CN112094361A - Agaricus blazei polysaccharide iron complex, preparation method and application - Google Patents

Abstract

The invention belongs to the technical field of polysaccharide complexes, and discloses a Agaricus blazei polysaccharide iron complex, a preparation method and an application. The complex, the content of iron element is 9.44~16.54%, and the content of Agaricus blazei polysaccharide is 61.67~68.55%. The present invention adopts Agaricus blazei polysaccharide, an active substance in medicinal and edible homologous Agaricus blazei, as the sugar base, and ferric inorganic salt as the glycosyl base, and through complexation reaction, a stable Agaricus blazei polysaccharide iron complex is formed, and its dissolution property is low and high. The invention has good stability, wide raw material sources, simple and stable preparation process, and can provide a novel iron supplement, which can inhibit colitis while supplementing iron. The Agaricus blazei polysaccharide iron complex of the present invention is suitable for use as an iron supplement for animals, and is also suitable for treating ulcerative colitis.

Description

Agaricus blazei polysaccharide iron complex, preparation method and application

technical field

The invention belongs to the technical field of polysaccharide complexes, and relates to a Agaricus blazei polysaccharide complex, in particular to a Agaricus blazei polysaccharide iron complex, a preparation method and an application.

Background technique

Iron is an essential trace element in living organisms and plays an extremely important role in the metabolic process. Iron is a necessary raw material for the synthesis of hemoglobin. When the body lacks iron, iron deficiency or iron deficiency anemia occurs. Iron deficiency anemia (IDA) is a common nutritional disorder. The traditional medicine for the treatment of IDA is ferrous sulfate, and taking ferrous sulfate has a stimulating effect on the gastrointestinal tract, which is not conducive to the digestion and absorption of iron. For the treatment of severe iron deficiency anemia, the new generation of ferrous sulfate sustained-release capsules or sustained-release tablets have the advantages of better absorption and less side effects than ferrous sulfate; Cell membrane damage caused by lipid peroxidation. For the supplementation of iron, the current research at home and abroad is a polysaccharide-iron complex (PIC) which is formed by using polysaccharide as a carrier and complexing with ferric ions. Polysaccharide iron complexes are composed of two parts: glycosyl and glycosyl (Fe ³⁺ ). The glycosyl group is ferric iron, but it does not exist in free state. , in the body can also be reduced to ferrous iron to be absorbed and utilized. The polysaccharide iron complex has the advantages of less side effects, good solubility and high iron content, and is an iron supplement with good application prospects.

Ulcerative colitis (Ulcerative Colitis) is a common chronic intestinal inflammation. The alternating relapse and remission characteristics of UC make it a lifelong disease, and its etiology and pathogenesis have not been fully elucidated. At present, domestic and foreign researches are mostly focused on the environment, genetic factors, external infection and immunity. Symptoms are mainly recurring intestinal ulcers with abdominal pain, diarrhea, and bloody mucus.

Agaricus Blazei Murill is native to Brazil, and is also known as bergamot mushroom and small pine mushroom. It is a rare medicinal and edible fungus. family, mushrooms. Agaricus blazei fruit body is rich in polysaccharides, glycoprotein complexes, sterols and other active substances. Agaricus blazei polysaccharides have antioxidant, immune regulation, hypoglycemic, anti-tumor, hypolipidemic and other effects.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a kind of Agaricus blazei polysaccharide iron complex, to provide a kind of novel iron supplement, and play the effect of inhibiting colitis while supplementing iron;

Another object of the present invention is to provide a kind of preparation method of above-mentioned Agaricus blazei polysaccharide iron complex;

Another object of the present invention is to provide the application of the above-mentioned Agaricus blazei polysaccharide iron complex.

In order to achieve the above object, the technical scheme adopted in the present invention is:

A kind of Agaricus blazei polysaccharide iron complex, the Agaricus blazei polysaccharide iron complex uses Agaricus blazei polysaccharide as a sugar base and ferric as a glycosyl base, through complexation reaction, the complex of Agaricus blazei polysaccharide and iron is formed. compound;

The content of iron element in the Agaricus blazei polysaccharide iron complex is 9.44-16.54%, and the content of Agaricus blazei polysaccharide is 61.67-68.55%;

Described Agaricus blazei polysaccharide iron is reddish brown powder;

The solubility of the Agaricus blazei polysaccharide iron at 25° C. per 100 g of water is 10-25 g.

The present invention also provides a preparation method of the Agaricus blazei polysaccharide-iron complex, which is to obtain the Agaricus blazei polysaccharide-iron complex by complexation reaction of Agaricus blazei polysaccharide and ferric inorganic salt.

As a limitation, the preparation method comprises the following steps carried out in sequence:

1) get Agaricus blazei polysaccharide and catalyst to be dissolved in distilled water, slowly drip the aqueous solution and alkali solution of ferric inorganic salt, maintain the pH value of gained reaction system all the time, after complexation reaction, cool, obtain system M;

2) After the system M is centrifuged, the obtained supernatant is then subjected to crystallization, washing, freeze-drying, and dialysis for desalting to obtain the Agaricus blazei polysaccharide iron complex.

As a further limitation, the temperature of the complexation reaction is 60-100° C. and the time is 1-5 h.

As a further limitation, in step 1), the pH value of the reaction system is 6-10.

As a further limitation, the molecular weight cut-off of the dialysis desalination is 8000-14000;

In the crystallization process, the amount of the organic solvent used is 3 to 5 times the volume of the supernatant.

As another limitation, the ferric inorganic salt is ferric chloride;

When the ferric inorganic salt is added to the reaction solution and the precipitate is no longer dissolved, the addition is stopped.

As a further limitation, the catalyst is sodium alginate, trisodium citrate or sodium tartrate; the weight ratio of the Agaricus blazei polysaccharide to the catalyst is 1:0.5-3;

The alkaline solution is an aqueous sodium hydroxide solution.

The invention also provides the application of the above-mentioned Agaricus blazei polysaccharide iron complex as a medicine for supplementing iron.

The present invention also provides the application of the above Agaricus blazei polysaccharide iron complex as a medicine for treating ulcerative colitis.

Owing to adopting the above-mentioned technical scheme, compared with the prior art, the technical progress achieved in the present invention is:

The present invention adopts Agaricus blazei polysaccharide, an active substance in medicinal and edible homologous Agaricus blazei, as the sugar base, and ferric inorganic salt as the glycosyl base, and through complexation reaction, a stable Agaricus blazei polysaccharide iron complex is formed, and its dissolution property is low and high. The stability is good, the nutritional value is high, the preparation process is simple and stable, and a new type of iron supplement can be provided;

The Agaricus blazei polysaccharide-iron complex obtained by the invention has few side effects, stable compatibility, and iron content is higher than most iron salts, and the Agaricus blazei polysaccharide-iron complex is more easily absorbed by the body, can effectively supplement the iron element in the body, and improve the iron content. protein content, improve hematopoietic function;

The Agaricus blazei polysaccharide iron complex obtained by the invention contains FePS, which has good scavenging activity against hydroxyl radicals, superoxide anions and lipid peroxides; Colitis has a certain anti-inflammatory effect.

The Agaricus blazei polysaccharide iron complex of the present invention is suitable for use as an iron supplement for animals, and is also suitable for treating ulcerative colitis.

Description of drawings

Fig. 1 is the o-filroline standard curve of gained in the embodiment of the present invention 1;

Fig. 2 is the glucose standard curve obtained in the embodiment of the present invention 1;

Fig. 3 is the physical comparison diagram of Agaricus blazei polysaccharide and Agaricus blazei polysaccharide iron complex PHW1 in Example 8 of the present invention;

Fig. 4 is the infrared spectrogram of Agaricus blazei polysaccharide and Agaricus blazei polysaccharide iron complex PHW1 determined in Example 8 of the present invention;

Fig. 5 is the XPS full scan spectrum of Agaricus blazei polysaccharide and Agaricus blazei polysaccharide iron complex PHW1 determined in Example 8 of the present invention;

Fig. 6 is the XPS scanning pattern of Fe2p of measuring Agaricus blazei polysaccharide iron complex PHW1 in Example 8 of the present invention;

Fig. 7 is the XPS scanning pattern of Fe2p of Agaricus blazei polysaccharide measured in Example 8 of the present invention;

Fig. 8 is the high-resolution Cls spectrum of Agaricus blazei polysaccharide iron complex PHW1 determined in Example 8 of the present invention;

Fig. 9 is the high-resolution Cls spectrum of Agaricus blazei polysaccharide determined in Example 8 of the present invention;

Fig. 10 is the X-ray diffraction analysis chart of Agaricus blazei polysaccharide and Agaricus blazei polysaccharide iron complex PHW1 determined in Example 8 of the present invention;

11 is a graph showing the DPPH free radical scavenging ability of Agaricus blazei polysaccharide iron complex PHW1 measured in Example 10 of the present invention;

Figure 12 is a graph showing the in vitro iron release performance of Agaricus blazei polysaccharide iron complex PHW1 measured in Example 10 of the present invention.

Detailed ways

The present invention will be further described in detail below through specific embodiments, and it should be understood that the described embodiments are only used to explain the present invention, but do not limit the present invention.

Embodiment 1 A kind of preparation method of Agaricus blazei polysaccharide iron complex

The present embodiment prepares a kind of Agaricus blazei polysaccharide iron complex, including the following steps that are carried out in turn:

1) Dissolve 2kg Agaricus blazei polysaccharide (off-white, with a purity of 87.83%) and 1kg trisodium citrate in 50L distilled water at 80°C (pH value is 9), keep stirring at 80°C, and slowly dropwise add 3L to the concentration of 2mol/L ferric chloride aqueous solution and 0.10L sodium hydroxide aqueous solution with a mass concentration of 20% were used to maintain the pH value of the obtained reaction system at 9 all the time, and the dropwise addition time was controlled at 1h. During the dropwise addition, the ferric chloride solution was just dropped to form a precipitate, and then the precipitate gradually dissolved again. When the dropwise addition to the precipitate no longer dissolved in the reaction, it indicated that the complexing ability of the reaction system was saturated, and the dropwise addition of trichloride was stopped. The iron compound aqueous solution and the sodium hydroxide aqueous solution were continuously stirred at 80 °C for complexation reaction for 2 h, and cooled to room temperature to obtain system M1;

2) System M1 was centrifuged at 8000 r/min for 20 min, the supernatant was taken, 3 times the volume of absolute ethanol was added for crystallization, a reddish-brown precipitate was precipitated, and the resulting precipitate was washed 3 times with absolute ethanol after centrifugation at 8000 r/min for 5 min , after centrifugation at 8000r/min for 5min, the resulting precipitate was freeze-dried to obtain 1.8kg of Agaricus blazei polysaccharide-iron complex crude product;

1.8kg of Agaricus blazei polysaccharide iron complex was redissolved in 50L of distilled water, and the salt was removed by dialysis with running water in a bag. The molecular weight cutoff was 10,000, and 1.6kg of reddish-brown powder Agaricus blazei polysaccharide iron complex [namely ABMP-Fe(Ⅲ) was obtained. )complex], marked as PHW1, its iron content is 15.62%, the Agaricus blazei polysaccharide content is 67.67%, and the solubility per 100g of water at 25°C is 22.15g.

In addition to iron and Agaricus polysaccharides, Agaricus blazei polysaccharide iron complex may also contain a small amount of protein, nucleic acid, and ash components, and these substances will not cause a burden to the organism.

Among them, the iron content determination method is:

Determination of iron content by phenanthroline spectrophotometry: take nine 50mL volumetric flasks, accurately add standard iron use solution (10μg/mL) 0.00, 1.00, 2.00, 4.00, 6.00, 8.00, 10.00, 12.00, 14.00mL, respectively, Then add 1.0 mL of 10% hydroxylamine hydrochloride solution, 2 mL of 0.15% o-phenanthroline chromogenic solution and 5.0 mL of 10% sodium acetate solution, dilute to the mark with distilled water, shake well, leave for 15 min, and use the reagent solution as blank at 510 nm. The absorbance value was measured by an ultraviolet-visible spectrophotometer, taking the mass concentration of iron as the abscissa and the absorbance as the ordinate, see Figure 1, and fitting the regression equation, y=1.2685x-0.0907, R2=0.9972.

The solution of Agaricus blazei polysaccharide iron complex with a mass concentration of 200μg·mL ^-1 was precisely prepared. Accurately pipette 5 mL of Agaricus blazei polysaccharide iron complex solution into a 25 mL volumetric flask and add 1 mL of hydroxylamine hydrochloride with a concentration of 10%, 5.0 mL of NaAc with a concentration of 1.0 mol·L ^-1 , and 2.0 mL of a 0.15% concentration of phenanthroline solution. , add distilled water to volume, and react at room temperature for 15 min. Taking distilled water and reagent solution as blank, the absorbance value was measured at 510nm, and the iron content in the iron complex of Agaricus blazei polysaccharide iron was calculated according to the standard curve.

Agaricus blazei polysaccharide content was determined by the phenol-sulfuric acid method, specifically:

Glucose standard curve drawing: Accurately prepare 2mL of standard glucose solution with mass concentration of 0, 0.008, 0.012, 0.016, 0.020, 0.024, 0.028, 0.032, 0.040mg/mL in a 15mL centrifuge tube, carry out color reaction, and measure at 490nm The absorbance y was measured, and the measurement was repeated 3 times. Referring to Figure 2, taking y as the ordinate and the mass concentration x of the standard glucose solution as the abscissa, perform linear regression fitting, and draw a standard curve. The equation of the standard curve is y=15.36x+0.0686, and R ² =0.9982.

Determination of Agaricus blazei polysaccharide content: Precisely weigh 0.1 g of the dried Agaricus blazei polysaccharide iron complex, prepare 2 mL of a certain concentration solution in a centrifuge tube, add 1 mL of a 6% phenol solution, and then add 1 mL of phenol solution with a mass fraction of 6%. Add 5 mL of concentrated sulfur solution dropwise to the surface and mix well, place it in a 40°C water bath for 30 minutes, then place it in an ice-water bath for 5 minutes, and measure the absorbance value at the absorption wavelength of 490 nm with a UV-Vis spectrophotometer.

Agaricus blazei polysaccharide content (%)=CV/m100% in Agaricus blazei crude polysaccharide iron complex; V is the diluted volume mL of Agaricus blazei crude polysaccharide iron complex solution; m is the mass of Agaricus blazei crude polysaccharide iron complex.

Embodiment 2～7 Preparation method of Agaricus blazei polysaccharide iron complex

Embodiments 2 to 7 are respectively a kind of preparation method of Agaricus blazei polysaccharide iron complex, and their steps are basically the same as those of embodiment 1, and the difference is only in the amount of raw materials and process parameters. For details, see Table 1:

List of various process parameters in Table 1 Examples 2 to 7

The contents of other parts of Examples 2 to 7 are the same as those of Example 1.

Example 8 Determination of properties of Agaricus blazei polysaccharide iron complex

(1) Determination of physical and chemical properties

Referring to FIG. 3 , the raw material Agaricus blazei polysaccharide (purity 87.83%) used in the present invention is off-white powder, and the prepared Agaricus blazei polysaccharide iron complex is red-brown powder.

Stability test of Agaricus blazei polysaccharide iron complex aqueous solution: respectively take the Agaricus blazei polysaccharide iron complexes PHW1-PHW7 prepared in Examples 1-7 and dissolve them in water according to their maximum solubility to prepare 50mL Agaricus blazei polysaccharide iron complex aqueous solution S-PHW1 ~S-PHW7. Add 0.5g of potassium ferrocyanide to the aqueous solutions S-PHW1～S-PHW7 respectively, stir, and the aqueous solution does not turn red. Therefore, it is proved that after the Agaricus blazei polysaccharide iron complex prepared by the present invention is dissolved in water, the aqueous solution does not Containing free Fe ³⁺ , the glycosyl iron elements in Agaricus blazei polysaccharide iron complexes are complexed with glycosyl Agaricus polysaccharides to form stable complexes.

(2) Infrared spectroscopy

A small amount of the Agaricus blazei polysaccharide iron complexes PHW1-PHW7 prepared in Examples 1-7 and the raw material Agaricus blazei polysaccharide were taken separately, compressed by KBr tablet method, and scanned by infrared spectrum in the wavelength range of 4000-500 cm ^-1 .

The infrared spectrum of the Agaricus blazei polysaccharide iron complex PHW1 prepared in Example 1 and the raw material Agaricus blazei polysaccharide is shown in Figure 4. It can be seen that the Agaricus blazei polysaccharide iron complex is similar to the typical absorption peak of Agaricus blazei polysaccharide, indicating that it is a glycosyl Agaricus blazei Murray. After the polysaccharide was complexed with iron, the basic structure did not change, but the absorption peak of hydroxyl stretching vibration of Agaricus blazei polysaccharide iron complex PHW1 around 3400-3300 cm ^-1 was significantly wider than that of Agaricus blazei polysaccharide, and the strongest absorption peak was Red-shifted from 3318cm ^-1 to 3334cm ^-1 , which indicates that Agaricus blazei polysaccharide is a sugar group in the iron complex of Agaricus blazei polysaccharide PHW1, in which -OH and C=O participate in the complexation reaction; C=O stretches at 1612cm ^-1 The increase of the vibration intensity indicates that after the iron element is coordinated with the carbonyl group, the electron-withdrawing effect of the iron element increases the polarization degree of the carbonyl group, which in turn causes the C=O vibrational dipole moment to increase. Therefore, it can be known that the Agaricus blazei polysaccharide iron complex PHW1 has a structure similar to FeOOH.

Infrared spectrum scanning was performed on the Agaricus blazei polysaccharide iron complexes PHW2-PHW7 prepared in Examples 2-7 respectively, and the obtained infrared spectra were the same as the Agaricus blazei polysaccharide iron complexes PHW1 prepared in Example 1, and will not be repeated here.

3) X-ray analysis

31) X-ray photoelectron spectroscopy analysis

Take a small amount of the Agaricus blazei polysaccharide iron complex PHW1 prepared in Example 1 and the raw material Agaricus blazei polysaccharide respectively for X-ray photoelectron spectroscopy analysis, using a monochromatic Al-Kα X-ray source (hv=1486.6eV), the main chamfer The vacuum of Ber was kept at 3 × ^10-6 Pa above the N1 region during XPS data acquisition. General measurement scans (binding energy range: 0-1400eV; pass energy: 160eV) and high-resolution spectroscopy (pass energy: 40eV) .

The measurement results of the above experiments are shown in Figures 5 to 7. It can be seen that there are no characteristic peaks of iron in the full scan spectrum of Agaricus blazei polysaccharide and the XPS scan spectrum of Fe2p, indicating that Agaricus blazei polysaccharide does not contain iron; In the full scan spectrum of Agaricus blazei polysaccharide iron complex PHW1 and the XPS scan spectrum of Fe2p, the characteristic electron binding energy peak of iron appeared at 711.0eV, indicating that Agaricus blazei polysaccharide iron complex PHW1 contains iron, which can It was deduced that the iron element was complexed with Agaricus blazei polysaccharide, and the Agaricus blazei polysaccharide was successfully modified.

X-ray photoelectron spectroscopy analysis was carried out on the Agaricus blazei polysaccharide iron complexes PHW2-PHW7 prepared in Examples 2-7 respectively, and the obtained full scan spectrum and Fe2p XPS scan spectrum were similar to the Agaricus blazei polysaccharide iron complex PHW1 prepared in Example 1. , which will not be repeated here.

32) X-ray diffraction analysis

A small amount of the Agaricus blazei polysaccharide iron complex PHW1 prepared in Example 1 and the raw material Agaricus blazei polysaccharide were respectively taken for X-ray diffraction spectrum analysis, and the Ultima IV-X-ray diffractometer was used to determine Agaricus blazei polysaccharide and Agaricus blazei polysaccharide iron complexes. , the test conditions are: copper target, LynxExe array detector, 40kV, 40mA, scanning step size 0.02 degrees, scanning speed 17.7 seconds/step, and collecting 0°-90° (2θ) diffraction data at room temperature. The specific measurement results are shown in the table. 2:

Table 2 Binding energy and element ratio of Agaricus blazei polysaccharide and Agaricus blazei polysaccharide iron complex PHW1

Referring to Table 2 and Figures 8 and 9, it can be seen that the Agaricus blazei polysaccharide changes the surface chemical composition of Agaricus blazei polysaccharide and introduces more carbon elements after the introduction of iron and complexation to form the Agaricus blazei polysaccharide iron complex PHW1.

Referring to Figure 10, Agaricus blazei polysaccharide iron complex PHW1 has characteristic absorption peaks at 0.25nm and 0.33nm, which are characteristic absorption peaks of iron oxides, according to Coe E M, Bowen L H, Bereman R D, etc. in "Journal of lnorganic Biochemistry" (1995, 57:63-71) published "A study of an iron dextran complex by mossbauer spectroscopy and X-Ray diffraction", which can prove that Agaricus blazei polysaccharide iron complex PHW1 contains iron oxides, which further confirms the existence of similar iron dextran complexes in Agaricus blazei polysaccharide iron. Square β-FeOOH structure.

X-ray diffraction analysis was carried out on the Agaricus blazei polysaccharide iron complexes PHW2-PHW7 prepared in Examples 2-7 respectively, and the obtained diffraction data, high-resolution Cls spectrum and X-ray diffraction analysis were the same as the Agaricus blazei polysaccharide iron complexes prepared in Example 1. The complex PHW1 is similar and will not be repeated here.

Example 9 Anti-enteritis performance test of Agaricus blazei polysaccharide iron complex

40 SPF female SD rats were selected and randomly divided into 5 groups with 8 rats in each group, namely blank group, DSS model group and DSS-Fe1-DSS-Fe3 group. Among them, the DSS-Fe1 group was given 50 mg/kg per day. The dosage of Agaricus blazei polysaccharide iron complex PHW1 prepared in Example 1, the DSS-Fe2 group was administered daily at a dose of 100 mg/kg The Agaricus blazei polysaccharide iron complex PHW1 prepared in Example 1; DSS-Fe3 group daily The Agaricus blazei polysaccharide iron complex PHW1 prepared in Example 1 was administered by gavage at a dose of 200 mg/kg; the blank group and the DSS model group were intragastrically administered with an equal volume of normal saline; it should be noted that the DSS-Fe1 to DSS-Fe3 groups were intragastrically administered The stomach was the same volume of water solution of Agaricus blazei polysaccharide iron complex with different concentrations; continuous gavage was given for 14 days, during which sufficient diet and drinking water were given. From the 15th day, the gavage was stopped. Except for the blank group, the drinking water was changed to an aqueous solution containing 4.5% dextran sulfate sodium DSS (DSS is used to induce ulcerative colitis in mice), and water was freely drank, and other conditions remained unchanged. Continuous feeding for 7d.

After the experiment, blood was collected from the retro-orbital venous plexus of the rats in different groups, and the serum was collected by standing and centrifuged, and stored at -80°C for future use. Rats were sacrificed, and some tissue fluids from fresh colonic segments were collected and frozen in liquid nitrogen.

(1) The effect of Agaricus blazei polysaccharide iron complex on the secretion of cytokines in rat colon tissue

Serum was taken separately to detect inflammatory cytokines (IL-1β, IL-6, TNF-α) in serum by ELISA. The specific detection method was as follows: at room temperature, the serum to be tested was diluted 1:1, and then coated with Add 100 μL of serum to be tested to the plate, incubate at 30°C for 100 min, wash 3 times, add 100 μL of biotin antibody, incubate for 60 min, wash 3 times, incubate 100 μL of enzyme conjugate for 25 min, wash 3 times, incubate 100 μL of chromogenic substrate for 20 min, Add 50uL of stop solution, and immediately measure the OD value with a microplate reader at a wavelength of 450nm. The secretion levels of IL-1β, IL-6, and TNF-α in the serum of the rats in each group were detected, the detection results of each group were counted, and the mean value was calculated. The specific results are shown in Table 4.

Table 3 List of IL-1β, IL-6, TNF-α secretion levels

group n IL-1β (pg/mL) IL-6 (pg/mL) TNF-α (pg/mL) DSS-Fe1 group 8 38.32±2.61ab 160.04±21.77a 35.40±4.48ab DSS-Fe2 group 8 35.62±4.10bc 148.99±9.99ab 33.73±4.40ab DSS-Fe3 group 8 34.22±2.18bc 140.55±12.85b 27.21±3.90c DSS Model Group 8 41.37±6.28a 172.72±25.92a 36.28±5.01a blank group 8 13.65±2.07d 77.43±13.73c 16.16±2.96d

Note: The data in the table are represented by the mean ± standard error; the same shoulder letters in the same row indicate insignificant (P>0.05); different ones indicate significant difference (P<0.05).

It can be seen from Table 3 that, compared with the blank group, after the DSS-induced ulcerative colitis experiment in mice, the levels of IL-1β, IL-6, and TNF-α inflammatory factors in the DSS model group and DSS-Fe1-DSS-Fe3 groups were increased. The expression levels were significantly increased. Among them, the expression levels of IL-1β, IL-6, and TNF-α in the DSS model group were significantly higher than those in the DSS-Fe1-DSS-Fe3 groups; and the DSS-Fe1 group was compared with the DSS-Fe2 and DSS-Fe3 groups. , with the increase of the dose of Agaricus blazei polysaccharide iron complex, the expressions of IL-1β, IL-6 and TNF-α were significantly decreased. Therefore, the concentrations of L-1β, IL-6 and TNF-α inflammatory factors in the model group were significantly higher than those in the Agaricus blazei polysaccharide iron intervention group (p<0.05), and decreased with the increase in the concentration of Agaricus blazei polysaccharide iron . The above experiments prove that the Agaricus blazei polysaccharide iron complex of the present invention has inhibitory effect on the secretion of IL-1β, IL-6 and TNF-α in rats with colitis, and the inhibitory effect is positively correlated with the concentration. The Agaricus blazei polysaccharide complexed with iron can inhibit and reduce the inflammation of colitis.

(2) Effects of Agaricus blazei polysaccharide-iron complex on the mRNA level of colonic tight junction protein

PremixEx Taq^TM说明书进行：反应流程为95℃预变性10min，95℃变性10s，60℃退火20s，72℃延伸15s，40个循环。基因的相对表达水平以2^-△△Ct进行分析，其中△Ct＝目的基因Ct-内参基因Ct；△△Ct＝△Ct DSS组-△Ct空白组，注：DSS组为DSS模型组及DSS-Fe1～DSS-Fe3组中的任一组。以GAPDH为内参基因，按2-△dct法比较大鼠紧密连接蛋白ZO-1、occludin、claudin1 mRNA的相对基因表达量，统计并计算均值，具体检测结果见表4：Refer to the instruction manual of EASYspinPlus Tissue/Cell RNA Kit to extract total RNA from some tissue fluids of colon segments respectively, and reverse it into cDNA using HiFiScript cDNA First Strand Synthesis Kit. GAPDH is used as the internal reference gene, and real-time fluorescence quantitative PCR method is used to detect gene expression. level differences. Real-time fluorescence quantitative PCR operation steps according to

PremixEx Taq ^TM instructions were carried out: the reaction process was pre-denaturation at 95°C for 10 min, denaturation at 95°C for 10s, annealing at 60°C for 20s, extension at 72°C for 15s, 40 cycles. The relative expression levels of genes were analyzed by 2- ^△△Ct , where △Ct=target gene Ct-internal reference gene Ct;△△Ct=△Ct DSS group-△Ct blank group, Note: DSS group is DSS model group and DSS - any of the Fe1 to DSS-Fe3 groups. Using GAPDH as the internal reference gene, the relative gene expression levels of rat tight junction proteins ZO-1, occludin, and claudin1 mRNA were compared according to the 2-△dct method, and the mean value was calculated and calculated. The specific test results are shown in Table 4:

Table 4 List of relative gene expression averages of ZO-1, occludin, and claudin1 mRNA

group n ZO-1 occludin claudin1 DSS-Fe1 group 8 0.105±0.016c 0.243±0.033c 0.134±0.016b DSS-Fe2 group 8 0.149±0.021bc 0.306±0.032bc 0.177±0.029b DSS-Fe3 group 8 0.158±0.007b 0.349±0.045b 0.200±0.016b DSS Model Group 8 0.101±0.015c 0.265±0.023c 0.154±0.035b blank group 8 1a 1a 1a

Note: The data in the table are expressed as the mean ± standard deviation; the same shoulders in the same line indicate insignificant (P>0.05); different ones indicate significant difference (P<0.05).

It can be seen from Table 4 that with GAPDH as the internal reference, the relative gene expression of tight junction protein ZO-1, occludin, and claudin1 mRNA in the two groups of rats was compared according to the 2-Δdct method. The results showed that the rats in each group were drinking water containing DSS freely. Compared with the blank group, the mRNA expressions of zo-1, Occludin and Claudin-1 in the colon were significantly decreased (p < 0.05), indicating that acute colitis in rats will cause the decrease of the expression of claudin-related mRNA in the colon tissue. ; Compared with the DSS model group, the zo-1 and occludin mRNA expressions in the DSS-Fe1-DSS-Fe3 group were significantly higher than those in the DSS model group (p<0.05), and increased with the dose of Agaricus blazei polysaccharide-iron complex. It proves that Agaricus blazei polysaccharide iron complex has a certain improvement effect on the decrease of tight junction protein expression in DSS-induced colitis; claudin 1: High dose Agaricus blazei polysaccharide iron complex can significantly increase the expression of claudin1 mRNA (p <0.05).

To sum up, the Agaricus blazei polysaccharide iron complex of the present invention can down-regulate pro-inflammatory factors and increase the expression of tight junction proteins, thereby reducing the intestinal mucosal permeability in rats with colitis, and achieving a protective effect on the intestinal mucosal barrier. Therefore, The Agaricus blazei polysaccharide iron complex of the invention has certain anti-inflammatory effect on ulcerative colitis.

Example 10 Test of Agaricus blazei polysaccharide iron complex on free radical scavenging ability and in vitro iron release performance

1) Agaricus blazei polysaccharide iron scavenging ability of DHHP free radicals

According to "Purification, characterization and antioxidant activity of polysaccharides from Flammulina velutipes residue" published in "Carbohydrate Polymers" (2016, 145:71-77) by Liu Y, Zhang B, Ibrahim S A, etc., Agaricus blazei polysaccharide iron complex PHW1 was dissolved in Deionized water was passed through a 0.45 μm filter membrane, and the obtained solution was 1.5 mL and 1.5 mL of an ethanol solution of DPPH with a concentration of 25 mg/L, mixed by vortexing, and incubated at 25°C for 30 min (the blank solution was replaced by deionized water. Matsutake polysaccharide iron complex, the positive solution replaces Agaricus polysaccharide iron complex with vitamin C), and the solution absorbance is measured at 517 nm.

Among them, DPPH free radical scavenging rate (%)=(A0-A1+A2)/A0×100, where A0, A1 and A2 are the initial and final absorbance of blank solution and Agaricus blazei polysaccharide iron complex solution, respectively.

Referring to Figure 11, it can be seen that Agaricus blazei polysaccharide iron has a certain scavenging effect on DPPH free radicals, and the scavenging rate increases with the increase of the concentration.

The DHHP free radical scavenging ability was measured for the Agaricus blazei polysaccharide iron complexes PHW2-PHW7 prepared in Examples 2-7 respectively, and the obtained scavenging rates were similar to the Agaricus blazei polysaccharide iron complexes PHW1 prepared in Example 1, and will not be repeated here.

2) Iron release properties of Agaricus blazei polysaccharide complexes in vitro

According to "Synthesis and in vitro behavior of iron-crosslinked N-methyl and N-benzyl hydroxamated derivatives of alginic" published by Al Khatib HS, Taha MO, Aiedeh KM, etc. in "European Polymer Journal" (2006, 42: 2464-2474), respectively acid as controlled release carriers", "Production kinetics of angiotensin-Iconverting enzyme inhibitory peptides from bonitomeat in artificial gastricjuice" and Dong X in Process Biochemistry (2006, 41:505-511) by Fida H, Megumi K, Yoichi K et al. , Fang C, Ming Z, etc. in "Materials Letters" (2007, 61:3154-3158) "New application of hierarchical zeolite in life science: Fast trappingnitrosamines inartificial gastric juice by alkaline-tailored HZSM-5", 1g Ji Matsutake polysaccharide iron complex PHW1 was placed in 1000mL artificial gastric juice (2.0g of NaCl, 3.2g of pepsin, 80mL of 1mol/L HCl deionized water diluted to 1000mL, pH=2.0) for 2h, and then changed its pH to make it The pH of the artificial intestinal juice was changed to 8 (add 5.0 mL of bile salt solution with a concentration of 25.0 g/L, 1.0 mL of pancreatic extract with a concentration of 4.0 g/L, and 1000 mL of NaHCO ₃ with a concentration of 0.1 mol/L to adjust the pH to 8) , placed for 3 hours, the solution temperature was always controlled at 37 °C, the first hour was sampled every 20 minutes, and the samples were taken every 30 minutes thereafter. The iron release rate is calculated according to the following formula: iron release rate (%)=F ₁ /F ₀ ×100, where F ₁ is the total amount of iron in the solution, and F ₀ is the iron content in the Agaricus blazei polysaccharide iron complex.

Referring to Figure 12, it can be seen that the iron release test was carried out in artificial gastric juice (pH=2.0) for 2 hours and artificial intestinal juice (pH=8.0) for 3 hours. Agaricus blazei polysaccharide iron complex has good drug release activity. It can reach 79.5% after 1h and 89.33% after 3h. The above experiments prove that Agaricus blazei polysaccharide iron complex has good iron activity.

The in vitro iron release properties of the Agaricus blazei polysaccharide iron complexes PHW2 to PHW7 prepared in Examples 2 to 7 were measured respectively, and the obtained iron ion release rates were similar to the Agaricus blazei polysaccharide iron complexes PHW1 prepared in Example 1, and will not be repeated here.

Claims (10)

1. a Agaricus blazei polysaccharide iron complex, is characterized in that, The Agaricus blazei polysaccharide iron complex is a complex of Agaricus blazei polysaccharide and iron element formed by using Agaricus blazei polysaccharide as a sugar base and ferric iron as a glycosyl base; The content of iron element in the Agaricus blazei polysaccharide iron complex is 9.44-16.54%, and the content of Agaricus blazei polysaccharide is 61.67-68.55%. 2. a kind of preparation method of Agaricus blazei polysaccharide iron complex according to claim 1, is characterized in that, it is to take Agaricus blazei polysaccharide and trivalent inorganic salt through complexation reaction to obtain the Agaricus blazei polysaccharide iron complex . 3. the preparation method of Agaricus blazei polysaccharide iron complex according to claim 2, is characterized in that, this preparation method comprises the following steps that carry out successively: 1) Dissolve Agaricus blazei polysaccharide and catalyst in water, add aqueous solution and alkali solution of ferric inorganic salt dropwise, maintain the pH value of the obtained reaction system all the time, and obtain system M after complexation reaction; 2) After the system M is centrifuged, the supernatant obtained is then subjected to crystallization, washing, drying, and dialysis to remove salts to obtain the Agaricus blazei polysaccharide iron complex. 4. The preparation method of Agaricus blazei polysaccharide iron complex according to claim 3, wherein the temperature of the complexation reaction is 60-100°C and the time is 1-5h. 5 . The preparation method of Agaricus blazei polysaccharide iron complex according to claim 3 , wherein, in step 1), the pH value of the reaction system is 6-10. 6 . 6. the preparation method of Agaricus blazei polysaccharide iron complex according to claim 3, is characterized in that, the molecular weight cut-off of described dialysis desalination is 8000～14000. 7. The preparation method of Agaricus blazei polysaccharide iron complex according to any one of claims 2-7, wherein the ferric inorganic salt is ferric chloride. 8. The preparation method of Agaricus blazei polysaccharide iron complex according to any one of claims 3-7, wherein, The catalyst is sodium alginate, trisodium citrate or sodium tartrate; the weight ratio of the Agaricus blazei polysaccharide to the catalyst is 1:0.5 to 3; The alkaline solution is an aqueous sodium hydroxide solution. 9. the application of the Agaricus blazei polysaccharide iron complex of claim 1 as the medicine of iron supplement. 10. The application of the Agaricus blazei polysaccharide iron complex according to claim 1 as a medicine for treating ulcerative colitis.

Images