Biological metabolism regulator based on radix paeoniae alba polysaccharide iron as well as preparation method and application of biological metabolism regulator

Abstract

The invention relates to a biological metabolism regulator based on radix paeoniae alba polysaccharide iron and a preparation method and application thereof.The preparation method comprises the following steps that radix paeoniae alba polysaccharide and trisodium citrate are completely dissolved in pure water, a ferric trichloride solution is slowly added under magnetic stirring, a sodium hydroxide solution is dropwise added at the same time, after the reaction is completed, centrifugation is conducted, and supernatant is obtained; and adding absolute ethyl alcohol to separate out the radix paeoniae alba polyferose. Centrifuging to take precipitate, repeating for three times, and dissolving with pure water; and dialyzing the solution with deionized water, then adding absolute ethyl alcohol for precipitation and centrifugation, repeating for three times, and freeze-drying to obtain the radix paeoniae alba polyferose product. The product can stably reach the intestinal tract in a targeted mode, remove ROS in the intestinal tract and regulate glycometabolism of macrophages, so that polarization typing of the macrophages is changed, the anti-inflammatory effect is achieved, and new possibility is provided for treatment of the inflammatory bowel disease. Animal experiments prove that the radix paeoniae alba polyferose enteric capsule has a better and more stable comprehensive treatment effect on mice with inflammatory bowel diseases than a radix paeoniae alba polyferose solution.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a biological metabolism regulator based on white peony polysaccharide iron, and a preparation method and application thereof. Background Art

[0002] Inflammatory bowel disease (IBD), which includes ulcerative colitis (UC) and Crohn's disease (CD), is a chronic, nonspecific inflammatory disease of the gastrointestinal tract characterized by recurrent episodes. The development and application of targeted immune therapy and the evolution of therapeutic targets have revolutionized the clinical treatment of IBD. However, current treatment focuses primarily on inducing and maintaining clinical remission, and a complete cure is not yet possible. 47% of patients still experience chronic, active inflammation, and 40% require surgical intervention, severely impacting their quality of life and placing a heavy burden on society. The pathogenesis of IBD remains incompletely understood. Existing literature suggests that its etiology may be closely related to multiple factors, including reactive oxygen species (ROS), intestinal microbiota, intestinal mucosal barrier function, innate and adaptive immune regulation, and external environmental factors. Therefore, in-depth research into the pathogenesis of IBD and the identification of new therapeutic targets to reduce the burden of healthcare on society are urgent.

[0003] Intestinal inflammatory reactions can lead to intestinal dysfunction, which in turn affects iron absorption. Intestinal inflammation destroys the structure and function of the intestinal lining, especially the absorption area of ​​the small intestine, which can lead to incomplete iron absorption and subsequently the occurrence of iron deficiency anemia. In addition, IBD patients usually receive some immunosuppressive treatments (such as aminosalicylic acid drugs, immunosuppressants, intestinal biological agents, etc.). These drugs may cause adverse gastrointestinal reactions, such as gastrointestinal irritation, loss of appetite, etc., further affecting iron intake and absorption.

[0004] White peony polysaccharide is mainly used to nourish blood and soften the liver, relieve pain. However, polysaccharides usually have a large molecular weight and complex structure, which makes their absorption in the body more difficult. By forming complexes with metal ions, their biocompatibility, stability and targeting can be improved, thereby increasing their bioavailability in the body. In addition, certain metal ions (such as iron, zinc, copper, etc.) themselves have certain biological activities and can regulate immune responses or anti-inflammatory responses through complexation with polysaccharides. Summary of the Invention

[0005] Technical problems to be solved: In response to the problems existing in the prior art such as poor efficacy, low biocompatibility, poor stability and targeting of some drugs, which aggravate intestinal inflammatory damage and lead to the occurrence of iron deficiency anemia, the present invention proposes a biological metabolic regulator based on white peony polysaccharide iron, and its preparation method and application. The prepared biological metabolic regulator based on white peony polysaccharide iron can stably target and reach the intestine, eliminate ROS in the intestine, regulate the sugar metabolism of macrophages, thereby changing their polarization typing, and then exert anti-inflammatory effects and repair intestinal damage, providing new possibilities for the treatment of inflammatory bowel disease.

[0006] Technical solution: The first object of the present invention is to provide a method for preparing a biological metabolic regulator based on white peony polysaccharide iron, the steps of which are as follows:

[0007] Step 1, dissolving white peony polysaccharide (PP) and trisodium citrate in pure water, stirring to obtain solution I, dissolving ferric chloride in pure water to prepare solution II, dissolving sodium hydroxide in pure water to prepare solution III, the mass ratio of white peony polysaccharide and trisodium citrate in solution I is (2-4): 1, the concentration of ferric chloride in solution II is 2-4 mol / L, and the concentration of sodium hydroxide in solution III is 10-20wt%;

[0008] Step 2, solution II is added dropwise to solution I with magnetic stirring, and solution III is continuously added during the period to maintain the pH between 8.5-9.5. When red insoluble matter appears in the reaction system, the dropwise addition of solution II is stopped. At this time, the solution is solution IV, and solution IV is placed on a rotary heating table and continued to stir and react. After the reaction is completed, solution IV is collected, centrifuged and the supernatant is taken. 3-10 times the volume of anhydrous ethanol is added to the supernatant to precipitate the crude white peony polysaccharide iron product. The added amount volume ratio of solution I to solution II is 10mL: (10-100) μL, the stirring reaction condition is 60-90 ° C, and the reaction time is 0.5-2h; the centrifugal speed is 5000-10000rpm, and the centrifugation time is 5-15min;

[0009] Step 3: dissolving the crude white peony polysaccharide iron product in pure water to prepare solution V, placing solution V into a dialysis bag and dialyzing with deionized water. When dialyzing solution V with deionized water, MW = 3000-4500, the dialysis time is 12-48h;

[0010] Step 4: After dialysis is completed, add 3-10 times the volume of anhydrous ethanol to solution V to precipitate the white peony polysaccharide iron product, centrifuge and obtain the precipitate. Repeat the above operation three times, then freeze-dry, grind and collect to obtain white peony polysaccharide iron (PPFeCs) powder, wherein the centrifugal speed is 5000-10000rpm, and the centrifugation time is 5-15min.

[0011] Furthermore, in the step 1, the mass ratio of white peony polysaccharide and trisodium citrate in solution I is 4:1, the concentration of ferric chloride in solution II is 3 mol / L, and the concentration of sodium hydroxide in solution III is 10 wt%.

[0012] Furthermore, the stirring reaction conditions in the step 2 are 70° C. and the reaction time is 1 hour; the centrifugal speed is 8000 rpm and the centrifugal time is 5 minutes.

[0013] Furthermore, in step three, MW=4500 and the dialysis time is 24 hours.

[0014] Furthermore, the centrifugal speed in step 4 is 8000 rpm and the centrifugal time is 5 min.

[0015] Furthermore, the freezing step in step 4 is as follows: pre-freeze the dialyzed white peony polysaccharide iron product at -80°C for 1-5 days, and then perform vacuum freeze-drying for 1-2 days.

[0016] The second object of the present invention is to provide a biological metabolism regulator based on white peony polysaccharide iron prepared based on the above method.

[0017] Preferably, the polysaccharide content in the biological metabolism regulator based on white peony polysaccharide iron is 83.11±1.234%, and the iron content is 16.89±0.271%.

[0018] The third object of the present invention is to provide the application of the above-mentioned biological metabolism regulator based on white peony polysaccharide iron in the preparation of a medicine for treating inflammatory bowel disease. The medicine for treating inflammatory bowel disease is anti-inflammatory, antioxidant and biological metabolism regulator, and the biological metabolism regulator based on white peony polysaccharide iron can be by regulating the sugar metabolism of macrophages, thereby changing its polarization typing, and then playing an anti-inflammatory effect. The biological metabolism regulator based on white peony polysaccharide iron can increase the content of intestinal tight junction proteins (ZO-1 and Occludin) so as to complete repairing intestinal damage, and by regulating the expression of factors TNF-α, IL-1β and IL-10, play an anti-inflammatory effect.

[0019] Preferably, the inflammatory bowel disease drug is a free radical scavenging drug, and the dosage of the biological metabolic regulator based on white peony polysaccharide iron for scavenging free radicals is 10-2000 μg / mL.

[0020] Furthermore, the dosage of the biological metabolism regulator based on white peony polysaccharide iron for scavenging free radicals is 20-1000 μg / mL.

[0021] As an example, the free radical scavenging effect is embodied as superoxide anion (O2 ·-), hydrogen peroxide (H2O2), hydroxyl radical (·OH) and 1,1-diphenyl-2-trinitrophenylhydrazine (DPPH·) contents.

[0022] Preferably, the inflammatory bowel disease drug is a drug for treating DSS-induced inflammatory bowel disease in C57BL / 6 mice.

[0023] Furthermore, the therapeutic effect of the biological metabolic regulator based on white peony polysaccharide iron is reflected in the recovery of the colon length, disease activity index (DAI) and weight change trend of each group of mice, as well as the callback of hematological indicators and the restoration of red blood cell morphology in iron deficiency anemia, a complication of DSS-induced inflammatory bowel disease C57BL / 6 mice.

[0024] Preferably, the biological metabolic regulator based on white peony polysaccharide iron is administered by oral administration of solution or oral administration of enteric-coated capsules. When the administration method is oral administration of solution, the effective dose is 10-40 mg / kg; when the administration method is oral administration of enteric-coated capsules, the effective dose is 2-20 mg / kg.

[0025] Furthermore, experiments have shown that the enteric-coated capsule gavage method is better than the solution gavage method. This is because when the solution is gavage administered, most of the drug is destroyed by gastric juice, resulting in low drug utilization.

[0026] The fourth object of the present invention is to provide a drug for treating inflammatory bowel disease, which comprises the above-mentioned biological metabolism regulator based on white peony polysaccharide iron and one or more pharmaceutically or food-acceptable excipients.

[0027] Beneficial effects:

[0028] (1) The white peony polysaccharide proposed in the present invention is an active ingredient extracted from the dried roots of the Ranunculaceae plant Paeonia lactiflora Pall., and has biological activities such as anti-inflammatory and antioxidant properties. Iron ions are a bioactive component and are not only an important component of hemoglobin, but also capable of transporting oxygen to various parts of the body and maintaining normal respiratory function. Iron ions are also involved in various metabolic processes in the body. In addition, the polysaccharide-metal complex exhibits improved biocompatibility, stability, and targeting, thereby improving its bioavailability in the body.

[0029] (2) The white peony polysaccharide iron prepared by the present invention has an iron content of up to 16.89±0.271%, which can effectively improve various body indicators of iron deficiency anemia, a complication of inflammatory bowel disease, including: callback of hematological indicators and recovery of red blood cell morphology.

[0030] (3) Experiments have shown that the biological metabolic regulator based on white peony polysaccharide iron provided by the present invention has a good therapeutic effect on the DSS-induced inflammatory bowel disease C57BL / 6 mouse model, and the mouse colon length, disease activity index (DAI) and body weight are restored; and the white peony polysaccharide iron provided by the present invention has a good therapeutic effect on the complication iron deficiency anemia of the DSS-induced inflammatory bowel disease C57BL / 6 mouse model, and can restore hematological indicators and red blood cell morphology. The biological metabolic regulator based on white peony polysaccharide iron provided by the present invention can change the sugar metabolism mode of macrophages, thereby changing their polarization typing, and then exerting an anti-inflammatory effect. By increasing the expression of intestinal tight junction proteins (ZO-1 and Occludin) in the DSS-induced inflammatory bowel disease C57BL / 6 mouse model, it can also regulate the expression of factors TNF-α, IL-1β and IL-10.

[0031] (4) Experiments have shown that the biological metabolic regulator based on white peony polysaccharide iron provided by the present invention can protect white peony polysaccharide iron from being destroyed by gastric acid by administering it through enteric-coated capsules, thereby improving the utilization rate and efficacy of the drug and reducing the amount of drug used. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the specific embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0033] Figure 1 This is a TEM image of the biological metabolism regulator based on white peony polysaccharide iron prepared in Example 1.

[0034] Figure 2 This is a biological metabolic regulator based on white peony polysaccharide iron and a particle size distribution diagram of white peony polysaccharide prepared in Example 1 (the illustration is the Tyndall phenomenon of PPFeCs). In the legend, PP is white peony polysaccharide, and PPFeCs is the biological metabolic regulator based on white peony polysaccharide iron prepared in Example 1.

[0035] Figure 3 This is an infrared spectrum of the biological metabolism regulator based on white peony polysaccharide iron and white peony polysaccharide prepared in Example 1.

[0036] Figure 4 These are the XRD spectra of the biological metabolism regulator based on white peony polysaccharide iron, white peony polysaccharide and ferric chloride prepared in Example 1.

[0037] Figure 5This is the XPS graph of the biological metabolism regulator based on white peony polysaccharide iron and white peony polysaccharide prepared in Example 1, where (a) is the overall spectrum and (b) is the high-resolution Fe2p peak.

[0038] Figure 6 The free radical scavenging performance comparison diagram of the biological metabolic regulator based on white peony polysaccharide iron and white peony polysaccharide prepared in Example 1, in which (a) is the superoxide anion (O2 ·- ), (b) is hydrogen peroxide (H2O2), (c) is hydroxyl radical (·OH), and (d) is 1,1-diphenyl-2-trinitrophenylhydrazine (DPPH·).

[0039] Figure 7 The figures show the therapeutic effects of the white peony polysaccharide iron-based biological metabolic regulator prepared in Example 1, white peony polysaccharide, 5-aminosalicylic acid, and ferric chloride on the DSS-induced inflammatory bowel disease C57BL / 6 mouse model by oral gavage. In the figure, (a) is the length and quantification of the colon of each group of mice, (b) is the disease activity index (DAI) of each group of mice, and (c) is the weight change trend of each group of mice.

[0040] Figure 8 The biological metabolic regulator based on white peony polysaccharide iron prepared in Example 1, white peony polysaccharide, 5-aminosalicylic acid and ferric chloride were gavage-treated in the DSS-induced inflammatory bowel disease C57BL / 6 mouse model. This figure is an H&E image of the colon of each group of mice.

[0041] Figure 9 The biological metabolic regulator based on white peony polysaccharide iron prepared in Example 1 was used for the treatment of iron deficiency anemia, a complication of DSS-induced inflammatory bowel disease C57BL / 6 mouse model, by oral gavage. The figure shows the blood routine test and Wright's staining of blood smears of mice in the Control group, DSS group, and DSS+PPFeCs group, where (a) is the blood routine test and (b) is the Wright's staining of blood smears.

[0042] Figure 10 The biological metabolic regulator based on white peony polysaccharide iron prepared in Example 1 regulated the glucose metabolism and macrophage typing of the DSS-induced inflammatory bowel disease C57BL / 6 mouse model by gavage. This figure shows the expression levels of 6-phosphofructo-2-kinase 2 (PFKFB2), 6-phosphofructo-2-kinase 3 (PFKEB3), the expression levels of M1 pro-inflammatory macrophage marker CD86 and M2 anti-inflammatory macrophage marker CD206 in the colon of mice in the Control group, DSS group and DSS+PPFeCs group.

[0043] Figure 11The biological metabolic regulator based on white peony polysaccharide iron prepared in Example 1 was used for the treatment of DSS-induced inflammatory bowel disease C57BL / 6 mouse model by oral gavage. This figure is an immunofluorescence image of colon tight junction proteins (ZO-1 and Occludin) in mice in the Control group, DSS group, and DSS+PPFeCs group, where (a) is ZO-1 and (b) is Occludin.

[0044] Figure 12 These are comparative graphs of the efficacy and release of the enteric-coated capsules and the solution-based biological metabolic regulator iron-based white peony polysaccharide prepared in Example 1 for the treatment of DSS-induced inflammatory bowel disease in a C57BL / 6 mouse model. In the figure, (a) is the release graph of the solution-based oral administration, (b) is the release graph of the enteric-coated capsules-based oral administration, (c) is the colon length comparison graph, (d) is the colon length quantification graph, (e) is the disease activity index (DAI) of each group of mice, and (f) is the weight change trend graph of each group of mice. DETAILED DESCRIPTION

[0045] The present invention will be further described below with reference to the accompanying drawings and specific embodiments.

[0046] Unless otherwise specified, the raw materials used in the examples of this specification are all from common commercially available products.

[0047] Example 1

[0048] This embodiment provides a method for preparing a biological metabolism regulator based on white peony polysaccharide iron, and the specific steps are as follows:

[0049] (1) 100 mg of white peony polysaccharide and 25 mg of trisodium citrate (molecular weight 258.069) were dissolved in 10 mL of pure water to prepare solution I;

[0050] (2) Dissolve 0.4875 g of ferric chloride in 1 mL of pure water to prepare solution II;

[0051] (3) Dissolve 1 g of sodium hydroxide in 10 mL of pure water to prepare solution III;

[0052] (4) Add 30 μL of solution II dropwise to solution I and stir. During this time, add solution III continuously to ensure that the pH of the solution is between 8.5 and 9.5. Stop adding dropwise when red insoluble matter appears in the reaction system to prepare solution IV.

[0053] (5) Stir solution IV at 70°C for 1 h;

[0054] (6) After the reaction is completed, centrifuge at 8000 rpm for 5 min, take the supernatant, and add ten times the volume of anhydrous ethanol to solution IV to precipitate the crude product of white peony polysaccharide iron. The alcohol precipitation time is 24 h;

[0055] (7) Dissolving the crude white peony polysaccharide iron product in pure water to prepare solution V;

[0056] (8) Solution V was placed in a dialysis bag and dialyzed against deionized water for 24 h (MW = 4500);

[0057] (9) After dialysis is completed, add ten times the volume of anhydrous ethanol to solution V to precipitate the white peony polysaccharide iron product. The alcohol precipitation time is 24 hours. The above operation is repeated three times. Centrifuge at 8000 rpm for 5 minutes and collect the precipitate.

[0058] (10) The PPFeCs was then freeze-dried, ground, and collected to obtain the PPFeCs powder. The freezing steps were as follows: the dialyzed PPFeCs was pre-frozen at -80°C for 5 days and then freeze-dried in a vacuum for 2 days. The prepared sample was subjected to inductively coupled plasma (ICP) analysis, and the iron content was detected to be 16.89±0.271%.

[0059] The prepared white peony polysaccharide iron powder was tested, and the results are shown in Figure 1-3 . Figure 1 It can be clearly seen from the TEM image that the size of white peony polysaccharide iron is about 200nm. Figure 2 It can be seen that the particle size of white peony polysaccharide (PP) becomes smaller after combining with iron ions. Figure 3 The FT-IR spectrum of white peony polysaccharide is between 3400 and 3500 cm -1 There are typical free hydroxyl peaks on the left and right, but in white peony polysaccharide iron, the peak disappears because the free hydroxyl groups of white peony polysaccharide coordinate with iron ions.

[0060] Figure 4 The XRD spectra of the biological metabolism regulator based on white peony polysaccharide iron, white peony polysaccharide and ferric chloride prepared in Example 1 show that PP shows a broad peak at about 20°, indicating that it is amorphous. 3+ In addition, it was observed that PPFeCs did not show the characteristic peak corresponding to FeCl3, which further confirmed that all Fe 3+ All are complexed with polysaccharides.

[0061] Figure 5 The XPS diagram of the biological metabolism regulator based on white peony polysaccharide iron and white peony polysaccharide prepared in Example 1, (a) is the total spectrum, (b) is the high-resolution Fe2p peak. The initial PP is at 3300 cm -1 、2983cm -1 、1715cm -1 and 1034cm- 1The peaks are significant, corresponding to ν(–OH), ν(–CH), ν(–C=O), and ν(–C–O) vibrations. After complexation with iron ions, the ν(–OH) peak is observed to weaken. This change may be due to the Fe 3+ The XPS spectrum of PPFeCs showed that C (284.6eV), O (532.1eV) and Fe (711.4eV) elements were present in PPFeCs, with atomic percentages of 39.08%, 49.11% and 8.11%, respectively. It is worth noting that no Fe atoms were detected in PP alone. 3+ This confirms that the Fe 3+ The signal originates from metal-polysaccharide coordination. The Fe 2p peak can be deconvoluted into three different iron species, corresponding to octahedral Fe 2+ , octahedral Fe 3+ and tetrahedral Fe 3+ This suggests the existence of a coordinated interaction in PPFeC.

[0062] Example 2

[0063] This embodiment provides a method for preparing a biological metabolism regulator based on white peony polysaccharide iron, and the specific steps are as follows:

[0064] Step 1, dissolving white peony polysaccharide and trisodium citrate in pure water, stirring evenly to prepare solution I, dissolving ferric chloride in pure water to prepare solution II, dissolving sodium hydroxide in pure water to prepare solution III, the mass ratio of white peony polysaccharide and trisodium citrate in solution I is 2:1, the concentration of ferric chloride in solution II is 2 mol / L, and the concentration of sodium hydroxide in solution III is 10 wt%;

[0065] Step 2: Solution II is added dropwise to solution I with magnetic stirring, and solution III is continuously added during the period to keep the pH between 8.5. When red insoluble matter appears in the reaction system, the dropwise addition of solution II is stopped. At this time, the solution is solution IV, and solution IV is placed on a rotary heating table and continued to stir and react. After the reaction is completed, solution IV is collected, centrifuged and the supernatant is taken. 3 times the volume of anhydrous ethanol is added to the supernatant to precipitate the crude white peony polysaccharide iron product. The added amount volume ratio of solution I to solution II is 10mL: 10μL, the stirring reaction condition is 60°C, and the reaction time is 0.5h; the centrifugal speed is 5000rpm, and the centrifugation time is 15min;

[0066] Step 3: dissolving the crude product of white peony polysaccharide iron in pure water to prepare solution V, placing solution V into a dialysis bag and dialyzing with deionized water. When dialyzing solution V with deionized water, MW = 3000, the dialysis time is 48 h;

[0067] Step 4: After dialysis is completed, add 3 times the volume of anhydrous ethanol to solution V to precipitate the white peony polysaccharide iron product, centrifuge and obtain the precipitate. Repeat the above operation three times, then freeze-dry, grind and collect to obtain white peony polysaccharide iron powder, wherein the centrifugal speed is 5000 rpm and the centrifugation time is 15 min.

[0068] Example 3

[0069] This embodiment provides a method for preparing a biological metabolism regulator based on white peony polysaccharide iron, and the specific steps are as follows:

[0070] Step 1, dissolving white peony polysaccharide and trisodium citrate in pure water, stirring to obtain solution I, dissolving ferric chloride in pure water to obtain solution II, and dissolving sodium hydroxide in pure water to obtain solution III, wherein the mass ratio of white peony polysaccharide to trisodium citrate in solution I is 4:1, the concentration of ferric chloride in solution II is 4 mol / L, and the concentration of sodium hydroxide in solution III is 20 wt%;

[0071] Step 2: Solution II is added dropwise to solution I with magnetic stirring, and solution III is continuously added during the period to keep the pH between 9.5. When red insoluble matter appears in the reaction system, the dropwise addition of solution II is stopped. At this time, the solution is solution IV, and solution IV is placed on a rotary heating table and continued to stir and react. After the reaction is completed, solution IV is collected, centrifuged and the supernatant is taken. 10 times the volume of anhydrous ethanol is added to the supernatant to precipitate the crude white peony polysaccharide iron product. The added amount volume ratio of solution I to solution II is 10mL:100μL, the stirring reaction condition is 90°C, and the reaction time is 2h; the centrifugal speed is 10000rpm, and the centrifugation time is 15min;

[0072] Step 3: dissolving the crude white peony polysaccharide iron product in pure water to prepare solution V, placing solution V into a dialysis bag and dialyzing with deionized water. When dialyzing solution V with deionized water, MW = 4500, the dialysis time is 48 h;

[0073] Step 4: After dialysis is completed, add 10 times the volume of anhydrous ethanol to solution V to precipitate the white peony polysaccharide iron product, centrifuge and obtain the precipitate. Repeat the above operation three times, then freeze-dry, grind and collect to obtain white peony polysaccharide iron powder, wherein the centrifugal speed is 10000rpm and the centrifugation time is 15min.

[0074] Application Example 1

[0075] Free radical scavenging effect:

[0076] 1) Superoxide anion (O2 ·- ) Free radical scavenging rate

[0077] 1mL deionized water was added to 2mL50 mM Tris-HCl buffer (pH8.2), mixed, and incubated at room temperature for 20min. Take 2.9mL of the above solution, add 1mL sample solution (take 1mL pure water to completely dissolve 2mg white peony polysaccharide iron (product prepared in Example 1), and still use pure water to prepare 15, 30, 60, 120, 240μg / mL white peony polysaccharide iron solution, and prepare white peony polysaccharide solution of the same concentration as above in the same way as sample solution) and 0.1mL 6mM pyrogallol solution, mix, and react at room temperature for 5min. 3 drops of 10mM HCl solution were added to the mixed solution to terminate the reaction, and its absorbance was measured at 320nm (n=3). The sample control group was a mixed solution in which 1mL deionized water replaced the sample solution, and the blank control group was a mixed solution in which 0.1mL deionized water replaced pyrogallol. The superoxide anion (O2 ·- ) Clearance rate:

[0078] Clearance rate (%) = [1-(Ax-Ax0) / A0] × 100, where Ax is the absorbance value of the sample group, Ax0 is the absorbance value of the sample control group, and A0 is the absorbance value of the blank control group.

[0079] 2) Hydrogen peroxide free radical (H2O2) scavenging rate

[0080] To use the pH meter: Turn on the power, remove it from the electrolyte, rinse it with water, wipe it dry, and then calibrate it according to the calibration solution pH = 4, 7, and 10. After calibration, rinse it with water, wipe it dry, place it in the test solution, and press "Read" to measure. After the measurement is completed, press and hold the power button to turn off.

[0081] The preparation methods of test solutions of different concentrations are shown in the table below:

[0082]

[0083] Note: Table C 材料 It is white peony polysaccharide solution or white peony polysaccharide iron solution.

[0084] Determination of hydrogen peroxide by oxygen electrode method:

[0085] Start the oxygen electrode and begin monitoring changes in oxygen concentration with the above concentrations. Hydrogen peroxide is broken down by catalase, and the oxygen concentration decreases during the reaction. Record the rate of oxygen consumption, or the rate of decrease in oxygen concentration, which is proportional to the activity of the CAT enzyme.

[0086] Record the curve of oxygen concentration changes and calculate the oxygen consumption rate (unit: μmol O2 / min). The activity of the enzyme can be inferred based on the oxygen consumption of the reaction.

[0087] The hydrogen peroxide (H2O2) removal rate of the sample was calculated according to the following formula:

[0088] CAT enzyme activity is usually expressed as the amount of oxygen consumed per unit time. Based on the oxygen consumption rate obtained in the experiment, the enzyme activity can be calculated using the following formula:

[0089] CAT enzyme activity = Δ[O2]·V / Δt·m, where: Δ[O2] is the change in oxygen concentration (unit: μmol / L), Δt is the duration of the reaction (unit: min), V is the volume of the reaction solution (unit: L), and m is the mass of the enzyme sample (unit: mg).

[0090] 3) Hydroxyl radical (·OH) scavenging rate

[0091] Reagent preparation: Ferrous sulfate solution: Weigh 0.2502g of ferrous sulfate and add 10ml of pure water. Salicylic acid solution: Weigh 0.1243g of salicylic acid and add 10ml of anhydrous ethanol. 1 / 1000 hydrogen peroxide solution: Dissolve 10μl of hydrogen peroxide in 9.99ml of pure water to make a 1 / 1000 hydrogen peroxide solution. Dissolve 2mg of white peony polysaccharide iron in 1mL of pure water. Prepare white peony polysaccharide iron solutions of 3, 6, 13, 26, and 53μg / mL using pure water. Prepare white peony polysaccharide solutions of the same concentrations as above in the same manner. Samples were either white peony polysaccharide iron solution or white peony polysaccharide solution, and the control was water.

[0092] Experimental steps:

[0093]

[0094] The amount of each solution added was as shown in the table. After heating in a 37°C water bath for 30 minutes, the solution was taken out and the OD value was measured. 510 value.

[0095] Clearance rate (%): [A0-(A X -A X0 )] / A0×100, where Ax is the absorbance value of the sample group within 30 min, Ax0 is the absorbance value of the sample control group, and A0 is the absorbance value of the blank control group.

[0096] 4) DPPH free radical scavenging rate

[0097] Reagent preparation: Working solution: 2mg DPPH + 20ml anhydrous ethanol; Calibration of the working solution: Add 200μl of the purple working solution to a 96-well plate, add three replicates, and measure the OD value at a wavelength of 517nm using a microplate reader. The OD value should be around 0.8. If the OD value is greater than 0.8, adjust it by adding a little more anhydrous ethanol. Sample preparation: Dissolve 2mg of white peony polysaccharide iron in 1mL of pure water. Prepare white peony polysaccharide iron solutions at 62.5, 125, 250, 500, and 1000μg / mL using pure water. Prepare white peony polysaccharide solutions of the same concentration as above in the same manner.

[0098] Experimental steps:

[0099] (1) Control group (A0): 2 ml working solution

[0100] (2) Material Group (A X ):1ml sample + 1ml working solution

[0101] (3) Material control group (A X0 ):1ml sample + 1ml anhydrous ethanol

[0102] Incubate the reagent in a water bath: Heat in a 37°C water bath, remove after 30 minutes, and measure OD 517 value.

[0103] Detect and calculate clearance rate:

[0104] Clearance rate (%) = [A0-(A X -A X0 )] / A0×100, where Ax is the absorbance value of the sample group within 30 min, Ax0 is the absorbance value of the sample control group, and A0 is the absorbance value of the blank control group.

[0105] The above superoxide anion (O2 ·- The results of the free radical scavenging rate, hydrogen peroxide free radical (H2O2) scavenging rate, hydroxyl free radical (·OH) scavenging rate and DPPH free radical scavenging rate are shown in the table. Figure 6 ,from Figure 6 It can be seen that starting from 30 μg / mL, white peony polysaccharide iron showed better free radical scavenging ability than white peony polysaccharide.

[0106] Application Example 2

[0107] The treatment of inflammatory bowel disease mice with the biological metabolic regulator based on white peony polysaccharide iron prepared in Example 1 includes the following specific steps:

[0108] 1) Establishment of an inflammatory bowel disease mouse model: All C57BL / 6 mice were adaptively housed in mouse cages, using sawdust as bedding. At room temperature (24±1)°C and 50% humidity, they were randomly divided into 6 groups, with 10 mice in each group. Mice given pure water served as a blank control group, and the other 5 groups were given 2% DSS for 7+4 days. They were divided into a model group, a positive drug group (5-aminosalicylic acid, abbreviated as 5-ASA), a ferric chloride (FeCl3) group, a white peony polysaccharide group, and a white peony polysaccharide iron group. After the 7-day modeling was completed, the above 5 groups of mice were gavaged with the corresponding dose of drugs for 4 days. After the end, 0.1 mL of blood was collected from the eyeballs of all mice, and blood routine data were measured using a hematology analyzer. During this period, weight changes, fecal occult blood, and fecal viscosity were recorded and plotted. After the experiment, the colons of each group were collected for H&E staining, ZO-1 and Occludin immunofluorescence, and PFKFB2, PFKFB3, NF-κB p65 and p-NF-κBp65 protein WB detection experiments.

[0109] 2) Administration method: Oral administration was performed at 8 pm every day for 4 consecutive days. During the oral administration period, each group ate normally. The blank control group and the model control group were given the same volume of distilled water. The mice in the positive drug group were given a drug with a 5-ASA content of 20 mg / kg; the mice in the FeCl3 group were given a drug with a Fe content of 3.378 mg / kg; the mice in the white peony polysaccharide group were given a drug with a white peony polysaccharide content of 16.622 mg / kg; and the mice in the white peony polysaccharide iron group were given a drug with a white peony polysaccharide iron content of 20 mg / kg. Except for the blank control group, the other 5 groups were given 2% DSS for 7+4 days. Body weight, fecal occult blood and fecal viscosity were tested once a day (see Figure 7 ) and record, observe the animal status, and calculate the DAI score of each group of mice on the same day.

[0110] 3) Sample collection: After the last oral administration, mice in each group were allowed to drink water freely for 12 hours, then sacrificed and whole blood was collected from the eyeballs. Various biochemical indices were measured, including hemoglobin content in erythrocytes (HGB), red blood cell count (RBC), hematocrit (HCT), mean corpuscular volume (MCV) ( Figure 9 ), TNF-α, IL-1β and IL-10. The colon was taken for H&E staining ( Figure 8 ) and ZO-1 and Occludin immunofluorescence ( Figure 11 ). Tissue lysates were collected, protein bands were separated on sodium dodecyl sulfate-polyacrylamide gel, and transferred to nitrocellulose membrane. Protein expression was analyzed using protein-specific antibodies and HRP-labeled anti-rabbit IgG whole antibody. Finally, luminol-based enhanced chemiluminescence HRP substrate was used for visualization by fluorescence chemiluminescence analysis system. PFKFB2, PFKFB3, CD86 and CD206 proteins ( Figure 10 ) of the Western blotting assay.

[0111] from Figure 7 As can be seen in the figure, during IBD modeling and treatment, healthy mice showed no signs of fecal occult blood, while the 2% DSS model induced the presence of fecal occult blood. After treatment with PPFeCs, a significant improvement in fecal occult blood was observed.

[0112] from Figure 8 As can be seen in the figure, H&E staining showed a complete absence of crypts in PBS-treated IBD mice, indicating significant immune cell infiltration. In contrast, the PPFeCs-treated group showed significant improvement, with intact crypt structure and minimal inflammatory infiltration.

[0113] from Figure 9 As seen in the figure, blood analysis was performed to assess differences in hematological parameters between normal and IBD mice. As indicated in the Intestinal Inflammation Guide, red blood cell count, HGB, hematocrit (HCT), and MCV levels decreased in the DSS group, indicating that IBD induces anemia. Following treatment with PPFeCs, these levels returned to normal, demonstrating that PPFeCs can effectively treat anemia.

[0114] from Figure 10 As can be seen in the figure, Western blot (WB) experiments were further performed in the colons of mice in the control (healthy), DSS, and DSS+PPFeCs groups. PFKFB2, PFKFB3, CD86, and CD206 proteins were significantly elevated in the DSS group, indicating that the colons of DSS mice were in a state of high inflammation and aerobic glycolysis. After PPFeCs treatment, the levels of these proteins decreased significantly, indicating that the inflammation of IBD was reversed and oxidative phosphorylation was activated.

[0115] from Figure 11 As can be seen in the figure, we investigated the effects of PPFeCs on colonic epithelial cells and the damaged colonic epithelial barrier. As shown by immunofluorescence staining, DSS treatment significantly reduced the expression of tight junction proteins ZO-1 and occludin in colonic tissue. These proteins are major components of tight junctions and are essential for maintaining the structure and function of the intestinal epithelium and the integrity of the intestinal barrier. PPFeCs treatment effectively restored the expression of (a) ZO-1 and (b) occludin proteins, repairing the damaged intestinal barrier.

[0116] Application Example 3

[0117] The release of white peony polysaccharide and iron ions in the biological metabolic regulator based on white peony polysaccharide iron was tested as follows:

[0118] (1) Preparation of simulated gastric fluid: 1 g of pepsin was dissolved in 100 mL of pure water, and the pH of the solution was adjusted to 1.5 with 1 mol / mL HCl. The solution was then filtered through a 0.2 mm filter to obtain simulated gastric fluid.

[0119] (2) Preparation of simulated intestinal fluid: Dissolve 1.36 g KH2PO4 and 1 g trypsin in 100 mL pure water, and adjust the pH of the solution to 6.8 with 0.4 wt% HCl. Then filter the solution through a 0.2 mm filter to obtain simulated intestinal fluid.

[0120] (3) The simulated colonic fluid was replaced with PBS.

[0121] (4) Experimental plan: 10 mg of white peony polysaccharide iron prepared in Example 1 was dissolved in 4 mL of pure water to obtain a white peony polysaccharide iron solution. This solution was placed in a dialysis bag (MW = 3500) to form a whole. The whole was placed in 30 mL of simulated gastric fluid for 2 hours; thereafter, the whole was placed in simulated intestinal fluid for 4 hours; and finally, it was placed in simulated colonic fluid for 24 hours. This process simulated in vivo digestion dialysis at 37°C and 180 rpm, and 5 mL of the solution outside the dialysis bag was taken at the following time points: 1, 2, 3, 4, 5, 6, 12, 18, and 24 hours. After taking out, the same volume of simulated solution was added.

[0122] (5) Determination of release of polysaccharides and iron ions: The contents of white peony polysaccharides and iron ions in the solution outside the dialysis bag were determined by the anthrone-sulfuric acid method and inductively coupled plasma (ICP), respectively.

[0123] Figure 12 (a) and (b) show the release of white peony polysaccharide and iron ions in different environments and at different times within 24 hours. As can be seen from the figure, white peony polysaccharide and iron ions are released in large quantities in gastric juice.

[0124] Comparative Example 1

[0125] The optimization of biological metabolic regulator based on white peony polysaccharide iron is as follows:

[0126] (1) 10 mg of the white peony polysaccharide iron prepared in Example 1 was wrapped with an enteric coating to form white peony polysaccharide iron enteric-coated capsules;

[0127] (2) The remaining steps are the same as those in Application Example 3;

[0128] (3) Determination of polysaccharide and iron ion release: The contents of white peony polysaccharide and iron ion in the solution outside the dialysis bag were determined by anthrone-sulfuric acid method and inductively coupled plasma (ICP) respectively. Figure 12The results of (a) and (b) show that approximately 48.13% of the white peony polysaccharide iron in (a) reached the intestine after dissociation by gastric acid, while approximately 98.58% of the white peony polysaccharide enteric-coated capsules in (b) reached the intestine. Comparing the release rate of the white peony polysaccharide iron enteric-coated capsules and the white peony polysaccharide iron, it can be tentatively considered that the enhanced efficacy is 2.05 times.

[0129] (4) Compare the therapeutic effects of the two different dosage forms based on the release effect. We tentatively determined that capsule gavage is 2.05 times more effective than solution gavage, so we reduced the solution gavage dose by 2.05 times and loaded it into the capsule, that is, the dose of white peony polysaccharide iron enteric-coated capsules was 9.76 mg kg -1 , and conduct another animal experiment.

[0130] (5) The results of animal experiments showed that after treatment with white peony polysaccharide iron and white peony polysaccharide iron enteric-coated capsules, Figure 12 (c) is the colon length between different groups. Compared with the healthy group, the colon length of IBD mice treated with PBS was significantly reduced by 28.2%, while the white peony polysaccharide iron and white peony polysaccharide iron enteric-coated capsule groups only decreased by 11.6% and 7.0%. (d) is a quantitative graph of the colon length of the above groups. (e) is the weight change curve of mice between different groups. The weight of mice gradually increased after treatment with white peony polysaccharide iron and white peony polysaccharide iron enteric-coated capsules, and the difference observed was very small compared with the healthy group. (f) is the DAI score curve showing results similar to the weight change curve. After treatment with white peony polysaccharide iron and white peony polysaccharide iron enteric-coated capsules, the DAI score of IBD mice was significantly reduced. As we expected, compared with the therapeutic effect of the solution gavage group, the drug dose used in the capsule gavage group was less than half, but achieved a therapeutic effect similar to that of the solution group. This result indicates that white peony polysaccharide iron enteric-coated capsules have better clinical performance than white peony polysaccharide iron solution.

[0131] Although the embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the description and implementation methods. They can be fully applied to various fields suitable for the present invention. For those familiar with the art, additional modifications can be easily implemented. Therefore, without departing from the general concept defined by the claims and the scope of equivalents, the present invention is not limited to the specific details and illustrations shown and described herein.

Claims

1. A method for preparing a biological metabolism regulator based on white peony polysaccharide iron, characterized in that: Here are the steps: Step 1, dissolving white peony polysaccharide and trisodium citrate in pure water, stirring evenly to prepare solution I, dissolving ferric chloride in pure water to prepare solution II, dissolving sodium hydroxide in pure water to prepare solution III, the mass ratio of white peony polysaccharide and trisodium citrate in solution I is (2-4): 1, the concentration of ferric chloride in solution II is 2-4 mol / L, and the concentration of sodium hydroxide in solution III is 10-20 wt%; Step 2, solution II is added dropwise to solution I with magnetic stirring, and solution III is continuously added during the period to keep the pH between 8.5-9.

5. When red insoluble matter appears in the reaction system, the addition of solution II is stopped. At this time, the solution is solution IV, and solution IV is placed on a rotary heating table and continued to stir and react. After the reaction is completed, solution IV is collected, centrifuged and the supernatant is taken. 3-10 times the volume of anhydrous ethanol is added to the supernatant to precipitate the crude white peony polysaccharide iron product. The volume ratio of the added amount of solution I to solution II is 10 mL: (10-100) mL, the stirring reaction conditions are 60-90 ° C, the reaction time is 0.5-2 h; the centrifugal speed is 5000-10000 rpm, and the centrifugation time is 5-15 min; Step 3, dissolving the crude white peony polysaccharide iron product in pure water to prepare solution V, placing solution V into a dialysis bag and dialyzing with deionized water, when dialyzing solution V with deionized water, MW = 3000-4500, and the dialysis time is 12-48 h; Step 4: After dialysis is completed, add 3-10 times the volume of anhydrous ethanol to solution V to precipitate the white peony polysaccharide iron product, centrifuge and take the precipitate, repeat the above operation three times, then freeze-dry, grind and collect to obtain white peony polysaccharide iron powder, wherein the centrifugal speed is 5000-10000 rpm, and the centrifugation time is 5-15min.

2. A biological metabolism regulator based on white peony polysaccharide iron prepared based on the method described in claim 1.

3. A biological metabolism regulator based on white peony polysaccharide iron according to claim 2, characterized in that, The polysaccharide content of the biological metabolism regulator based on white peony polysaccharide iron is 83.11±1.234%, and the iron content is 16.89±0.271%.

4. Use of a biological metabolic regulator based on white peony polysaccharide iron as claimed in claim 2 in the preparation of a drug for treating inflammatory bowel disease.

5. The use according to claim 4, characterized in that The inflammatory bowel disease drug is a free radical scavenging drug, and the dosage of the biological metabolic regulator based on white peony polysaccharide iron for scavenging free radicals is 10-2000 mg / mL.

6. The use according to claim 5, characterized in that The free radical scavenging effect is reflected in the decrease of the contents of superoxide anion, hydrogen peroxide, hydroxyl radical and 1,1-diphenyl-2-trinitrophenylhydrazine.

7. The use according to claim 4, characterized in that The inflammatory bowel disease drug is a drug for treating DSS-induced inflammatory bowel disease C57BL / 6 mice.

8. The use according to claim 4, characterized in that The administration method of the biological metabolism regulator based on white peony polysaccharide iron is oral administration of solution or oral administration of enteric-coated capsules.

9. The use according to claim 8, characterized in that When the administration method is oral administration of solution, the effective dose is 10-40 mg / kg; when the administration method is oral administration of enteric-coated capsules, the effective dose is 2-20 mg / kg.

10. A drug for treating inflammatory bowel disease, comprising the biological metabolism regulator based on white peony polysaccharide iron according to claim 2 and one or more pharmaceutically or food-acceptable excipients.

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