CN115819633A - Nitraria polysaccharide BDP-I (B) and application thereof - Google Patents

Abstract

The invention separates and identifies a brand new polysaccharide compound BDP-I from yellow thorns by a specific separation and purification means. Meanwhile, the invention also researches the biological activity of BDP-I, 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.

Description

Nitraria polysaccharide BDP-I (B) and application thereof

Technical Field

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

Background

Yellow thorn, the scientific name Berberis Amurensis (Berberis dasystachya M.), 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 hasVarious active ingredients including phytosterols, organic acids, polysaccharides, polyphenols, 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 is more suitable to be used as food, health care products or medicines for treating and preventing diabetes due to natural safety and wider application range of the yellow thorn fruit powder.

For many years, the research on yellow thorns at home and abroad is mostly focused on the alkaloid component berberine, which is considered as 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.

Specifically, the invention provides a polysaccharide from Nitraria tangutorum bobr, which has the following structure:

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 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.

Meanwhile, the infrared spectrum of the polysaccharide is shown in figure 5, and the infrared spectrum can also be used as a quality control map of the polysaccharide.

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 content of the arabinose, the galactose, the glucose and the galacturonic acid in percentage by mole is 10.8%, 9.9%, 8.9% and 70.4% in sequence.

Based on the polysaccharide compound, the invention also provides a cell protection composition, and the active ingredient of the composition comprises the xanthium polysaccharide BDP-I (B).

In the present invention, the cells include islet cells, and particularly, islet β cells.

The invention also provides application of the polysaccharide in preparing an antioxidant product. The research of the invention finds that the polysaccharide compound can reduce H ₂ O ₂ Damage to cells.

For example, the present inventors have discovered that the polysaccharide compound reduces oxidative damage to pancreatic islet cells, particularly pancreatic islet beta cells.

The pancreatic islets are endocrine cell masses distributed between acini of the exocrine part of the pancreas, and are distributed unevenly on each part of the pancreas, with the largest number of pancreatic tails. Islets vary in size, with small cells being only a few, and large cells being hundreds. There is also a sporadic supply of endocrine cells located near the acini and ducts. About 50 thousands of islets of langerhans occupy 1-2% of the pancreas.

Islet cells were classified as follows:

b cells (beta cells) account for about 60-80% of islet cells, are mainly located in the central part of the islets, and can secrete insulin to regulate blood sugar content. Pancreatic islet B cell function is impaired and insulin secretion is absolutely or relatively inadequate, thus causing diabetes.

A cell (alpha cell) accounts for about 24-40% of islet cells, secretes glucagon, which acts in contrast to insulin and increases blood sugar.

D cells (delta cells) accounting for about 6-15% of the total islet cells secrete somatostatin.

Pancreatic islet PP cells, which comprise approximately 1% of the pancreatic islet cells, secrete pancreatic polypeptide.

The invention also provides application of the polysaccharide in preparing a product for preventing or treating diseases caused by islet cell injury.

In the invention, the disease caused by the damage of the islet B cells is mainly prevented or treated.

Such damage, including but not limited to oxidative damage.

Diabetes mellitus is a metabolic disorder caused by insulin deficiency or (and) a decrease in the biological effects of insulin (e.g., insulin resistance), and is a common disease characterized by persistent elevation of blood glucose and the appearance of diabetes.

Because islet cells contain a low level of antioxidant enzymes, diabetic patients are prone to have islet beta cell oxidative stress reaction under the action of high-sugar environment, cytokines, islet amyloid polypeptide and the like, and the latter inhibits insulin gene transcription by activating signal transduction pathways, such as a c-Jun amino terminal kinase (JNK) pathway, an hexosamine pathway and the like, so that islet beta cells are damaged, insulin secretion is reduced, and diabetes is further aggravated.

The diseases of the invention include, but are not limited to diabetes, hyperlipidemia, obesity and other diseases related to insulin deficiency or insulin resistance.

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 and regulating related signalsPassage and regulation of intestinal flora ^[13,14] And the like. Related research shows that glucotoxicity and lipotoxicity can also promote the generation 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 oxidation 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] The low-concentration hydrogen peroxide (100 mu mol/L) is used 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 prevent H to some extent ₂ O ₂ Resulting in damage to human breast epithelial cells. Insulinomas, the most common of the endocrine tumors of the pancreas, produce or release a large amount of insulin without depending on the change of blood glucose, and thus can be widely used clinically as an in vitro model of islet beta cells for studying the mechanism of treating diabetes ^[21] 。

The invention firstly adopts modern chromatographic means to clarify the structure of the uniform polysaccharide BDP-I (B) of the nitraria tangutorum bobr, and secondly adopts H with different concentrations ₂ O ₂ Inducing RIN-m5F cells, establishing an oxidative damage model, intervening model cells by using BDP-I (B) with different concentrations, detecting cell survival rate, and determining activityExploring BDP-I (B) for H by taking oxygen (ROS) level and Malonaldehyde (MDA) content as indexes ₂ O ₂ The protective action of inducing the oxidative damage of 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, and discussing protection H of yellow spine polysaccharide through molecular biological level ₂ O ₂ 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 yellow thorn berries and the research and development of yellow thorn polysaccharide health food.

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 Compared with the model group, the cell survival rate is increased after the BDPs of 0.0625-0.5 mg/mL and the polysaccharide BDP-I (B) of 0.0625-0.5 mg/mL are dried (P)<0.05 ROS, MDA levels are in a downward trend. In conclusion, the polysaccharide BDP-I (B) of the yellow spine can improve the cell survival rate within a certain dosage range, and can improve the cell survival rate of the yellow spine polysaccharide BDP-I (B) of the yellow spine polysaccharide in the aspect of 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.

Drawings

FIG. 1 chromatography elution curve of polysaccharide DEAE anion column

FIG. 2Sephadex 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 polysaccharide BDP-I (B) and monosaccharide standards (B) purified from Nitraria tangutorum bobr, 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. 8Dept135 map

FIG. 9HSQC (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 Induction of the effect on cell viability for 3 h; (B) H ₂ O ₂ Effect of 6H Induction 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 cell ROS, note: * P is<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 group (250. Mu. Mol/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 cellsNote that: 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 the SOD enzyme activity of 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 crushed yellow thorn powder with petroleum ether (60-90 ℃) by 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 with the protein removed 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) 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 taken. 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 purified polysaccharide separated 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 the retention time (x, min) of each standard as the horizontal axis and the Log of the 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. And (3) accurately absorbing the acid hydrolysis solution, transferring the acid hydrolysis solution into a tube, blowing the tube to dry by nitrogen, adding 5ml of water, uniformly mixing by vortex, absorbing 100uL of the acid hydrolysis solution, adding 900uL of deionized water, and centrifuging the solution 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 sample and 200mg of potassium bromide, pressing into tablets, and pressing the blank control by 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 uronic 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 under reduced pressure to dryness 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. Measuring an acetylation product sample by using a Shimadzu GCMS-QP 2010 gas chromatography-mass spectrometer;

GC-MS conditions: RXI-5SIL MS chromatography 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 600MHz NMR spectrometer.

2. Analysis of results

2.1 purification of Nitraria polysaccharide by DEAE anion column chromatography

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%. The BDP-I component with the highest yield is selected for further purification due to the requirement of subsequent cell experiments.

2.2Sephadex G-200 gel column chromatography purification of Nitraria polysaccharide

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). Taking glucan with the molecular weights of 5000, 11600, 23800, 48600, 80900, 148000, 273000, 409800, 670000 and 3693000Da as a standard substance and taking elution time as an abscissa, obtaining lgMp-RT (peak position molecular weight), lgMw-RT (weight average molecular weight), lgMn-RT (number average molecular weight) correction curves, wherein fitting equations are y = -0.195x +12.375 and R2=0.9913 respectively; 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 shows a single absorption peak with good peak type symmetry, and indicates the purificationThe BDP-I (B) has higher purity. 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 the monosaccharide composition of the xanthatin BDP-I (B) by adopting an 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 the 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 end group 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 glycosidic linkages in both the alpha and beta configuration 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, dept135 profile analysis found that δ 62.64, 68.27, 67.79, 68.25, 70.76, 70.5, 61.6 inverted peaks, indicated as 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 to assign the hydrocarbon of each cross peak. 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. As a result, BDP-I (B) contains → 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 → 4 → 10 monosaccharide residues.

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

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) 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, BDP-i (B) was shown to contain → 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 → these 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 Bertoni polysaccharide BDP-I (B) vs. H ₂ O ₂ Protective Effect of injured RIN-m5F cells

1. Materials and instruments

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 devices 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) Adding 1mL of trypsinized solution (0.25% Trypsin-0.53Mm EDTA) into the culture flask, digesting in an incubator at 37 deg.C for 2 minutes, observing the digestion condition of the cells under a microscope, if the cells are mostly rounded and dropped off, quickly taking back the operation, and adding a small amount of complete medium 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 incubator 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 cell 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

2.2.1H ₂ O ₂ Influence on cell survival

To investigate the safe concentration of H2O2 on RIN-m5F cells, cell viability was determined by the CCK-8 method, whereby yellowish WST-8 was reduced to a highly water-soluble yellow formazan dye by dehydrogenase in the mitochondria of cells under the action of an electron carrier (1-Methoxy PMS), and absorbance at 450nm indirectly reflected the number of living cells.

Grouping cells: blank group, serum-free RPMI-1640 culture medium; control group, RPMI-1640 medium containing 10% fetal bovine 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 1 × 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, 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 set up for each set, the wells were slowly added to avoid air bubble formation, the tips were changed for each row, the plates were incubated in a constant temperature incubator for 0.5h, and the assay at 450nm was performed 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 And (4) concentration.

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

2.2.2H 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.

Selection of 2.2.3BDP-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) vs. 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 containing 10% FBS + cells + optimal concentration H2O2 culture optimal time; 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 After induction of cells at concentrations and times, cells were cultured for 24h with various concentrations of BDP-I (B) intervention (0.0625, 0.125, 0.25, 0.50, 1,2 mg/mL) and cell viability was determined according to the procedure of CCK-8 method 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 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 reference, the grouping and processing of cells were performed in the same manner as 2.3, and after the completion of the processing, the cells were collected, washed with PBS, and then cell lysate was added, and cell supernatant was collected by centrifugation. 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..1H ₂ O ₂ Establishment of model for inducing oxidative stress of RIN-m5F cells

Islet beta cell effortlessnessDamaged by ROS, islet beta cells are apoptotic, 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, 200-500 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 same 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). From this, it was confirmed that 0.0625 to 1mg/mL are a safe concentration of BDP-I (B) for the cells.

Protection of H2O 2-induced RIN-m5F cells by 3 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 Injured cell survival rate, on 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 and in the form of aggregated spheres or spindle, and the model group cells were cultured in 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.

3.5ROS 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, inhibition of 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. As shown in FIG. 18c, the results show 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; and (3) detecting the influence of BDP on the ROS level and MDA content in the 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 according to 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 cells, marked increase in cell viability (. About.P)<0.05 The intracellular ROS level is also reduced, the activities of SOD and CAT are improved, and the MDA content is reducedDescription of 0.0625-0.25 mg/mL BDP-I (B) vs. 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.

Cited documents:

[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 P2015-10-21, china.

[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-2diabetes 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, the composition analysis of Acanthopanax trifoliatus polysaccharide and the mechanism of action of acanthopanax trifoliatus polysaccharide in reducing blood sugar are studied [ D ]. Guangzhou, university of Guangdong 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 pharmacokinetic study of Mangiferin in 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 method for in vitro evaluation of antioxidant activity by cell model [ J ]. Proceedings of 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 (10)

1. A polysaccharide from yellow spine has the following structure:

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.

2. The xanthane polysaccharide of claim 1, characterized in that: 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.

3. The xanthane polysaccharide of claim 1, characterized in that: the polysaccharide has an infrared spectrum of 3397cm ^-1 、2927cm ^-1 、1740cm ^-1 、1612cm ^-1 、1421cm ^-1 、1105cm ^-1 、1020cm ^-1 Has characteristic peaks.

4. The xanthane polysaccharide of claim 1, characterized in that: the IR spectrum of the polysaccharide is shown in FIG. 5.

5. The xanthum polysaccharide according to claim 1, characterized in that: in monosaccharide composing the polysaccharide, the molar ratio of arabinose, galactose, glucose and galacturonic acid is 10.8:9.9:8.9:70.4.

6. the xanthane polysaccharide of claim 1, characterized in that: 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.

7. A composition for protecting a cell, comprising: the active ingredient of which comprises the xanthane polysaccharide according to any one of claims 1 to 6.

8. Use of the polysaccharide of any one of claims 1 to 6 for the preparation of an antioxidant product.

9. Use of the polysaccharide of any one of claims 1 to 6 for the preparation of a product having islet cell protective effect.

10. Use of the polysaccharide of any one of claims 1 to 6 for the preparation of a product for the prevention or treatment of a disease caused by islet cell damage.

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