according to the molar mass of the monosaccharides.

Sample preparation

About 5mg of each sample was placed in an ampoule, and 2M TFA (10 ml) was added thereto, followed by hydrolysis at 120 ℃ for 3 hours. Accurately absorbing the acid hydrolysis solution, transferring the acid hydrolysis solution into a tube, blowing and drying the acid hydrolysis solution by nitrogen, adding 5ml of water, uniformly mixing the acid hydrolysis solution and the tube by vortex, absorbing 100uL of the acid hydrolysis solution, adding 900uL of deionized water, and centrifuging the mixture at 12000rpm for 5min. The supernatant was taken for HPIC analysis.

Chromatographic process

A chromatographic column: dionex carbopac TMPA20 (3 × 150); mobile phase: a is H2O; b, 250mM NaOHC;50mM NaOH and 500mM NaOAC; flow rate: 0.3ml/min; sample introduction amount: 5 mu L of the solution; column temperature: 30 ℃; a detector: an electrochemical detector.

1.6 Fourier Infrared Spectroscopy

Precisely weighing 2mg of the sample and 200mg of potassium bromide, and pressing the sample into tablets, wherein the blank control is formed by pressing potassium bromide powder into tablets. The samples were respectively subjected to scanning and recording by a Fourier transform infrared spectrometer FT-IR650 (Tianjin Hongkong science and technology development Co., ltd.).

1.7 uronic acid reduction

And (3) carrying out a reduction experiment on the sample by adopting an uronic acid reduction instrument, namely weighing 80mg of polysaccharide sample into a beaker, adding distilled water for dissolving, then adding an aldehydic acid activator, setting the pH value of the reduction instrument to be 4.6, and reacting for 3 hours. The pH was then set to 6.8 and the reaction was continued for 2h. The sample was concentrated and dialyzed against a 1000Da dialysis bag. Repeating the above steps for 3-5 times, and lyophilizing. The obtained sample is subjected to monosaccharide composition verification and subsequent methylation experiments are carried out.

1.8 polysaccharide ligation assay

The connection mode of polysaccharide samples is measured by GC-MS after methylation and other derivatization.

After methylation, hydrolysis and acetylation, the sample is determined by GC-MS and compared with a standard mass spectrum library.

Weighing a polysaccharide sample (2-3 mg), placing the polysaccharide sample in a glass reaction bottle, adding 1mL of anhydrous DMSO, quickly adding a methylation reagent solution A, sealing, dissolving under the action of ultrasound, and then adding a methylation reagent solution B. Reacting for 60min in a magnetic stirring water bath at 30 ℃. Finally, 2mL of ultrapure water was added to the above mixture to terminate the methylation reaction.

The methylated polysaccharide was hydrolyzed with 1ml of 2M trifluoroacetic acid (TFA) for 90min and evaporated to dryness in a rotary evaporator. Adding 2ml of double distilled water into residues, reducing 60mg of sodium borohydride for 8 hours, adding glacial acetic acid for neutralization, performing rotary evaporation, drying in a 101-DEG oven, adding 1ml of acetic anhydride for acetylation at 100 ℃, reacting for 1 hour, and cooling. Then 3mL of toluene was added, concentrated to dryness under reduced pressure, and repeated 4-5 times to remove excess acetic anhydride.

The acetylated product was treated with 3mL CH ₂ Cl ₂ After dissolution, the mixture was transferred to a separatory funnel, and after adding a small amount of distilled water and shaking sufficiently, the upper aqueous solution was removed, and this was repeated 4 times. CH (CH) ₂ Cl ₂ The layer was dried over an appropriate amount of anhydrous sodium sulfate, and the volume was adjusted to 10mL, and the mixture was placed in a liquid phase vial. The sample of the acetylated product is determined by a Shimadzu GCMS-QP 2010 gas chromatography-mass spectrometer;

GC-MS conditions: RXI-5SIL MS chromatographic column 30m 0.25mm 0.25um; the temperature programming conditions are as follows: the initial temperature is 120 ℃, and the temperature is increased to 250 ℃/min at the speed of 3 ℃/min; keeping for 5min; the temperature of the sample inlet is 250 ℃, the temperature of the detector is 250 ℃/min, the carrier gas is helium, and the flow rate is 1mL/min.

1.9 nuclear magnetic resonance analysis

A polysaccharide sample of 50mg was weighed, dissolved in 0.5ml of heavy water and freeze-dried. And then dissolving the freeze-dried powder in 0.5ml of heavy water again, continuously freezing and drying, and repeating the processes to fully exchange active hydrogen. The sample was then dissolved in 0.5ml of heavy water and the 1H NMR spectrum, the 13C NMR spectrum, the DEPT135 one-dimensional spectrum and the two-dimensional spectrum were measured at room temperature at 25 ℃ on a nuclear magnetic resonance apparatus at 600 MHz.

2. Analysis of results

2.1 DEAE anion column chromatography for purifying polysaccharide from Xanthoceras

Redissolving crude polysaccharide BDP of radix seu folium Spiraeae Fortunei, purifying by DEAE anion column chromatography, eluting with distilled water, 0.2M NaCl, 0.5M NaCl, and 1.0M NaCl, tracking the sugar content of each gradient eluate by phenol-sulfuric acid method, and drawing elution curve to obtain four absorption peaks (shown in figure 1) as water elution component (BDPs-w), 0.2M NaCl elution component (BDP-1), 0.5M NaCl elution component (BDP-2), and 1M NaCl elution component (BDP-3). When the NaCl concentration is more than 0.5mol/L, the peak shape of elution is small, so that only distilled water, 0.2M NaCl eluate is collected. Mixing eluates obtained from absorption peaks, concentrating, dialyzing, and freeze drying to obtain distilled water eluate containing polysaccharide BDP-W and 0.2M NaCl eluate containing polysaccharide BDP-I, with yield of 13.6% and 20.45%. And selecting the BDP-I component with the highest yield for further purification due to the requirement of subsequent cell experiments.

2.2 Sephadex G-200 gel column chromatography for purifying polysaccharide of yellow spine

Further purifying the polysaccharide BDP-I from the yellow spine by Sephadex G-200, drawing an elution curve (as shown in figure 2), and obtaining three absorption peaks which are relatively uniformly and symmetrically distributed and are respectively named as BDP-I (A), BDP-I (B) and BDP-I (C). Collecting BDP-I (B), and concentrating, dialyzing, and vacuum freeze-drying to obtain polysaccharide sample for subsequent structure identification and activity analysis.

2.3 determination of molecular weight

Performing purity identification and molecular weight determination on the polysaccharide BDP-I (B) separated and purified in two steps by using high performance liquid gel permeation chromatography (HPGPC). Using dextran with molecular weight of 5000, 11600, 23800, 48600, 80900, 148000, 273000, 409800, 670000 and 3693000Da as standard, and using elution time as abscissa, obtaining lgMp-RT (peak molecular weight), lgMw-RT (weight average molecular weight) and lgMn-RT (number average molecular weight) calibration curves, and fitting the calibration curves to a methodThe strokes are respectively y = -0.195x +12.375, R2=0.9913; y = -0.2078x +12.968 ² ＝0.993；y＝-0.181x+11.734，R ² =0.9972. The elution curve obtained by detection is shown in figure 3, which is a single absorption peak with good peak type symmetry, and shows that the purity of the purified BDP-I (B) is high. The peak molecular weight of BDP-I (B) was 39.469kDa, the weight average molecular weight was 47.714kDa, and the number average molecular weight was 32.638kDa, calculated from dextran standard curves.

2.4 monosaccharide composition determination

Measuring monosaccharide composition of polysaccharide BDP-I (B) by ion chromatography. As shown in fig. 4, the monosaccharide composition in the purified polysaccharide consisted of arabinose (Ara), galactose (Gal), glucose (Glc), and galacturonic acid (GalA) compared to the HPLC profile of the monosaccharide standard, and the molar percentages of the monosaccharides were 10.8%, 9.9%, 8.9%, and 70.4%, respectively, calculated from the molar concentrations and peak areas of the monosaccharide standard and the internal standard, with arabinose (Ara) and galacturonic acid (GalA) as the main monosaccharide components.

2.5 Infrared Spectroscopy

The infrared spectrum of the polysaccharide BDP-I (B) is shown in FIG. 5, and the absorption band is 3600-3200cm ^-1 Is a stretching vibration absorption peak of-OH, and an absorption peak in this region is a characteristic peak of the glucide. The method comprises the following specific steps: 3397cm ^-1 Is the absorption peak of stretching vibration of O-H and is the characteristic peak of saccharide. At 2927cm ^-1 Has an absorption peak which is attributed to C-H stretching vibration. At 1740cm ^-1 The absorption peak at (B) is attributed to C = O stretching vibration, indicating that the BDP-i (B) structure contains an acetyl group. At 1612cm ^-1 The absorption peak at (A) may be a characteristic peak of crystal water. At 1421cm ^-1 There is an absorption peak attributed to C-O stretching vibration. At 1105cm ^-1 Variable angle vibration, 1020cm, attributed to O-H ^-1 The absorption peak at (a) is due to stretching vibrations of the pyranose ring.

2.6 methylation analysis

Methylation analysis is one of the important means for exploring the primary structure of natural polysaccharides. The information of the connection point of each monosaccharide residue in the polysaccharide molecule can be obtained through methylation analysis, and meanwhile, the proportion information of monosaccharide components with different connection modes in the polysaccharide molecule can also be obtained. Because the polysaccharide BDP-I (B) contains uronic acid, the uronic acid is reduced before methylation, and monosaccharide composition verification of the obtained sample shows that the uronic acid is successfully reduced.

After BDP-I is methylated, hydrolyzed and acetylated, partially methylated sugar alcohol acetyl ester derivatives (PMAAs) are obtained, a GC-MS total ion flow diagram (shown in figure 6) of PMAAs of the BDP-I (B) of the xanthum polysaccharide is obtained through GC-MS measurement, the GC-MS total ion flow diagram is compared with a standard mass spectrum diagram library to determine the types of methylated sugar residues, finally the types and the mole percentages of glycosidic bonds of polysaccharide components are obtained, and specific statistical results are shown in table 1. As shown in Table 1, BDP-I (B) has complicated glycosidic linkage, and comprises 11 different glycosidic linkages, wherein arabinose (Araf) exists in five linkages of terminal sugar (1-Araf), 1,5-Araf, 1,3,5-Araf, 1,2,5-Araf and 1,3-Arap; galactose (Gal) exists in five connection forms of terminal sugar (1-Galp), 1,4-Galp, 1,3-Galp, 1,6-Galp, 1,3,6-Galp; glucose (Glc) exists as a1,4,6-Glcp linkage. Wherein the proportion of galactose is the largest, and the proportion of 1,4-Galp and 1,3,6-Galp is respectively 18.03 percent and 35.00 percent.

TABLE 1 results of methylation analysis

2.7 nuclear magnetic resonance analysis

The structure of BDP-I (B) was further analyzed by 1D NMR (C/H spectrum) and 2D NMR (HSQC/HMBC). Of BDP-I (B) as shown in FIG. 7 ¹ H-NMR and ¹³ the C-NMR spectrum showed from FIG. 7 (a) that the BDP-I (B) anomeric hydrogen signals were mainly concentrated between 3.0 and 6.0 ppm. Delta 3.2-4.0ppm is sugar ring proton signal, and the signal peaks of main terminal proton peaks delta 5.17, 5.14, 5.11, 5.01, 4.94, 4.71, 4.65, 4.55, 4.47, 4.4 and 4.37 are distributed in the region of 4.3-6.0 ppm. Chemical shifts of the anomeric hydrogens were both less than 5.0ppm and greater than 5.0ppm, indicating the presence of both alpha and beta configuration glycosidic linkages in BDP-I (B).

The BDP-I (B) anomeric carbon signals are mainly concentrated between 100-110ppm as shown by FIG. 7 (B), and 10 distinct anomeric carbon signal peaks are respectively delta 110.62, 108.89, 108.88, 107.8, 105.39, 104.9, 104.69, 104.48, 101.49 and 100.38. As shown in fig. 8 by Dept135 profile analysis, it was found that δ 62.64, 68.27, 67.79, 68.25, 70.76, 70.5, 61.6 had inverted peaks, indicating signal peaks for C6 or C5.

Binding by HSQC mapping (FIG. 9 a) ¹ H-NMR analysis shows that the anomeric carbon has anomeric hydrogen signals corresponding to the anomeric carbon in the spectrum, and the chemical shifts are respectively 5.17, 5.01, 5.11, 5.14, 4.47, 4.37, 4.55, 4.4, 4.65 and 4.94ppm, specifically delta 110.62/5.17, 108.88/5.01, 108.89/5.11, 107.8/5.14, 104.69/4.47, 104.9/4.37, 105.39/4.55, 104.48/4.4, 101.49/4.65 and 100.38/4.94. In conjunction with methylation analysis results, they were assigned to glycosidic bond → 5) - α -L-Araf- (1 →, → 5) - α 0-L-Araf- (1 →, → 3,5) - α -L-Araf- (1 →, → 2,5) - α -L-Araf- (1 →, → 3,6) - β -D-Galp- (1 →, → 6) - β -D-Galp- (1 →, → 3) - β -D-Galp- (1 →, → 4) - β -D-Manp- (1 →, → 4) - α -D-Galp- (1 →, respectively corresponding to A, B, C, D, E, F, G, H, I in qc.

FIGS. 9B, c, d are HMBC, HH-COSY and NOESY profiles of BDP-I (B), combined ¹ H-NMR and HSQC spectra were further analyzed, and the hydrocarbons of each cross peak were assigned. Taking the cross peak A as an example, the anomeric carbon signal is delta 108.8, the corresponding anomeric hydrogen signal in HSQC spectrum is delta 5.01, and the signal of H1/H2 is 5.00/4.06ppm by HH-COSY analysis; the H2/H3 signal was 4.06/3.93ppm; signal 3.93/4.14ppm for H3/H4; the signal for H4/H5a was 4.14/3.80ppm; it can be concluded that H1, H2, H3, H4, H5a are δ 5.00, 4.06, 3.93, 4.14, 3.80, respectively, and that the corresponding C1-C5 are 108.87, 82.17, 78.11, 83.67, 68.26. The remaining sugar residues were resolved in the same manner to obtain assignment results of chemical shifts of carbon and hydrogen of the monosaccharide residue in BDP-I (B), as shown in Table 2. The results showed that BDP-i (B) contained → 5) - δ 2-L-Araf- (1 →, → 5) - α -L-Araf- (1 →, → 3,5) - α -L-Araf- (1 →, → 2,5) - α -L-Araf- (1 →, → 3,6) - δ 0-D-Galp- (1 →, → 6) - δ 1-D-Galp- (1 →, → 3) - β -D-Galp- (1 →, → 4) - β -D-Manp- (1 →, → 4) - α -D-GalAp- (1 → these 10 monosaccharide residues.

TABLE 2 of BDP-I (B) monosaccharide residues ¹ H-NMR and ¹³ C-NMR chemical shift assignment

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In summary, due to the low content of other sugars. Polysaccharides composed primarily of galactose and arabinose, we can conclude that the polysaccharide has the predominant glycosidic linkage structure of → 6) - β -D-Galp- (1 → glycosidic linkage, and the branched segments are linked to the backbone by 3,6) - β -D-Galp- (1 → O-3 linkage as follows:

4. small knot

(1) The polysaccharide of the yellow thorn is separated, purified and analyzed in a primary structure by adopting various analysis means. Four kinds of preliminary purified polysaccharides of BDPs-w, BDP-I, BDP-II and BDP-III are obtained by DEAE-52 separation, and the BDP-I with higher yield is selected to be purified by Sephadex G-200 to obtain uniform polysaccharide.

(2) The purity identification and molecular weight determination of the two-step separation and purification of the polysaccharide BDP-I (B) of the yellow spine are carried out by HPGPC, and the weight average molecular weight of the BDP-I (B) is 47.714kDa. The monosaccharide composition of BDP-I (B) consists of arabinose (Ara), galactose (Gal), glucose (Glc) and galacturonic acid (GalA), and the mole percentages of the monosaccharides are respectively 10.8%, 9.9%, 8.9% and 70.4%. The infrared spectrum shows that BDP-I (B) has a characteristic polysaccharide absorption peak (3397 cm) ^-1 )，1020cm ^-1 The peak at (A) indicates the presence of pyranose in BDP-I (B).

(3) Combined with methylation and nmr analysis, the results showed that BDP-i (B) contains → 5) - α -L-Araf- (1 →, → 3,5) - α -L-Araf- (1 →, → 2,5) - α -L-Araf- (1 →, → 3,6) - β -D-Galp- (1 →, → 6) - β -D-Galp- (1 →, → 3) - β -D-Galp- (1 →, → 4) - β -D-Galp- (1 →, → 4) - α -D-GalAp- (1 → 10 monosaccharide residues.

The homogeneous polysaccharide BDP-I (B) of the yellow spine is preliminarily analyzed by a plurality of analysis methods and means, and a theoretical basis is provided for the subsequent research of the antioxidant mechanism of BDP-I (B) on oxidative damage islet cells.

Example 2 Nitraria berry polysaccharides BDP-I (B) vs. H ₂ O ₂ Protection of injured RIN-m5F cells

1. Materials and apparatus

1.1 Experimental materials

Yellow thorn berries, which are collected from ripe fruits of Xining City, qinghai province; RIN-m5F cells (rat islet beta-cell tumor cells) were purchased from Sainbur Biotechnology GmbH.

1.2 Experimental reagents

TABLE 3 Main reagents List

1.3 Experimental instruments

Table 4 Instrument and Equipment List

2. Experimental methods

2.1 cell culture and related techniques

2.1.1 preparation of related Agents

RPMI-1640 complete medium: under aseptic conditions, 10% FBS and 1% double antibody were added to RPMI-1640 medium, and the mixture was sealed and stored at 4 ℃ until use.

H ₂ O ₂ Mother liquor (1 mM): 1 μ L H ₂ O ₂ Make up to 10mL with medium. The mother liquor was diluted to various concentrations at the time of the experiment.

BDP-I (B) mother liquor (2 mg/mL): 20mg of BDP-I (B) solid powder is accurately weighed and dissolved in 10mL of culture medium, and the mixture is evenly mixed by vortex oscillation.

Alpha-lipoic acid (1 mmol/L): weighing 2.06mg of lipoic acid, adding 10ml of PRM 1640 cell culture solution to obtain lipoic acid mother liquor with final concentration of 1mmol/L, diluting the mother liquor to 300 mu M as positive control when in use, and performing sterile filtration for later use.

2.1.2 cell culture

RIN-m5F cells were cultured in PRMI1640 medium containing 10% fetal bovine serum, 1% penicillin and streptomycin mixture at a relative humidity of 95%,37 ℃ and 5% CO ₂ Culturing in a constant humidity incubator, and changing the culture solution every other day.

2.1.3 cell passages

The cell density reaches 80-90%, and subculture can be carried out.

(1) The culture supernatant was discarded, and the cells were rinsed 1-2 times with PBS containing no calcium or magnesium ions.

(2) 1mL of trypsinized solution (0.25% Trypsin-0.53Mm EDTA) was added to the flask, the flask was incubated at 37 ℃ for 2 minutes, and then the digestion of the cells was observed under a microscope, and if the cells were mostly rounded and dropped, the procedure was quickly returned, and a small amount of complete medium was added to stop the digestion. (to avoid cell clumping, do not hit or shake the flask while waiting for the cells to separate.)

(3) Add 4mL complete medium and gently blow and mix well. The cell suspension was inoculated at a suitable ratio of 1:2-1:3 for subculture, then supplemented with fresh complete medium to 5mL, and placed in a cell culture chamber at 37 ℃ and 5% CO2 saturation humidity for static culture.

(4) Culturing and observing after the cells are completely attached to the wall; fresh complete medium was then replaced every 2-3 days. Cells in logarithmic growth phase after passage 4 were used for the experiment.

2.1.4 cell cryopreservation

When the cell growth state is good, the cells can be frozen.

1) After discarding the medium, the PBS was washed once and 1mL of pancreatin was added, after the cells became round and detached, 1mL of serum-containing medium was added to stop digestion, and the cells were counted using a cell counting plate.

2) Centrifuging at 1500rpm for 5min to remove supernatant, adding frozen stock solution, and gently and uniformly blowing. The final concentration of DMSO is 10%, and the cell density is not less than 1 × 10 ⁶ and/mL, freezing and storing 1mL of cell suspension in each freezing and storing tube, and paying attention to the marked date, cell name and name of the freezing and storing tube.

3) And (4) program freezing: placing the freezing tube in a refrigerator at 4 deg.C for 30min, placing in a refrigerator at-20 deg.C for 1h, placing in a refrigerator at-80 deg.C overnight, and placing in a liquid nitrogen tank the next day.

2.1.5 cell Resuscitation

The tube containing 1mL of cell suspension was thawed by rapid shaking in a 37 ℃ water bath, transferred to a sterile centrifuge tube, and 5mL of medium was added and mixed well. The cell suspension was transferred to a culture flask for overnight culture. The next day the fluid was changed and cell density was checked.

2.2 establishing an oxidative stress model

2.2.1 H ₂ O ₂ Influence on cell survival

In order to investigate the safe concentration of H2O2 on RIN-m5F cells, the cell viability was determined by CCK-8 method, WST-8 in yellowish color was reduced to yellow formazan dye with high water solubility by dehydrogenase in the mitochondria of cells under the action of electron carrier (1-Methoxy PMS), and absorbance at 450nm indirectly reacted to the number of living cells.

Grouping cells: blank group, serum-free RPMI-1640 culture medium; control group, RPMI-1640 medium containing 10% fetal calf serum + cells; model group, RPMI-1640 medium containing 10% fetal calf serum + cells + H2O2 with different concentrations; positive control group (α -lipoic acid): 300 mu mol/L alpha-lipoic acid.

The method comprises the following specific operations: at 1X 10 ⁵ Density inoculation per mL cells were seeded in 96-well plates at 100 μ l per well and cultured for 24h until cells attached. Adding different concentrations of H according to cell groups ₂ O ₂ After induction (10, 50, 100, 150, 200, 250, 300, 400) of μmol/L for 3,6, 12, and 24 hours, 100 μ L of CCK-8 solution (CCK-8 reagent: serum-free culture =1 10) was added to each well, six wells were provided for each set, the wells were slowly loaded so as not to generate air bubbles, the tips of each column were changed, the plate was incubated in a constant temperature incubator for 0.5h, and the measurement was performed at 450nm after completion. Calculation of cell viability according to equation 1, determined to result in half-lethal cell (IC) ₅₀ ) H of (A) to (B) ₂ O ₂ Concentration of。

Wherein A1 is the absorbance of a well having cells, a CCK-8 solution, and a drug solution;

a2 is the absorbance of a well with medium and CCK-8 solution without cells;

a3 is the absorbance of a well with cells, CCK-8 solution, and no drug solution;

2.2.2 H ₂ O ₂ effect on cellular ROS levels

ROS level detection by DCFH-DA fluorescent probe method at 1X 10 ⁵ The cells per mL were inoculated into a 96-well plate at a density of 100. Mu.l per well and cultured under the conditions of 37 ℃ and 5% CO2 (v/v) for 24 hours, the cells of each treatment group were harvested, the medium was aspirated and discarded, after washing with PBS for 2 times, 100. Mu.L of a serum-free medium containing DCFH-DA (10. Mu. Mol/L1:1000 dilution) was added, incubated for 1 hour at 37 ℃ in the absence of light, and subjected to detection on a machine using a full-wavelength microplate reader, wherein the excitation wavelength was 488nm and the emission wavelength was 525nm.

2.2.3 Selection of BDP-I (B) concentration

Grouping cells: blank group: serum-free RPMI-1640 medium; control group: RPMI-1640 medium + cells containing 10% fetal bovine serum; BDP-I (B) group: RPMI-1640 medium containing 10% fetal bovine serum + cells + different concentrations of BDP-I (B).

Cells in logarithmic growth phase were grown at 1X 10 ⁵ The cells were inoculated into 96-well plates at a density of one/mL for 24h, treated with BDP-I (B) at different concentrations (0.0625, 0.125, 0.25, 0.50, 1,2 mg/mL), cultured for 24h, 48h, 72h, respectively, and the cell viability was measured according to the CCK-8 method of 3.3.2.1 to determine the optimal time and concentration.

2.3 BDP-I (B) to H ₂ O ₂ Protective effects of induced oxidative damage of RIN-m5F cells

Grouping cells: blank group: serum-free RPMI-1640 medium; control group: 10% FBS in RPMI-1640 medium + cells; model group: RPMI-1640 medium + cells + optimal concentration H2O2 culture optimum time containing 10% FBS; BDP-I (B) group: 10% FBS-containing RPMI-1640 medium + cells + optimal concentration H2O2 culture optimal time + different concentrations of BDP-I (B).

2.3.1 detection of cell viability

With H after successful modeling ₂ O ₂ Cells were induced at concentrations and times followed by various concentrations of BDP-I (B) intervention (0.0625, 0.125, 0.25, 0.50, 1,2 mg/mL) for 24h, and cell viability was determined according to the procedure of CCK-8 method at 2.2.1.

2.3.2 detection of cellular ROS levels

With H after successful modeling ₂ O ₂ Concentration and time after induction of cells, different concentrations of BDP-I (B) intervention (0.0625, 0.125, 0.25, 0.50, 1,2 mg/mL) were incubated for 24h and ROS levels were measured according to the procedure of DCFH-DA fluorescence probe method at 2.2.2.

2.4 morphological Observation of cells

The number of cells and the growth state were observed by an inverted microscope. Cells grown in log phase at a density of 1X 10 ⁵ The cells were seeded in six well plates and used for the experiments after the cells were adherent. Cells were treated in groups according to 2.3 experiments, observed under the mirror and images were left.

2.5 Detection of SOD, GSH-PX, CAT activity and MDA content

In the reference, the grouping and processing of cells were performed in the same manner as in 2.3, and after the completion of the processing, the cells were collected, washed with PBS, added to a cell lysate, and centrifuged to collect the cell supernatant. The procedures were performed according to the kit instructions of SOD, GSH-PX, CAT, and MDA.

2.6 data analysis

The experimental data are all expressed as mean ± sem. Statistical data were obtained using SPSS 20.0 software, single-factor analysis of variance was performed, and mapping was performed using originPro 2019 software. * P <0.05 indicated significant difference, and P <0.01 indicated very significant difference.

3. Analysis of results

3..1 H ₂ O ₂ Establishment of model for inducing oxidative stress of RIN-m5F cells

Islet beta cells are vulnerable to ROS, causing islet beta cell apoptosis, leading to the development of diabetes. Large scale study adopted H ₂ O ₂ And establishing a cell oxidative damage model to induce beta cells to generate oxidative damage. As shown in fig. 10, with H ₂ O ₂ The cell viability decreased with increasing concentration. Compared with a control group, 200 to 500 mu mol/L H ₂ O ₂ After the cells are incubated for 3-6 h, the survival rate of the cells is obviously reduced (P)<0.05 250 μmol/L H) of the series of combinations ₂ O ₂ After incubation of cells, cell viability decreased very significantly (. About.P)<0.01 250. Mu. Mol/L H) ₂ O ₂ Treatment to half the inhibition rate IC ₅₀ . Compared with a control group, 50-500 mu mol/L H ₂ O ₂ After the cells are incubated for 12-24 h, the survival rate of the cells is obviously reduced (P)<0.05 200. Mu. Mol/L H) and ₂ O ₂ treatment to half the inhibition rate IC ₅₀ 。

As shown in FIG. 11, 10-500. Mu. Mol/L H is compared to the blank group ₂ O ₂ Cells were induced and their ROS levels increased with prolonged incubation time, indicating H ₂ O ₂ Cellular ROS levels can be affected. In comparison with cell control group, via H ₂ O ₂ After 3h and 6h of induction, the ROS level is in an ascending trend, and the ROS level is significant when the concentration reaches 100 mu mol/L ([ P ])<0.05 ); warp H ₂ O ₂ After 12h and 24h of induction, the intracellular ROS level (P) is remarkably increased when the concentration reaches more than 100 mu mol/L<0.05)。

The result analysis and the cell survival rate are considered, 250 mu mol/L H is selected ₂ O ₂ Induction of 3h, 200. Mu. Mol/L H ₂ O ₂ Induction for 12h served as the model condition for the subsequent experiments.

3.2 Effect of different concentrations of BDP-I (B) on cell survival

As shown in FIG. 12, in the graph A, the effect of BDP-I (B) on cell survival rate is shown for 24h, and the cell survival rate is gradually increased when the BDP-I (B) concentration is in the range of 0.0625-0.125 mg/mL, and gradually decreased when the BDP-I (B) concentration is higher than 0.125 mg/mL. In the range of 0.0625-1 mg/mL, after BDP-I (B) treats the cells for 24 hours, 48 hours and 72 hours, the survival rate of the cells is not obviously different compared with that of a control group; at a concentration of 2mg/mL, cell viability decreased significantly compared to the control group (. P < 0.05). Thus, 0.0625-1 mg/mL was determined as the safe concentration of BDP-I (B) to the cells.

3.3 Protection of H2O 2-induced RIN-m5F cells by BDP-I (B)

As shown in FIG. 13, 250. Mu. Mol/L H ₂ O ₂ Cell viability was significantly reduced after 3h induction of cells compared to normal control group (. About.P)<0.05 ); after the BDP-I (B) concentration is 0.0625-0.25 mg/mL, the cell survival rate is obviously increased compared with that of a model group after the cell is intervened<0.05 ); compared with the control group, when the BDP-I (B) concentration is between 0.0625 and 0.5mg/mL, the cell survival rate is significant ( ^# P<0.05 ); therefore, 0.0625-0.25 mg/mL BDP-I (B) can increase H ₂ O ₂ Viability of injured cells, for H ₂ O ₂ The damaged cells have protective effect.

3.4 Effect of BDP-I (B) on cell morphology

As shown in FIG. 14, the control group was RIN-m5F cells cultured in normal medium, which were in the form of aggregated spheres or spindle, and the model group was 250. Mu. Mol/L H ₂ O ₂ Shrinkage, cell density reduction and edge blurring after stimulation; cell morphology slowly recovered and cell density increased following various concentrations of BDP-I (B) intervention. BDP-I (B) Low dose group (0.125 mg/mL) intervention H ₂ O ₂ The number of cells after the stimulated cells is obviously increased, and the cell boundaries become smooth gradually.

3.5 ROS level determination

As shown in FIG. 15, 250 μ M H ₂ O ₂ After induction of cells, model group ROS levels were significantly higher than control group (. About.p)<0.01 ); ROS levels were elevated compared to the cell control group; after 0.0625-0.25 mg/mL BDP and BDP-I (B) intervention for 24 hours, the ROS level of the cells tends to be remarkably reduced compared with that of a model group (P)<0.05 Explain H) ₂ O ₂ Induction of increased ROS levels in RIN-m5F cells, BDP-I (B) can lower H ₂ O ₂ ROS levels in RIN-m5F cells after injury, inhibit H ₂ O ₂ Induced oxidative stress of the cells.

3.6 Detection of SOD and CAT activity and MDA content

The results of the activity of BDP-I (B) on SOD and CAT in RIN-m5F cells and the determination of MDA content are shown in FIGS. 16, 17 and 18. SOD and CAT can effectively eliminate excessive ROS and hydroxyl-induced lipid peroxide in vivo, thereby protecting the integrity of cell structure and function. As can be seen from FIGS. 16-a and b, H ₂ O ₂ The SOD and CAT activity of the treated cells is obviously reduced compared with that of a control group ( ^* P<0.05 After BDP-I (B) dry prognosis, the antioxidant enzyme activity is gradually increased and is dose-dependent, the SOD activity of BDP-I (B) reaches (19.29 +/-2.09) U/mg at 0.25mg/mL, and the CAT activity reaches (17.45 +/-1.70) U/mg.

MDA is one of the end products of the oxidation process of peroxidized lipids, and it can attack unsaturated fatty acids in cell membranes to cause cell damage. The result, shown in FIG. 18c, shows that H ₂ O ₂ Induction can increase MDA content in cells ( ^* P<0.05 And the content of MDA in the cells is reduced after intervention of BDP and BDP-I (B), the content of MDA in a BDP-I (B) high-dose group (0.25 mg/mL) is close to that of a positive control group, and the BDP-I (B) can inhibit the release of MDA and protect cells damaged by oxidative stress.

4. Small knot

Determining suitable H by detecting cell survival rate by CCK-8 method ₂ O ₂ Inducing concentration and safe treatment concentration of BDP-I (B), and observing the influence of BDP-I (B) on the morphology of the oxidative damage cells under a mirror; detecting the influence of BDP on ROS level and MDA content in oxidative damage cells and the activities of SOD, GSH-PX and CAT enzymes, and judging the oxidative stress level in the cells. The conclusion is drawn from the study results:

H ₂ O ₂ the treatment induced oxidative damage to RIN-m5F cells, 250. Mu. Mol/L H ₂ O ₂ After 3h of induction of RIN-m5F cells, the cell morphology is damaged, the cell survival rate is reduced to 50%, and the ROS level is obviously increased (P)<0.05)；

BDP-I (B) can intervene in cells with oxidative damage and can reduce H in a certain concentration range ₂ O ₂ Oxidative damage to cellsCell viability was significantly increased (. About.P)<0.05 The intracellular ROS level is also reduced, the activities of SOD and CAT are improved, the MDA content is reduced, which indicates that 0.0625-0.25 mg/mL BDP-I (B) is used for H ₂ O ₂ The induced oxidative damage of RIN-m5F cells has protective effect, and the BDP-I (B) high-dose group (0.25 mg/mL) has optimal anti-oxidative stress effect.

[1] Li Yaojie, woody plant of Qinghai, M, qinghai, renminbu Press, 1987.

[2] The forward wins, wang Ning, zhao Yue, and so on, the research on the change rule of berberine in different parts of Berberis plants at different latitudes in Qinghai province [ J ]. Western forestry science, 2015,44 (02): 136-140.

[3] Sophora odorata fruit and extract thereof can be used for reducing blood sugar, such as Han Lijuan, yang Fang, and the like, CN104906163A [ P ],2015-09-16.

[4] Sophora root, han Lijuan, yang Fang, a Qinghai three thorn fruit powder composition and its tablet and use, CN105661392A [ P ].2016-06-15.

[5] Zhaojun, han Lijuan, she Ying, et al, a hypoglycemic tablet and its use, CN104983770A [ P ].2015-10-21.

[6] Shanyongkai, cui Guiping, wang Shengxin, development of composite wild thorn juice health drink [ J ] Chinese food and nutrition, 2003 (11): 46-48.

[7]Wang H Y,Guo L X,Hu W H,et al.Polysaccharide from tuberous roots of Ophiopogon japonicus regulates gut microbiota and its metabolites during alleviation of high-fat diet-induced type-2 diabetes in mice[J].Journal of Functional Foods,2019,63:103593.

[8] Ji Lanju analysis of free amino acids and ascorbic acid from fruits of Hippophae rhamnoides, nitraria tangutorum [ J ]. Journal of Integrated Plant Biology,1989 (6): 487-488.

[9] Zheng Shijia study of composition analysis and hypoglycemic mechanism of Acanthopanax trifoliatus polysaccharide [ D ] Guangzhou, guangdong university of pharmacy, 2019.

[10]Han L J,Suo Y R,Yang Y J,et al.Optimization,characterization,and biological activity of polysaccharides from Berberis dasystachya Maxim[J].International Journal of Biological Macromolecules,2016,85:655-666.

[11] Meng Zhaojun, han Lijuan, soyleigh fruit polysaccharide, its preparation method and application [ P ]. Qinghai: CN104945525A,2015-09-30.

[12] Zhou Hong pharmacokinetics study of mangiferin INS-1 islet beta cells [ D ]. Guangzhou, guangzhou university of traditional Chinese medicine, 2020.

[13]Federici M,Hribal M,Perego L,et al.High glucose causes apoptosis in cultured human pancreatic islets of langerhans.Diabetes,2001,50(6):1290-1301.

[14] Sun Huan the molecular mechanism of berberine to improve high-sugar, high-lipid-induced apoptosis of pancreatic islet NIT-1 [ D ]. University of science and technology, 2008.

[15]Xu L,YANG F,WANG J,et al.Anti-diabetic effect mediated by Ramulus mori polysaccharides[J].Carbohydrate Polymers,2015,117:63-69

[16] The protective effect and mechanism of rush-aminobutyric acid on RIN-m5f cells are explored to [ D ] no tin: jiangnan university, 2014.

[17] Source of Hangqi, huang Qinghua, li Raoling research review by experimental methods for in vitro evaluation of antioxidant activity by cell models [ J ]. Proceedings of the Guangdong college of pharmacy 2012,28 (02): 208-211.

[18] Zheng Yansong, li Yuan, zhang Shangong, etc. a myocardial cell oxidative damage model is established with low concentration hydrogen peroxide [ J ]. Fourth university of military medicine, 2001 (20): 1849-1851+1854.

[19]Ankur,Maheshwari,Man,et al.Pathways involved in testicular germ cell apoptosis induced by H2O2 in vitro[J].FEBS Journal,2009.

[20]Fernando Warleta,María Campos,Yosra Allouche,et al.Squalene protects against oxidative DNA damage in MCF 10Ahuman mammary epithelial cells but not in MCF7 and MDA-MB-231human breast cancer cells[J].Food and Chemical Toxicology,2010(48):1092～1100.

[21] Yang Yan, liu Dongbo, kang Xincong, et al H2O2 induces pancreatic islet RIN-m5F cells to construct an oxidative stress model [ J ]. Food and machinery, 2014,30 (02): 40-43.

Claims

1. A preparation method of polysaccharide of yellow thorn shown as formula BDP-I (B),

the method comprises the following operation steps:

2. The method of claim 1, wherein: the weak base anion exchange cellulose column chromatography is selected from diethylaminoethyl cellulose-52 column.

3. The method of claim 1, wherein: the sephadex is selected from sephadex G-200.

4. The method of claim 1, wherein: the crude polysaccharide of the yellow thorn is prepared by the following method:

5. The method of claim 1, wherein: the polysaccharide has a peak molecular weight of 39.469kDa, a weight average molecular weight of 47.714kDa and a number average molecular weight of 32.638kDa.

6. The method of claim 1, wherein: the polysaccharide has an infrared spectrum of 3397cm ^-1 、2927cm ^-1 、1740cm ^-1 、1612cm ^-1 、1421cm ^-1 、1105cm ^-1 、1020cm ^-1 Has characteristic peaks.

7. The method of claim 1, wherein: in monosaccharide composing the polysaccharide, the molar ratio of arabinose, galactose, glucose and galacturonic acid is 10.8:9.9:8.9:70.4.

8. the method of claim 1, wherein: the monosaccharide composing the polysaccharide comprises 10.8 mol percent of arabinose, 9.9 mol percent of galactose, 8.9 mol percent of glucose and 70.4 mol percent of galacturonic acid.