CN113234179B

CN113234179B - Preparation method of selenized pectin, product and application thereof

Info

Publication number: CN113234179B
Application number: CN202110513678.XA
Authority: CN
Inventors: 何静仁; 安孝宇; 陶雯; 杨宁; 张瑞; 叶树芯
Original assignee: Wuhan Polytechnic University
Current assignee: Wuhan Polytechnic University
Priority date: 2021-05-11
Filing date: 2021-05-11
Publication date: 2022-07-08
Anticipated expiration: 2041-05-11
Also published as: CN113234179A

Abstract

The invention relates to a preparation method of selenized pectin, and a product and application thereof, wherein the preparation method comprises the following steps: dissolving pectin in nitric acid, adding sodium selenite, and mixing to obtain a mixed system; carrying out ultrasonic treatment, microwave treatment and ultraviolet treatment on the mixed system simultaneously in the reaction process; evaporating and concentrating the crude product system; adding 3-4 times of anhydrous ethanol into the concentrated solution, precipitating with ethanol overnight, taking out, centrifuging, washing the obtained precipitate with anhydrous ethanol for 2-3 times, and dissolving with a small amount of distilled water to obtain a solution; and filtering, concentrating and freeze-drying the dissolved solution to obtain the selenized pectin. The invention can successfully carry out selenylation modification on pectin, has little influence on the structure of the pectin due to the selenylation modification, and simultaneously improves the selenium content and the yield of the selenylation pectin; the selenized pectin has the effect of preventing and treating ulcerative colitis.

Description

Preparation method of selenized pectin, product and application thereof

Technical Field

The invention relates to the technical field of food science, in particular to a preparation method of selenized pectin, a product and application thereof.

Background

Pectin is a natural high-molecular polysaccharide with a complex structure, is mainly derived from cell walls of plants such as oranges, apples, grapes and sugar beets, is used as water-soluble dietary fiber, and has the effects of improving gastrointestinal tract functions, reducing blood sugar and blood fat, preventing cardiovascular and cerebrovascular diseases, losing weight, resisting bacteria and the like. Pectin is considered as a natural food additive, is non-toxic and harmless, has no dosage limit when being used as the food additive except for special requirements in production, and is widely applied to the fields of food, medicine and cosmetics.

Meanwhile, selenium is a trace element necessary for human bodies, has very important effects on human health, has multiple effects of resisting oxidation and cancers, improving immunity, reducing blood sugar and the like, and plays an important role in treating keshan disease and Kaschin-Beck disease, preventing and treating cardiovascular diseases and the like. However, selenium cannot be synthesized spontaneously in human bodies and can only be taken in vitro, but the selenium content in natural foods is low, so that proper selenium supplement is beneficial to body health. Inorganic selenium has high toxicity and certain limitation in use, while organic selenium has the characteristics of high absorption rate and bioactivity, low toxicity, small environmental pollution and the like. However, at present, no technical scheme for selenizing pectin exists.

Disclosure of Invention

The invention aims to provide a preparation method of selenized pectin, a product and an application thereof, which can successfully carry out selenizing modification on the pectin, has little influence on the structure of the pectin by the selenizing modification, and simultaneously improves the selenium content and the yield of the selenized pectin; the selenized pectin has the effect of preventing and treating ulcerative colitis.

In order to solve the technical problems, the technical scheme adopted by the invention is as follows:

on the one hand, the preparation method of the selenized pectin comprises the following steps:

s1, dissolving pectin in nitric acid with the volume fraction of 0.5%, adding sodium selenite, and fully mixing to obtain a mixed system;

s2, placing the mixed system at 55-75 ℃ for reaction to obtain a crude product system; in the reaction process, ultrasonic treatment, microwave treatment and ultraviolet treatment are simultaneously carried out on the mixed system, and the reaction time is 4-8 h;

s3, cooling the mixed system to room temperature after the reaction is finished, and then adding Na into the crude product system₂CO₃Adjusting the pH value of a crude product system to 5-6, and then carrying out evaporation concentration on the crude product system by using a rotary evaporator to concentrate the volume of the crude product system to 15-20% of the original volume, and stopping the evaporation concentration to obtain a concentrated solution;

S4, adding 3-4 times volume of anhydrous ethanol into the concentrated solution, putting the concentrated solution into a refrigerator at 4 ℃ for alcohol precipitation overnight, taking out the concentrated solution, centrifuging the concentrated solution for 20-30min under the condition of 3500-4500r/min, washing the obtained precipitate for 2-3 times by using the anhydrous ethanol, and adding a small amount of distilled water for dissolving to obtain a dissolved solution;

and S5, filtering, concentrating, freezing and drying the dissolved solution to obtain the selenized pectin.

Preferably, in step S1, the weight ratio of pectin to sodium selenite is 1 (0.8-1.5).

Preferably, in step S2, the process of simultaneously performing the ultrasonic treatment, the microwave treatment and the ultraviolet treatment on the mixed system includes:

performing magnetic stirring on the mixed system, and simultaneously performing ultrasonic treatment, microwave treatment and ultraviolet treatment on the mixed system;

wherein the ultrasonic treatment conditions are as follows: the ultrasonic power is 180-300W (preferably 240W), and the ultrasonic working frequency is 25KHz-30 KHz;

the microwave treatment conditions were: microwave power 180-300W (preferably 270W);

the ultraviolet treatment conditions were: the wavelength of the ultraviolet radiation light source is 365nm, and the power of the ultraviolet radiation is 180-300W (preferably 270W).

Therefore, the high-efficiency separation and recombination of the micro-components such as molecules, electrons and the like in the components in the mixed system can be promoted by three different energy sources of ultraviolet irradiation, microwave radiation and ultrasonic oscillation, the reaction process is accelerated, the selenylation modification effect of the pectin is remarkably improved, and the yield of the selenylation pectin is improved.

Preferably, step S5 includes: and (3) filtering the dissolved solution by a microfiltration ceramic membrane at 40-60 ℃, filtering by a roll-type ultrafiltration membrane at 45-55 ℃, concentrating by a roll-type high-pressure reverse osmosis membrane at 35-45 ℃, and freeze-drying the obtained concentrated solution to obtain the selenylation pectin.

Preferably, the filter pore diameter of the microfiltration ceramic membrane is 0.5-1.0 μm, the cut-off molecular weight of the roll-type ultrafiltration membrane is 100-200kDa, and the cut-off molecular weight of the roll-type high-pressure reverse osmosis membrane is 150-1000 Da.

On the one hand, the selenized pectin prepared by the method is also provided.

On one hand, the method for establishing the model of the influence of the selenized pectin on the DSS-induced ulcerative colitis mouse is also provided, and the method comprises the following steps:

(1) grouping: several mice were divided into treatment and prevention groups, and the treatment and prevention groups were each randomly divided into 7 groups: a normal group, a DSS model group, a positive group, a pectin high dose group, a pectin low dose group, a pectin selenide high dose group, and a pectin selenide low dose group; each group was acclimatized for one week, during which time the mice had free access to water;

(2) modeling and dosing:

(2-1) taking 750mg of mesalazine, adding a CMC (sodium carboxymethylcellulose) solution with the mass fraction of 1% to a constant volume of 25mL, and preparing a 30mg/mL mesalazine solution;

respectively taking 250mg and 500mg of pectin, respectively adding distilled water to a constant volume of 25mL, and preparing into 10mg/mL pectin solution and 20mg/mL pectin solution;

respectively taking 250mg and 500mg of the selenylation pectin, respectively adding distilled water to a constant volume of 25mL, and preparing 10mg/mL selenylation pectin solution and 20mg/mL selenylation pectin solution;

(2-2) on days 1 to 7 of the treatment group, the other groups except the normal group freely drink DSS solution with mass fraction of 3%;

on the 8 th to 21 th days, except for the normal group and the DSS model group, the positive group, the pectin low dose group, the pectin high dose group, the pectin selenide high dose group and the pectin selenide high dose group respectively correspond to a 30mg/mL mesalazine solution, a 10mg/mL pectin solution, a 20mg/mL pectin solution, a 10mg/mL pectin selenide solution and a 20mg/mL pectin selenide solution;

each group is perfused for 1 time every day, and the perfused volume is 10 mL/kg;

meanwhile, in the prevention group, except for the normal group, the DSS model group and the positive group, the pectin low dose group, the pectin high dose group, the pectin selenide high dose group and the pectin selenide high dose group correspond to a 10mg/mL pectin solution, a 20mg/mL pectin solution, a 10mg/mL pectin selenide solution and a 20mg/mL pectin selenide solution in intragastric administration respectively on days 1-14; each group is perfused for 1 time every day, and the perfused volume is 10 mL/kg;

On day 15-21, except for the normal group which freely drinks distilled water, the DSS model group, the positive group, the pectin low dose group, the pectin high dose group, the pectin selenide high dose group and the pectin selenide high dose group freely drink DSS solution with the mass fraction of 3%; meanwhile, except for the normal group and the DSS model group, the positive group, the pectin low dose group, the pectin high dose group, the pectin selenide high dose group and the pectin selenide high dose group respectively correspond to a mesalamine solution of 30mg/mL, a pectin solution of 10mg/mL, a pectin solution of 20mg/mL, a pectin selenide solution of 10mg/mL and a pectin selenide solution of 20 mg/mL; each group was intragastrically administered 1 time per day, with the intragastrically administered volume being 10 mL/kg;

(3) sampling: fasting mice of each group of the treatment group and the prevention group for 16 hours without water supply, and then carrying out eyeball blood sampling and anatomical sampling on each group;

(4) index detection: and (5) carrying out index detection on the sample.

Preferably, in the step (two), the Disease Activity Index (DAI) is calculated by recording the body weight, stool characteristics and hematochezia of the mice in each group from days 1 to 21 in the treatment group and days 15 to 21 in the prevention group.

Preferably, in the step (iii), the eyeball bleeding process includes: blood is taken from eyeball, and then standing for 30min, centrifuging for 15min at 4 deg.C and 3000r/min, separating serum, and storing in refrigerator at-20 deg.C for use.

Preferably, in step (iii), the anatomical sampling process includes: when in dissection, heart, liver, spleen, kidney and intestine tissues are quickly taken out and weighed, the colon is separated, the colon tissue from the cecum to the position 1cm above the anus is cut off and divided into 4 sections, each section is about 1cm, the colon tissue at the far end is placed in paraformaldehyde fixing solution with the mass fraction of 4 percent and stored at 4 ℃ to be tissue slices, and the rest tissues are quickly frozen in liquid nitrogen after being subpackaged and then transferred to a refrigerator at 80 ℃ below zero to be stored for later use.

Preferably, in step (iii), the anatomical sampling process further includes: weighing appropriate amount of colon tissue, cutting into pieces, adding precooled PBS buffer solution according to a ratio of 1:9, homogenizing on ice, centrifuging at 3500r/min for 5min, and storing supernatant at-80 deg.C for use.

Preferably, the mouse is a healthy male SPF ICR mouse, and the weight of the mouse is 20-22 g.

On the one hand, the application of the selenized pectin in preparing foods, medicines and health-care products for preventing/treating colitis is also provided.

The invention adopts HNO₃-Na₂SeO₃The method and the composite treatment assist in preparing the selenylation pectin, the pectin can be successfully selenylated and modified, the selenylation modification has little influence on the structure of the pectin, and meanwhile, the finally prepared selenylation pectin has high safety and no obvious toxic or side effect; the treatment effect of the selenized pectin is better than that of pectin under the same dosage, so that the selenized pectin has the effects of preventing and treating ulcerative colitis, can be used as a potential colitis adjuvant treatment medicament/food/health-care product, and can be used for appropriately supplementing, preventing and relieving colon inflammation diseases.

Drawings

FIG. 1 is a graph showing the effect of mass ratio of pectin to sodium selenite on the selenium content and yield of selenized pectin;

FIG. 2 is a graph of the effect of reaction temperature on selenium content and yield of pectin selenide;

FIG. 3 is a graph of the effect of reaction time on selenium content and yield of pectin selenide;

FIG. 4a is a graph showing the effect of different treatment regimes on the selenium content of pectin selenide;

FIG. 4b is a graph showing the effect of different treatment regimes on the selenium yield of pectin selenide;

FIG. 5a is a graph of the effect of pectin selenide on the body weight of female mice in various groups of mice;

figure 5b is the effect of pectin selenide on the body weight of male mice in each group of mice;

FIG. 6 is an infrared spectrum of pectin and pectin selenide;

FIG. 7a is a NMR of pectin;

FIG. 7b is a NMR chart of selenized pectin;

FIG. 8a is a graph of the molecular weight distribution of pectin;

FIG. 8b is a molecular weight distribution plot of pectin selenide;

FIG. 9 is a distribution diagram of the particle size of pectin and selenized pectin;

FIG. 10a is a 15mg/mL pectin and selenized pectin rheology plot;

FIG. 10b is a 20mg/mL pectin and selenized pectin rheology plot;

FIG. 10c is a rheology plot of 30mg/mL pectin and selenized pectin;

FIG. 11 is a scanning electron micrograph of pectin and pectin selenide wherein (a), (b) and (c) are magnified 100, 500 and 1000 times respectively; (d) (e) (f) are respectively magnified 100, 500 and 1000 times of electron micrographs of the pectin selenide;

FIG. 12a is the DAI score of treated mice;

FIG. 12b is the DAI score of the treated mice;

FIG. 13a is colon of mice in the treatment group;

FIG. 13b is colon of mice in prevention group;

FIG. 13c is colon length of treated mice;

FIG. 13d is colon length of mice in the prevention group;

FIG. 14a is the MPO content in colon tissue of mice in the treatment group;

FIG. 14b is the MPO content in colon tissue of the mice in the prevention group;

FIG. 15a is the activity of GSH-Px in colon tissue of treated mice;

FIG. 15b is a graph of GSH-Px activity in colon tissue of prevention group mice;

FIG. 16 is a photograph of a pathological section of a mouse (magnified 200 times).

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, embodiments of the present invention will be further described with reference to the accompanying drawings.

Optimized preparation method of selenized pectin

1.1 preparation method of selenized pectin

s2, placing the mixed system at 55-75 ℃ for reaction to obtain a crude product system; and in the reaction process, the mixed system is simultaneously subjected to ultrasonic treatment, microwave treatment and ultraviolet treatment;

S3, cooling the mixed system to room temperature after the reaction is finished, and then adding Na into the crude product system₂CO₃Adjusting the pH value of a crude product system to 5-6, and then carrying out evaporation concentration on the crude product system by using a rotary evaporator to concentrate the volume of the crude product system to 15-20% of the original volume, and stopping to obtain a concentrated solution;

and S5, filtering and concentrating the dissolved solution, and freeze-drying to obtain the selenized pectin.

1.2 Single factor test design

(1) Influence of mass ratio of pectin and sodium selenite on selenium content and yield

1g of pectin is accurately weighed and dissolved in 100mL of nitric acid with volume fraction of 0.5%, the influence on the selenium content and yield of the synthesized selenized pectin is researched when the mass ratio of the pectin to the sodium selenite is 1:0.8, 1:1, 1:1.2 and 1:1.4 respectively, other process steps from S1 to S5 are the same, and the selenium content and yield of the selenized pectin are determined as shown in FIG. 1.

As can be seen from FIG. 1, pectin and Na are included in the starting materials ₂SeO₃Increase of the ratioAnd the selenium content and the yield of the selenized pectin show a trend of increasing and then decreasing, wherein pectin and Na are contained in the selenized pectin₂SeO₃When the mass ratio is 1:1, the selenium content and the yield are respectively 198.1121 μ g/g and 91.35%, so in step S1 of the preparation method, the weight ratio of the pectin to the sodium selenite is preferably 1: 1.

(2) Effect of reaction temperature on selenium content and yield

1g of pectin is accurately weighed and dissolved in 100mL of nitric acid with a volume fraction of 0.5%, the weight ratio of the pectin to sodium selenite is 1:1 in the step S1, the influence of the reaction temperature of the mixed system in the step S2 at 55, 60, 65, 70 and 75 ℃ on the selenium content and yield of the synthesized selenized pectin is researched, other process steps in the steps S1-S5 are the same, and the selenium content and yield of the selenized pectin are determined as shown in FIG. 2.

As can be seen from fig. 2, the reaction temperature of the mixed system in step S2 has a significant influence on the preparation of selenized pectin. The selenium content is continuously increased along with the rise of the temperature within the range of 55-75 ℃, the minimum selenium content is 82.9790 mu g/g at 55 ℃, the selenium content is obviously improved from 65 ℃, and the selenium content reaches 240.0736 mu g/g at 75 ℃; the yield also shows the same change trend, and when the temperature is 75 ℃, the yield is higher and is 92.9 percent, and gradually becomes gentle. Therefore, with the change of temperature, the selenium content and the yield of the selenized pectin show a certain correlation, the selenium content and the yield are reduced due to overhigh temperature, the degradation of the polysaccharide is accelerated probably due to high temperature, and the hydroxyl of the polysaccharide is oxidized by the nitric acid at high temperature to damage the polysaccharide structure, and the stability of the organic selenium compound is possibly influenced. Therefore, the reaction temperature cannot be excessively high, and in step S2 of the above production method, it is preferable to carry out the reaction by subjecting the mixed system to 65 to 75 ℃ (preferably 70 ℃).

(3) Effect of reaction time on selenium content and yield

1g of pectin is accurately weighed and dissolved in 100mL of nitric acid with the volume fraction of 0.5%, the weight ratio of the pectin to sodium selenite in the step S1 is 1:1, the reaction temperature of the mixed system in the step S2 is 70 ℃, the influence of the reaction time of 4, 5, 6, 7 and 8 hours in the step S2 on the selenium content and yield of the synthesized selenized pectin is researched, and the selenium content and yield measurement results of the selenized pectin are shown in FIG. 3.

As can be seen from fig. 3, the selenium content of the selenized pectin tends to increase and then decrease with the increase of the reaction time, the selenium content is obviously increased with the increase of the reaction time at first, the selenium content reaches to 194.0447 μ g/g at the maximum when the reaction is carried out for 6 hours, and the selenium content is gradually reduced after the reaction time is prolonged; the yield is increased continuously with the time, and the reaction time gradually becomes gentle after reaching 7 hours. Pectin and Na due to too short reaction time₂SeO₃The combination can not be sufficiently and effectively combined, and the pectin can be degraded due to the long reaction time, so that the selenization reaction is influenced. In addition, in step S2 of the above preparation method, the reaction time of the mixed system is preferably 6 hours in order to save production cost.

(4) Effect of different treatments on selenium content and yield

Accurately weighing 1g of pectin, dissolving the pectin in 100mL of nitric acid with a volume fraction of 0.5%, wherein the weight ratio of the pectin to sodium selenite in step S1 is 1:1, the reaction temperature of the mixed system in step S2 is 70 ℃, and the reaction time is 6 hours, researching the influence on the selenium content and yield of the synthesized pectin selenide when the mixed system is subjected to ultrasonic treatment + ultraviolet treatment, microwave treatment + ultraviolet treatment and ultrasonic treatment, microwave treatment and ultraviolet treatment (namely composite treatment) simultaneously in step S2, and obtaining results as shown in FIGS. 4a-4 b.

Wherein the ultrasonic treatment conditions are as follows: the ultrasonic power is 180, 210, 240, 270 and 300W respectively, and the ultrasonic working frequency is 25KHz-30 KHz;

the microwave treatment conditions are as follows: the microwave power is 180, 210, 240, 270 and 300W respectively;

the ultraviolet treatment conditions were: the wavelength of the ultraviolet radiation light source is 365nm, and the power of the ultraviolet radiation is 180W, 210W, 240W, 270W and 300W respectively.

Meanwhile, magnetic stirring is carried out during treatment.

When the ultrasonic treatment, the ultraviolet treatment and the microwave treatment are respectively carried out at the power of 180W, 210W, 240W, 270W and 300W, the selenium content and the yield change trend of the selenized pectin prepared in different modes are shown in fig. 4a-4 b. The selenium content of the selenized pectin is highest and the yield is second in a composite treatment mode combining the three treatment modes, and in the ultrasonic treatment and ultraviolet treatment, when the power is 240W, the selenium content is highest and 289.5512 mug/g, and then the selenium content is reduced to a certain extent and tends to be gentle; the yield was at most 93.88% at a power of 270W. In the microwave treatment and the ultraviolet treatment, the selenium content is 298.1936 mug/g at the maximum when the power is 270W, then the selenium content is reduced and leveled off, and the yield is 92.65 percent at the maximum. In the ultrasonic treatment, the microwave treatment and the ultraviolet treatment, when the power is 270W, the selenium content is 370.1805 mu g/g at most, the yield is 93.57% at most, and the selenium content is obviously improved. Probably, when the power is low, the molecular motion in the reaction system is weak, and the collision and combination can not be fully realized, and when the power is high, the molecules are always in a strong motion state and are difficult to react. When the power is in the range of 180-300W, the yield generally fluctuates about 90%, the difference range is small, and the influence on the selenium content is more obvious. In contrast, the synergistic effect of the ultrasonic treatment, the microwave treatment and the ultraviolet treatment is the best, so that the ultrasonic treatment, the ultraviolet treatment and the microwave treatment are all determined to be suitable for 270W.

1.3 safety evaluation of pectin selenide

Acute toxicity test: 32 healthy ICR mice were selected, half female and half male, and the weight was 20-22 g. The groups were randomly divided into 4 groups of 8 individuals (4 individuals for each male and female), i.e., control group, high-, medium-, and low-dose groups of selenized pectin. Meanwhile, the selenized pectin prepared in the application is prepared into a high-dose selenized pectin solution, a medium-dose selenized pectin solution and a low-dose selenized pectin solution with corresponding concentrations.

The test was fasted for 16h (without restriction of water). And (3) performing intragastric administration on distilled water of a control group, wherein high, medium and low dosage components of the selenylation pectin respectively correspond to the intragastric administration high-dosage selenylation pectin solution, the medium-dosage selenylation pectin solution and the low-dosage selenylation pectin solution, so that the dosage requirements of 2000mg/kg, 1000mg/kg and 500mg/kg are respectively met correspondingly, performing intragastric administration twice in one day, wherein the interval between the two intragastric administrations is 6 hours, the intragastric administration amount is 0.2mL/10g · bw each time, and recovering normal diet 2-3 hours after the last intragastric administration. The mice were observed periodically for the first 24 hours after gavage to see if stress reaction occurred, and then observed once a day, the animal weight, toxic manifestation and death were recorded, continuously observed for 14 days, weighed on day 15, fasted for 16 hours (without restriction of drinking water), dissected, observed for organs, and the organ index was calculated according to the following formula, and the results are shown in table 1 and fig. 5a-5 b.

As shown in fig. 5a-5b, mice were dosed after one week of acclimatized feeding, with no stress within 24h and a significant weight rise in the first three days, probably due to fasting prior to dosing and an increase in diet. After continuously observing for 14 days, the mice in each group have no death phenomenon, and have normal hair color, free movement, good mental status and normal stool character. The body weight of each group of mice shows the same rising trend during the test period, and the body weight is increased normally.

After the dissecting on the 15 th day, the color of the organs of each group of mice is normal according to the observation, and as shown in table 1, compared with the normal group, the organs of the group with high, medium and low dosages of the selenylation pectin have no obvious lesion, and the indexes of heart, liver, spleen, kidney and intestine of each group of mice are not greatly different (P is more than 0.05). Therefore, the selenized pectin has no obvious toxicity to mice in the dosage range of 0-2000 mg/kg, and the LD50 is proved to be more than 2000 mg/kg. Therefore, the selenated pectin prepared by the method has high safety and no obvious toxic or side effect.

TABLE 1 organ index of each group of mice

Note: different letters represent significance of difference between groups (P < 0.05).

Structure representation and physicochemical property research of selenized pectin

2.1 measurement of the content of galacturonic acid

The galacturonic acid content of pectin and of the selenized pectin of the present application was determined using the carbazole-sulfuric acid method and the results are shown in table 2.

Drawing a standard curve: preparing standard galacturonic acid solutions with mass concentrations of 20, 40, 60, 80 and 100g/mL respectively. Taking 6 test tubes with plugs, respectively adding 1mL of galacturonic acid standard solution of 0, 20, 40, 60, 80 and 100g/mL into the test tubes, adding 6mL of high-grade concentrated sulfuric acid into each tube, quickly shaking up and cooling in an ice water bath, heating in a boiling water bath for 15min, cooling to room temperature, adding 0.5 mL0.15% carbazole reagent into each tube, shaking up, standing at room temperature for 30min, measuring absorbance at 530nm wavelength by taking tube No. 0 as a blank control, and drawing a standard curve by taking absorbance A as an abscissa and galacturonic acid concentration as an ordinate.

And (3) sample determination: preparing pectin solution with mass concentration of 50g/mL, and the determination steps are the same as above.

The galacturonic acid content calculation is as follows.

Galacturonic acid content (%) (C/50) 100%

In the formula: and C-is substituted into the standard curve to calculate the concentration of the galacturonic acid.

TABLE 2 galacturonic acid content

As can be seen from Table 2, the galacturonic acid content of the selenized pectin was increased compared to the protopectin.

2.2 Fourier Infrared Spectroscopy

Taking a proper amount of pectin and selenized pectin dried at 105 ℃ and KBr according to the proportion of 1:100, uniformly grinding in an agate mortar, tabletting, and scanning conditions are as follows: the wavelength range is 4000-400 cm^-1Scanning 32 times with resolution of 4cm^-1The results are shown in FIG. 6.

As can be seen from fig. 6, both pectin and the selenized pectin of the present application have characteristic absorption peaks typical of polysaccharides. At 3400cm^-1The strong absorption peak near the absorption peak is generated by O-H stretching vibration, and is 2920-2940 cm^-1Absorption peaks in the range ascribed to C-H stretching vibration (CH, CH)₂And CH₃)，1730～1770cm^-1The absorption peak is the characteristic absorption peak of carboxyl in uronic acid, which indicates that pectin and selenized pectin both contain uronic acid, 1620cm^-1The nearby absorption peak is the C ═ O vibration generated by free carboxyl groups in pectin. 1300-1450 cm^-1Deformation vibration at O-H; 1000-1300 cm^-1The polysaccharide fingerprint area is nearby, and the absorption peak in the range is generated by C-O stretching vibration. 1231.56, 1099.61 and 1051.64cm^-1The absorption peak shows that the peak types of the pectin and the selenized pectin are similar, wherein the sugar residue is in a pyranose form and is an asymmetric vibration peak of a glycosidic bond C-O-C, and the basic structures before and after selenization are not greatly changed. And selenized pectin at 829.27 and 759.56cm ^-1The absorption peaks appeared in the position are deduced to be respectively classified into the stretching vibration of Se ═ O and the stretching vibration of C-O-Se, which shows that the preparation method can complete selenizing modification, forms selenium ester bonds, and red shift appears in the pyran ring and respectively moves to 1230.63, 1096.97 and 1020.36cm^-1Selenium was also shown to be successfully introduced.

2.3 nuclear magnetic resonance spectrum

Dissolving a proper amount of dried pectin and selenized pectin samples in D₂In O, the sample was transferred to a nuclear magnetic tube, and one-dimensional 1H NMR was measured by a 600M nuclear magnetic resonance spectrometer and scanned 256 times at 25 ℃ to obtain results as shown in FIGS. 7a to 7 b.

As can be seen from FIGS. 7a-7b, the formants of most protons of pectin and selenized pectin appear at 3.2-5.2 ppm, which are characteristic signal peaks of polysaccharide, and the maximum signal peak at 4.47ppm is attributed to D₂Solvent peak O.¹H-NMR can judge the configuration of glycosidic bonds in polysaccharide structures, usually by chemical shift of anomeric hydrogens, according to literature reports,¹the anomeric hydrogen signal peak in H-NMR is more than 5.0ppm of alpha-type glycoside, less than 5.0ppm of beta-type glycoside, and the chemical shift of all protons is less than 5.4ppm, indicating that both are pyranose. It can be seen that pectin and selenized pectin have major chemical shifts of anomeric H-1 of 5.02 and 5.03, respectively, and thus are predominantly in the alpha configuration. The signal peak at 4.89ppm of H-5 was derived from the non-esterified galacturonic acid residue, the signal peak at 4.11ppm was due to arabinose H-4, and the strong peak at 3.74ppm was attributed to The signal generated from the methoxy group attached to the carboxyl group of galacturonic acid, the signal peaks at 1.25ppm and 1.19ppm were generated from rhamnose linked to the 1, 4-and 1, 2-glycosidic linkages, respectively, and the signal peak at 2.00ppm was due to acetylation substitution of galacturonic acid residues. The signal peaks of pectin and selenized pectin are basically consistent, which shows that the preparation method can complete the selenizing modification of pectin, and the basic structure of pectin is not changed.

2.4 determination of molecular weight

The molecular weight distribution of pectin and selenized pectin was determined by size exclusion chromatography-differential detection-18 angle laser light scattering coupled technology (SEC-RI-MALLS). And (3) testing conditions: and (3) chromatographic column: an agent PL aquagel-OH gel chromatography column; mobile phase: 0.2mol/L ammonium acetate; flow rate: 0.7 mL/min; sample introduction amount: 20L; column temperature: at 30 ℃. The results are shown in FIGS. 8a-8b, tables 3-4.

TABLE 3 molecular weight distribution of pectin

TABLE 4 molecular weight distribution of pectin selenide

From FIGS. 8a-8b, tables 3-4, it can be seen that the molecular weight of pectin is mainly concentrated at 5.165X 10⁵Da, 56.1%, secondly at 6.491X 10⁴23.6% of Da, 1.881 × 10⁴17.4% at Da; the molecular weight of the selenized pectin of the application is mainly concentrated in 6.027 x 10 ⁵Da, the percentage of occupation is up to 91.5%, and the peak types are symmetrical, which shows that the purity of the selenized pectin after treatment is higher, the molecular weight is slightly improved, which shows that the selenized pectin retains the basic structure of the pectin, and the addition of selenium has little influence on the pectin structure.

2.5 particle size and potential measurements

Preparing 1mg/mL pectin and selenylation pectin solution, and measuring the particle size and the potential of a sample by using a Malvern laser particle sizer. The refractive index was 1.543, the density was 1.508, and the absorption was 0.001. The results are shown in FIG. 9 and Table 5.

TABLE 5 particle size and potential analysis

As shown in fig. 9 and table 3, the particle size distribution of pectin and selenized pectin solutions is relatively uniform, mainly aggregating at about 900nm and 700nm, and the particle size of selenized pectin is reduced compared with that of protopectin, which indicates that the aggregation degree of pectin solution is relatively high, and selenized pectin solution has less aggregation and better stability. Pectin and selenized pectin are negatively charged, which shows that the pectin and the selenized pectin are acidic polysaccharides, and the surfaces of the selenized pectin liquid drops are more negatively charged, so that the electrostatic repulsion enhanced aggregation is reduced, the size of the liquid drops is reduced, and the stability is better. It is possible that the pectin contains more acidic groups due to the formation of selenate, thereby reducing the electronegativity and improving the dispersion stability.

2.6 determination of rheological Properties

Preparing pectin and selenized pectin with the concentrations of 10, 20 and 30mg/mL, and placing in a refrigerator at 4 ℃ for 12h to be tested. The static shearing mode is selected, a flat plate-flat plate measuring system is adopted, the diameter of a parallel plate is 40mm, and the gap distance is 1 mm. Placing a proper amount of sample in the center of a flat plate of a rheometer, pressing the parallel plates downwards and uniformly spreading the parallel plates, setting the scanning temperature to be 25 ℃, the strain to be 1 percent and measuring the shear rate to be 5-200 s^-1Viscosity and shear stress changes. And performing regression fitting on data points of the relation between the shear stress and the shear rate by using a Power-low model, wherein the shear force calculation formula is as follows. The results are shown in FIGS. 10a to 10c, Table 6.

σ＝Kγn

In the formula: σ represents shear stress, Pa; k represents the consistency coefficient, Pa.sn; gamma denotes shear rate, s^-1(ii) a n represents the fluid index.

TABLE 6 fluid index of different concentrations of pectin and of pectin selenide

From FIGS. 10a-10c, Table 6, it can be seen that both pectin and selenized pectin exhibited shear thinning at different concentrations, consistent with pseudoplastic fluid characteristics. With the increase of the concentration, the apparent viscosity of the pectin and the selenized pectin is increased, and the shear thinning phenomenon is more obvious, because with the increase of the concentration, the intermolecular entanglement and aggregation capability is enhanced, and the strong electrostatic repulsion force enables pectin molecules to extend along the chain direction, thereby promoting the cross-linking between the molecules and leading the flow resistance to be increased. And the viscosity of the pectin is obviously higher than that of the selenized pectin under the same concentration, which shows that the viscosity of the pectin can be reduced by selenizing modification.

2.7 scanning Electron microscope

Fixing the dried pectin and pectin selenide samples on a sample table adhered with conductive adhesive, spraying gold, observing the surface micro-morphology of the samples under a scanning electron microscope, and selecting proper positions and multiples to take photos for analysis. The results are shown in FIG. 11.

As can be seen from fig. 11, the microscopic morphology of the pectin particles is irregular fiber rod-like accumulation, and the surface is rough and has protrusions; the selenized pectin obtained after selenization preparation and freeze-drying has a sheet structure, relatively smooth surface and clear layers. The microstructure and the morphology difference between the pectin and the pectin are larger, and the part (b) in fig. 11 shows that pectin molecules are mutually cross-linked and wound, so that the structure is tighter, the protopectin is easier to aggregate, and the solubility is lower; and part (e) of fig. 11 shows that the sheet structure of the selenized pectin is relatively regular, the edges are branched, the area is large, the selenized pectin is thin, and the selenized pectin is stacked, so that the crosslinking degree is reduced, and the selenized pectin has better solubility. The structural morphology is more apparent after the magnification of the parts (c) and (f) of FIG. 11 is increased.

Research on treatment and prevention effects of pectin selenide on mice with DSS (dextran sulfate sodium) induced ulcerative colitis

3.1 test animals

Healthy male SPF grade ICR mice, weight 20 ~ 22g, purchase from the sbefu biotechnology limited company, license number: SCXK (Jing) 2019-.

3.2 mouse grouping and modeling

(2) modeling and administration, comprising the steps of:

(2-1) adding a CMC (sodium carboxymethylcellulose) solution with the mass fraction of 1% into 750mg of mesalazine, and fixing the volume to 25mL to prepare a mesalazine solution with the mass fraction of 30 mg/mL;

respectively taking 250mg and 500mg of the above-mentioned selenized pectin, respectively adding distilled water to a constant volume of 25mL, and preparing into a selenized pectin solution of 10mg/mL and a selenized pectin solution of 20 mg/mL;

On the 8 th to 21 th days, except for the normal group and the DSS model group, the positive group, the pectin low dose group, the pectin high dose group, the pectin selenide high dose group and the pectin selenide high dose group respectively correspond to a mesalazine solution with the weight of 30mg/mL, a pectin solution with the weight of 10mg/mL, a pectin solution with the weight of 20mg/mL, a pectin selenide solution with the weight of 10mg/mL and a pectin selenide solution with the weight of 20 mg/mL;

each group was intragastrically administered 1 time per day, with the intragastrically administered volume being 10 mL/kg;

meanwhile, in the prevention group, except for the normal group, the DSS model group and the positive group, the pectin low dose group, the pectin high dose group, the pectin selenide high dose group and the pectin selenide high dose group correspond to a 10mg/mL pectin solution, a 20mg/mL pectin solution, a 10mg/mL pectin selenide solution and a 20mg/mL pectin selenide solution in intragastric administration respectively on days 1-14; each group was intragastrically administered 1 time per day, with the intragastrically administered volume being 10 mL/kg;

on day 15-21, except the normal group, which drunk distilled water freely, the DSS model group, the positive group, the pectin low dose group, the pectin high dose group, the pectin selenide high dose group and the pectin selenide high dose group all drunk DSS solution with a mass fraction of 3% freely; meanwhile, except for the normal group and the DSS model group, the positive group, the pectin low dose group, the pectin high dose group, the pectin selenide high dose group and the pectin selenide high dose group respectively correspond to a mesalamine solution of 30mg/mL, a pectin solution of 10mg/mL, a pectin solution of 20mg/mL, a pectin selenide solution of 10mg/mL and a pectin selenide solution of 20 mg/mL; each group is perfused for 1 time every day, and the perfused volume is 10 mL/kg;

The phenomena of weight loss, loose stool, bloody stool, laziness in movement and the like of the mice indicate that the acute colitis model is successfully modeled. The modeling and dosing profiles for each group of mice are shown in tables 7-8.

(3) Sampling: the mice in the treatment group and the prevention group are fasted for 16 hours without water supply, and then eyeball blood sampling and anatomical sampling are carried out on each group;

wherein, the eyeball blood sampling process comprises: standing eyeball blood for 30min, centrifuging at 4 deg.C and 3000r/min for 15min, separating serum, and storing in refrigerator at-20 deg.C;

the anatomical sampling process includes: when in dissection, heart, liver, spleen, kidney and intestine tissues are quickly taken out and weighed, the colon is separated, the colon tissue from the cecum to the position 1cm above the anus is cut and divided into 4 sections, each section is about 1cm, the colon tissue at the far end is placed in paraformaldehyde fixing solution with the mass fraction of 4 percent and stored at 4 ℃ to be tissue slices, and the rest tissues are quickly frozen in liquid nitrogen after being subpackaged and then transferred to a refrigerator at-80 ℃ to be stored for later use;

weighing appropriate amount of colon tissue, cutting into pieces, adding precooled PBS buffer (protein inhibitor is added before use) at a ratio of 1:9, homogenizing on ice, centrifuging at 3500r/min for 5min, and storing the supernatant at-80 deg.C.

(4) And (3) detection: and carrying out index detection and section observation on the sample, wherein the indexes comprise organ indexes, colon length, IL-6, IL-10 and TNF-alpha contents in serum, MPO content in colon tissues and GSH-Px activity in the colon tissues.

TABLE 7 modeling and dosing profiles for treatment groups of mice

Table 8 modeling and dosing status of prevention group mice

3.3 disease Activity index score

The body weight, fecal characteristics and hematochezia of the mice in each group were recorded from days 1-21 in the treatment group and days 15-21 in the prevention group, and the Disease Activity Index (DAI) was calculated according to the following formula, with the results shown in FIGS. 12a-12 b. The scoring criteria are shown in Table 9.

DAI ═ 3 (mouse weight loss score + fecal trait score + hematochezia condition score)

TABLE 9 DAI scoring criteria

The Disease Activity Index (DAI) in mice is an important indicator for assessing the severity of colitis, with higher DAI scores leading to more severe colonic lesions. As can be seen from FIG. 12a, in the treatment experiment, the DAI score is continuously increased along with the increase of the modeling days, and the phenomena of weight loss, loose stool and bloody stool of the mouse are serious after 7 days, which indicates the success of modeling. The gavage treatment is started on day 8, the DAI scores of the mice in the sample group and the positive group are gradually reduced, the DAI score of the pectin selenide under the same dosage is lower, and the reduction of the pectin selenide high-dosage group is more obvious and is close to the positive group. The DAI score of the mice in the model group is reduced slowly, which indicates that the model has certain recovery capability. As shown in fig. 12b, in the prevention test, the DAI score of the model group mice is increased sharply, while the DAI score of the sample group mice is increased continuously, but is slowed down to different degrees, and the ascending trend of the DAI score is reduced with the increase of the gavage dose, and the high-dose selenizing pectin can obviously inhibit the increase of the DAI score caused by DSS, so as to alleviate the severity of colitis.

3.4 visceral indices

After the mouse was dissected, the weight of the heart, liver, spleen, kidney and colon was measured, and the index of each organ was calculated according to the following formula, and the results are shown in tables 10 to 11.

TABLE 10 index of organs in mice of the treatment group

TABLE 11 index of organs in prevention group mice

Spleen is an important immune organ of the body, and pathological enlargement occurs when the body is inflamed. As shown in tables 10-11, in both experimental groups, DSS modeling significantly increased the spleen index (P <0.05) in the model group mice compared to the normal group, resulting in enlarged spleen; the colon index of the model group is obviously improved, which indicates that the colon generates edema hyperplasia. In the treatment group, compared with the model group, the spleen index of the pectin high dose group, the selenized pectin low dose group and the high dose group is obviously reduced, and the colon index of the selenized pectin high dose group is obviously reduced (P <0.05) and is closest to the normal value. In the prevention group, compared with the model group, the spleen index of the pectin high-dose group and the colon index of the pectin selenide high-dose group are obviously reduced, and the colon index of the pectin selenide high-dose group is also obviously reduced (P <0.05), wherein the spleen index and the colon index of the mouse can be restored to normal levels by the high-dose pectin selenide for prevention, which indicates that the enema prevention can relieve the colonic inflammation to a certain extent.

3.5 Colon Length

The colon length of the mice was measured and recorded and averaged for each group. The results are shown in FIGS. 13a-13 d.

Colon length is an intuitive method of evaluating disease, and ulcerative colitis usually causes the colon length of mice to shorten. As shown in FIGS. 13a-13b, the colon surface of the normal group of mice was smooth, the stool particles were formed, and after DSS modeling, the colon of the mice appeared hyperemia and edema, and the stool was not formed. As shown in fig. 13c-13d, in the treatment and prevention test group, the colon length of the model group mice was significantly shortened (P <0.05) compared to the normal group. Compared with the model group, after the mice in the treatment group are subjected to intragastric administration of corresponding samples, the colon length is obviously increased (P is less than 0.05), and the symptoms are relieved to a certain extent. The colon length increase was most significant in the high dose group of pectin selenide, with no significant difference from the normal and positive groups (P > 0.05). The mice in the prevention group have a certain protection effect on the intestinal tract through the gavage prevention in 14 days before modeling, compared with the model group, the intestinal tract shortening phenomenon is obviously reduced (P is less than 0.05), the effect of the high-dose group of the pectin selenide is even slightly higher than that of a positive drug, and the pectin selenide has a better protection effect on the intestinal tract shortening.

3.6 determination of IL-6, IL-10 and TNF- α in serum

The IL-6, IL-10 and TNF-alpha content in mouse serum was determined strictly according to the kit instructions. The results are shown in tables 12 to 13.

TABLE 12 serum IL-6, IL-10 and TNF-alpha levels in the treated mice

TABLE 13 content of IL-6, IL-10 and TNF-alpha in serum of mice in prevention test group

Studies have shown that colitis occurs in association with the immune response of the immune system, activating and amplifying the inflammatory response, and that dysregulation between pro-inflammatory and anti-inflammatory cytokines in the intestinal system is an important factor in causing the inflammatory response. IL-6 can induce B cells to differentiate and generate antibodies, and induce T cells to activate, proliferate and differentiate, participate in immune response of organisms, is a promoter of inflammatory response, TNF-alpha is an important proinflammatory cytokine, can activate neutrophils and lymphocytes, promote synthesis and release of other cytokines, and activate inflammatory cascade reaction; IL-10 is an anti-inflammatory cytokine with immunosuppressive effects. DSS induction can improve IL-6 and TNF-alpha levels of mice with colitis, reduce IL-10 levels, participate in signal pathways such as cell apoptosis and protein synthesis and induce barrier dysfunction of intestinal mucosa.

As shown in tables 12-13, in the treatment and prevention test group, compared with the normal group, the IL-6 and TNF-alpha content in the serum of the mice in the model group is obviously improved, and the IL-10 content is obviously reduced (P <0.05), which indicates that DSS modeling is successful and colitis mice have obvious inflammatory reaction. In the treatment group, compared with the model group, the IL-6 and TNF-alpha contents of the positive group and each sample group are obviously reduced, the IL-10 content is obviously improved (P is less than 0.05), the colonic inflammation is relieved to different degrees, and the effect of the pectin selenide under the same dosage is better than that of the pectin. The high-dose selenylation pectin has the best treatment effect, the IL-6 and TNF-alpha levels are higher than those of other sample dose groups, the IL-10 level is lower than those of other sample dose groups (P is less than 0.05), and the high-dose selenylation pectin has no significant difference with the treatment effect of a positive medicament, so that the selenylation pectin has a certain treatment effect and is dose-related. In the prevention group, compared with the model group, the IL-6 and TNF-alpha content of the positive group and each sample group is also obviously reduced, the IL-10 content is obviously improved (P is less than 0.05), and the effect is more obvious along with the increase of the dosage. The IL-6 and TNF-alpha levels of the seleno-pectin high-dose group are obviously higher than those of other sample dose groups (P is less than 0.05), the IL-10 level is lower than those of other sample dose groups but is not obviously different from the pectin high-dose group, so that both pectin and seleno-pectin have a certain prevention effect, and the high-dose seleno-pectin has a better prevention effect.

3.7 determination of MPO in colonic tissue

MPO content in mouse colon tissue was determined strictly according to the kit instructions. The results are shown in FIGS. 14a-14 b.

MPO is an important enzyme in neutrophils, is widely used as an indicator of neutrophil infiltration, and can be used as a biochemical indicator of inflammatory reaction to judge the severity of inflammation. Studies have shown that DSS induction can increase MPO levels in the colon of mice, reflecting intestinal epithelial mucosal injury. As shown in FIGS. 14a-14b, the MPO content in colon tissue of the model group was significantly higher than that of the normal group (P <0.05) in both the treatment and prevention test groups, indicating that DSS caused cellular infiltration of colon mucosa in mice. In the treatment groups, the MPO content of the positive group and each sample dose group is obviously improved (P <0.05) compared with that of the model group, thereby relieving the colonic inflammatory reaction to a certain extent, wherein the MPO content of the pectin selenide high dose group is obviously lower than that of other sample groups (P <0.05), and the MPO content is equivalent to the treatment effect of mesalazine; in the prevention group, compared with the model group, the MPO content of the positive group and each sample dosage group is also obviously improved (P is less than 0.05), the MPO content of the selenized pectin is obviously lower than that of the pectin under the same dosage, the selenized pectin with high dosage has the best effect of preventing the colonic inflammation, and the colonic inflammation degree can be reduced.

3.8 determination of GSH-Px in Colon tissues

GSH-Px activity in mouse colon tissue was determined strictly according to the kit instructions. The results are shown in FIGS. 15a-15 b.

Research has shown that oxidative stress is closely related to inflammatory reaction, and excessive reactive oxygen radicals cause oxidative damage to colonic epithelial cells, playing an important role in the pathogenesis of colitis. GSH-Px is an important peroxide decomposing enzyme, selenium is an active center of GSH-Px, and can catalyze the reduction reaction of GSH to reduce harmful peroxide into harmless hydroxyl compounds, thereby protecting the structure and function of cell membranes from being damaged by the peroxide. Thus, increased GSH-Px activity can protect colon tissue.

The effect of pectin selenide on GSH-Px activity in colon tissues of mice is shown in FIGS. 15a-15b, and in the treatment and prevention test group, the GSH-Px activity in colon tissues of mice in the model group is obviously reduced compared with that in the normal group (P <0.05), which indicates that DSS induction causes oxidative stress to cause damage to colon mucosa. In the treatment group, compared with the model group, the GSH-Px activity of the positive group and each sample dosage group is obviously improved (P is less than 0.05), the antioxidant capacity can be improved to different degrees, and the colonic inflammation caused by oxidative stress is improved. The activity of GSH-Px of the pectin selenide high-dose group is obviously higher than that of other sample groups (P is less than 0.05), and the pectin selenide shows better treatment effect. In the prevention group, the gavage positive drugs and each dosage sample can obviously improve the activity of the GSH-Px (P <0.05) compared with the model group, the selenizing pectin effect is better under the same dosage, and the high-dosage selenizing pectin GSH-Px activity in the gavage is obviously improved (P < 0.05). Therefore, the selenized pectin can protect the oxidative damage of colon tissues by up-regulating the activity of the antioxidant enzyme, thereby relieving the DSS-induced colonic inflammation.

3.9 histopathological section

(1) Tissue dehydration: the tissue was dehydrated using gradient alcohol.

(2) The tissue is transparent: the tissue block must be transparent after being dehydrated by alcohol, the clearing agent (dimethylbenzene) can be simultaneously mixed with the dehydrating agent and the paraffin, and the paraffin can smoothly permeate into the tissue after the dehydrating agent is replaced by the clearing agent.

(3) Wax dipping: the transparent tissue blocks were sequentially waxed with 3 times of paraffin (60 ℃).

(4) Embedding: embedding is the encapsulation of the wax-impregnated tissue block in a paraffin block. The waxed colon tissue is quickly put into melted paraffin, cooled and solidified. The temperature of the paraffin for embedding is slightly higher than the temperature of paraffin immersion, so that the tissue block and the embedded paraffin are completely integrated.

(5) Slicing and baking: and slicing the embedded wax block by using a Leica pathological microtome, spreading the sliced tissue slice in a water bath kettle at 40 ℃, obliquely inserting the glass slide into the water surface to fish the slice, attaching the slice to the proper position of the glass slide, and baking the slice in an oven at 60 ℃ for 3 hours.

(6) Slicing and dewaxing: the paraffin section is sequentially placed into xylene I (20min), xylene II (20min), xylene III (20min), absolute ethyl alcohol I (5min), absolute ethyl alcohol II (5min), 95% ethyl alcohol (5min), 90% ethyl alcohol (5min), 80% ethyl alcohol (5min) and 70% ethyl alcohol (5min), and then the paraffin section is washed by distilled water for 5 min.

(7) Placing hematoxylin into the slices, dyeing for 2min, differentiating for 15s with hydrochloric acid ethanol, and washing with tap water to turn blue; dyeing with 1% water-soluble eosin dye solution for 3min, washing with tap water, and rinsing for 30 s; anhydrous ethanol I (5min), anhydrous ethanol II (5min), n-butanol (5min), xylene I (5min), xylene II (5min)

(8) After air drying, the gel was mounted on neutral gum and examined under a microscope (200X photograph), the results are shown in FIG. 16.

As can be seen from FIG. 16, the colon tissue structure of the normal mice in the two test groups is complete, the intestinal mucosa and the villi are normal, the goblet cells are rich, the crypt and the gland are normal in shape, and the inflammatory cell infiltration phenomenon is not seen; and the colon tissue structure of the model group mice is disordered, glands and crypts are damaged, goblet cells are deleted, and a large amount of inflammatory cell infiltration appears in a mucous layer. In the treatment group, the colonic tissue inflammation of each group of mice was improved to a different extent after the gavage treatment compared with the model group. The positive group, the pectin high-dose group and the pectin selenide high-dose group have complete colon tissue structures, the glands are arranged regularly, most goblet cells and visible crypt structures appear, the cell infiltration is obviously reduced, the high-dose pectin selenide has the best effect on the recovery of inflammation, and the treatment effect is obvious. In the prevention group, through early gastric lavage prevention, the colon mucosa and villus structures of each group of mice are slightly damaged compared with the model group, the structures are basically complete, the colon mucosa and villus structures have crypt and gland structures, the inflammatory infiltration is obviously reduced, the effect of reducing the colon inflammatory reaction of the selenized pectin high-dose group is best, the colon tissue morphology is similar to that of the positive group, and the colon inflammatory diseases of the mice can be well prevented and relieved.

In conclusion, the pectin is used as the raw material, and HNO is adopted₃-Na₂SeO₃The method and the composite treatment assist in preparing the selenylation pectin, and the optimal technological parameters for preparing the selenylation pectin are determined, the preparation method can successfully carry out selenylation modification on the pectin, the introduced selenium mainly exists in the form of selenium ester, the selenylation modification has little influence on the structure of the pectin, and meanwhile, the finally prepared selenylation pectin has high safety and no obvious toxic or side effect;

further, for a mouse model of ulcerative colitis established by 3% DSS, the selenized pectin has better treatment effect than pectin under the same dosage, and the selenized pectin with high dosage has the best effect in all sample groups and can reduce the inflammatory response of the colon of a mouse, so that the selenized pectin has prevention and treatment effects on the ulcerative colitis, can be used as a potential colitis adjuvant treatment medicament/food/health-care product, and can appropriately supplement, prevent and relieve the inflammatory disease of the colon.

The embodiments and features of the embodiments described herein above may be combined with each other without conflict.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and should not be taken as limiting the scope of the present invention, which is intended to cover any modifications, equivalents, improvements, etc. within the spirit and scope of the present invention.

Claims

1. The preparation method of the selenized pectin is characterized by comprising the following steps of:

s1, dissolving pectin in nitric acid, adding sodium selenite, and fully mixing to obtain a mixed system; the weight ratio of pectin to sodium selenite is 1 (0.8-1.5);

s4, adding 3-4 times volume of anhydrous ethanol into the concentrated solution, putting the concentrated solution into a refrigerator at 4 ℃ for alcohol precipitation overnight, taking out the concentrated solution, centrifuging the concentrated solution for 20-30 min under the condition of 3500-4500 r/min, washing the obtained precipitate for 2-3 times by using the anhydrous ethanol, and adding a small amount of distilled water for dissolving to obtain a dissolved solution;

2. The method of claim 1, wherein the step S2, the step of simultaneously performing the ultrasonic treatment, the microwave treatment and the ultraviolet treatment on the mixed system comprises:

wherein the ultrasonic treatment conditions are as follows: the ultrasonic power is 180-300W, and the ultrasonic working frequency is 25KHz-30 KHz;

the microwave treatment conditions are as follows: the microwave power is 180W and 300W;

the ultraviolet treatment conditions were: the wavelength of the ultraviolet radiation light source is 365nm, and the ultraviolet radiation power is 180-300W.

3. The method of claim 1, wherein step S5 includes: and (3) filtering the dissolved solution by a microfiltration ceramic membrane at 40-60 ℃, filtering by a roll-type ultrafiltration membrane at 45-55 ℃, concentrating by a roll-type high-pressure reverse osmosis membrane at 35-45 ℃, and freeze-drying the obtained concentrated solution to obtain the selenylation pectin.

4. The method according to claim 3, wherein the microfiltration ceramic membrane has a pore size of 0.5-1.0 μm, the molecular weight cut-off of the rolled ultrafiltration membrane is 100-200kDa, and the molecular weight cut-off of the rolled high-pressure reverse osmosis membrane is 150-1000 Da.

5. A selenized pectin prepared by the method of any one of claims 1-4.

6. A method for modeling the effect of pectin selenide on DSS-induced ulcerative colitis according to claim 5, comprising the steps of:

(1) grouping: several mice were divided into treatment and prevention groups, and the treatment and prevention groups were each randomly divided into 7 groups: a normal group, a DSS model group, a positive group, a pectin low dose group, a pectin high dose group, a pectin selenide low dose group, and a pectin selenide high dose group; each group was acclimatized for one week, during which time the mice had free access to water;

(2) modeling and dosing:

(2-1) respectively preparing a mesalazine solution of 30 mg/mL, a pectin solution of 10 mg/mL, a pectin solution of 20 mg/mL, a selenized pectin solution of 10 mg/mL and a selenized pectin solution of 20 mg/mL;

on the 8 th to 21 th days, except for the normal group and the DSS model group, the positive group, the pectin low dose group, the pectin high dose group, the pectin selenide low dose group and the pectin selenide high dose group respectively correspond to a 30 mg/mL mesalazine solution, a 10 mg/mL pectin solution, a 20 mg/mL pectin solution, a 10 mg/mL pectin selenide solution and a 20 mg/mL pectin selenide solution;

meanwhile, on the 1 st to 14 th days of the prevention group, except for the normal group, the DSS model group and the positive group, the low-dosage group, the high-dosage group, the low-dosage group and the high-dosage group of pectin selenide correspond to a 10 mg/mL pectin solution, a 20 mg/mL pectin solution, a 10 mg/mL pectin selenide solution and a 20 mg/mL pectin selenide solution respectively; each group was intragastrically administered 1 time per day, with the intragastrically administered volume being 10 mL/kg;

on day 15-21, except the normal group, which drunk distilled water freely, the DSS model group, the positive group, the pectin low dose group, the pectin high dose group, the pectin selenide low dose group and the pectin selenide high dose group all drunk DSS solution with a mass fraction of 3% freely; meanwhile, except for the normal group and the DSS model group, the positive group, the pectin low dose group, the pectin high dose group, the pectin selenide low dose group and the pectin selenide high dose group respectively correspond to a mesalamine solution of 30 mg/mL, a pectin solution of 10 mg/mL, a pectin solution of 20 mg/mL, a pectin selenide solution of 10 mg/mL and a pectin selenide solution of 20 mg/mL; each group was intragastrically administered 1 time per day, with the intragastrically administered volume being 10 mL/kg;

(3) sampling: eyeball blood collection and anatomical sampling are carried out on mice in each group of the treatment group and the prevention group;

(4) Index detection: and (5) carrying out index detection on the sample.

7. The method according to claim 6, wherein in the step (2), the body weight condition, the fecal characteristics and the fecal blood condition of the mice in each group are recorded and the disease activity index is calculated from days 1 to 21 in the treatment group and days 15 to 21 in the prevention group.

8. The method of claim 7, wherein the mouse is a healthy male SPF grade ICR mouse weighing 20-22 g.

9. Use of the selenized pectin of claim 5 in the preparation of a food/drug/health product for the prevention/treatment of colitis.