A notoginseng polysaccharide iron nano preparation, a preparation method and application thereof

Abstract

A Panax notoginseng polysaccharide iron nanoparticle preparation, its preparation method, and its application are disclosed. This invention can effectively prepare Panax notoginseng polysaccharide iron nanoparticles, solving the application problem in the preparation of drugs for treating iron deficiency anemia, inflammatory bowel disease-related anemia, iron deficiency anemia-related colitis, inflammatory anemia, or inflammatory bowel disease. The specific steps are as follows: The crude Panax notoginseng polysaccharide solution is purified using conventional purification methods to obtain purified Panax notoginseng polysaccharide. This purified polysaccharide is dissolved in ultrapure water, and an iron source is added under inert gas protection. After co-precipitation and chemical reduction reactions, the pH value is adjusted, and iron nanoparticles are loaded onto the Panax notoginseng polysaccharide to obtain Panax notoginseng polysaccharide-coated iron nanoparticles. The preparation method of this invention is simple, uses abundant raw materials, and has low production costs. It represents a major innovation in drugs for treating iron deficiency anemia and inflammatory bowel disease-related iron deficiency anemia, and has practical application value.

Description

Technical Field

[0001] This invention relates to the pharmaceutical field, and in particular to a Panax notoginseng polysaccharide iron nanoparticle preparation, its preparation method, and its application. Background Technology

[0002] Iron deficiency anemia (IDA) is a global health problem characterized by low hemoglobin levels, microcytic hypochromic red blood cells, and insufficient iron reserves. IDA not only affects multiple systems throughout the body but is also a complication of many diseases, such as inflammatory bowel disease. The first-line treatment for IDA is oral iron supplements, such as ferrous sulfate and ferrous succinate; however, these are not suitable for treating IDA with intestinal inflammation. This is because intestinal inflammation not only reduces the bioavailability and effectiveness of oral iron supplements but also amplifies their irritant side effects on the gastrointestinal tract, further exacerbating intestinal inflammation and oxidative stress. Therefore, there is an urgent need to develop new, highly effective oral iron supplements for better management and treatment of IDA.

[0003] Iron nanoparticles exhibit diverse biological activities due to their unique physical size and chemical characteristics. In the biomedical field, iron nanoparticles, as a novel oral iron supplement for intradermal abscess (IDA), possess a higher specific surface area and lower iron ion leakage compared to traditional FeSO4, and can improve intestinal iron absorption to some extent. However, the low bioavailability of iron nanoparticles has also attracted widespread attention from researchers. For example, the clinical therapeutic effect of iron nanoparticles on IDA is far less than that of FeSO4, and the oxidative stress response occurring in the intestine further limits its absorption. Furthermore, oral iron preparations are not suitable for the treatment of all types of IDA because they may cause or aggravate intestinal inflammation. Therefore, improving the bioavailability of oral iron nanoparticles in the intestine and reducing oxidative stress are urgent technical problems that need to be solved.

[0004] In recent years, natural polysaccharides derived from plants have attracted much attention due to their unique advantages and have been widely explored as efficient platforms for oral delivery of nanomedicines. Panax notoginseng polysaccharide (PNP), extracted from the traditional Chinese medicine Panax notoginseng, has become a commonly used polysaccharide drug delivery carrier. Firstly, PNP has been listed in the Chinese Pharmacopoeia as a safe, edible, and medicinal natural active ingredient with various applications in the biopharmaceutical field, particularly in the treatment of intestinal diseases, where it plays important roles in antioxidation, immunomodulation, anti-inflammation, and intestinal barrier repair. Secondly, PNP possesses high biocompatibility and biodegradability, and is rich in surface functional groups such as hydroxyl and carboxyl groups, which can form five- or six-membered chelate rings with iron, resulting in a tightly encapsulated structure with the iron core inside and the polysaccharide outside. This structural feature helps to solve the problem of iron nanoparticles easily agglomerating, thereby improving bioavailability. Based on this, a novel oral formulation of iron nanoparticles has been constructed using PNP-loaded iron nanoparticles, which is expected to become a good drug delivery system for improving the bioavailability of iron nanoparticles in the intestine, reducing their oxidative stress, and enhancing their activity; however, no publicly reported studies have been conducted to date. Summary of the Invention

[0005] In view of the above situation and to overcome the shortcomings of the existing technology, the purpose of this invention is to provide a Panax notoginseng polysaccharide iron nanoparticle preparation, its preparation method and application, which can effectively prepare Panax notoginseng polysaccharide iron nanoparticle preparation and solve the application problems in the preparation of drugs for treating iron deficiency anemia, inflammatory bowel disease-related anemia, iron deficiency anemia-related colitis, inflammatory anemia or inflammatory bowel disease. The technical solution provided by this invention is a method for preparing Panax notoginseng polysaccharide iron nanoparticles. The specific steps are as follows: the crude polysaccharide solution of Panax notoginseng is purified by conventional purification methods to obtain purified Panax notoginseng polysaccharide, which is dissolved in ultrapure water. Under the protection of inert gas, an iron source is added, and after co-precipitation and chemical reduction reaction, the pH value is adjusted to load iron nanoparticles onto Panax notoginseng polysaccharide to obtain iron nanoparticles coated with Panax notoginseng polysaccharide. The conventional purification methods mentioned refer to the CTAB method, the Sevag method, or the trichloroacetic acid method; The inert gas is nitrogen or argon; The iron source is FeCl3·6H2O (ferric chloride hexahydrate), FeSO4·7H2O (ferrous sulfate hexahydrate), FeSO4 (ferrous sulfate), or a combination thereof; The iron nanoparticles are one or more of iron oxide nanoparticles, iron tetroxide nanoparticles, or multivalent iron nanoparticles, with a particle size of 40-200 nm and a mass ratio of 1-3:10 with Panax notoginseng polysaccharide.

[0006] The application of the Panax notoginseng polysaccharide iron nanoparticles prepared by the method of this invention in the preparation of drugs for treating iron deficiency anemia, inflammatory bowel disease-related anemia, iron deficiency anemia-related colitis, inflammatory anemia, or inflammatory bowel disease.

[0007] The aforementioned Panax notoginseng polysaccharide iron nanoparticle preparation is an oral formulation.

[0008] The preparation method of this invention is simple, the raw materials are abundant, and the production cost is low. The Panax notoginseng polysaccharide iron nanoparticles prepared by this invention can significantly improve the stability and absorption efficiency of iron nanoparticles in the intestine and reduce their irritation to the gastrointestinal tract. This not only helps to improve the oral bioavailability of iron nanoparticles, but also effectively reduces the level of inflammation in the body. It is a major innovation in the treatment of iron deficiency anemia and iron deficiency anemia associated with inflammatory bowel disease, and has practical application value. Detailed Implementation

[0009] The specific implementation of the present invention will be described in detail below with reference to examples and specific circumstances. Example

[0010] This invention discloses a method for preparing Panax notoginseng polysaccharide iron nanoparticles, characterized by the following specific steps: Panax notoginseng crude polysaccharide solution and hexadecyltrimethylammonium bromide solution are mixed evenly in equal volumes, allowed to stand at 4°C, centrifuged, the supernatant is discarded, and the precipitate is collected to obtain purified Panax notoginseng polysaccharide. The purified polysaccharide is dissolved in ultrapure water, and under nitrogen protection, FeCl3·6H2O (ferric chloride hexahydrate) and FeSO4·7H2O (ferrous sulfate hexahydrate) are added at a molar ratio of 1-1.2:1. After stirring and reacting at room temperature, the pH of the solution is adjusted to 9-10, and the reaction is continued with stirring and allowed to stand. The precipitate is then magnetically separated, washed, and freeze-dried to obtain Panax notoginseng polysaccharide-coated Fe3O4 magnetic nanoparticles. Example

[0011] This invention discloses a method for preparing Panax notoginseng polysaccharide iron nanoparticles, characterized by the following steps: An equal volume of Sevag reagent is added to a Panax notoginseng polysaccharide solution. The Sevag reagent is prepared by mixing chloroform and n-butanol in a volume ratio of 4:1. The solution is allowed to stand overnight, centrifuged, and the supernatant is collected. Five volumes of anhydrous ethanol are added, and the solution is allowed to stand overnight. The supernatant is then discarded, and the precipitate is collected to obtain purified Panax notoginseng polysaccharide. This precipitate is dissolved in ultrapure water. Under argon protection, FeSO4 and NaBH4 are added to the solution in a molar ratio of 1:0.8-1.0. The reaction continues until no bubbles are generated. The solution is then centrifuged, the supernatant is discarded, and the precipitate is resuspended in ultrapure water and stirred at room temperature. Argon gas is introduced, and the same mass of FeSO4 is added again to adjust the pH to 10-12. The mixture is stirred, allowed to stand, centrifuged, and the supernatant is discarded. The precipitate is washed with deionized water and freeze-dried to obtain Fe@Fe3O4 core-shell nanoparticles coated with Panax notoginseng polysaccharide. Example

[0012] This invention discloses a method for preparing Panax notoginseng polysaccharide iron nanoparticles, characterized by the following specific steps: Trichloroacetic acid is added to a Panax notoginseng polysaccharide solution to adjust the pH to 2-3, the solution is allowed to stand overnight, centrifuged, and the supernatant is collected. Four times the volume of anhydrous ethanol is added, the solution is allowed to stand overnight, centrifuged, the supernatant is discarded, and the precipitate is collected to obtain purified Panax notoginseng polysaccharide. This polysaccharide is dissolved in ultrapure water, and FeSO4 is added under argon protection. The pH is adjusted to 10-12, the solution is heated to 80°C and stirred to react, allowed to stand, centrifuged, the precipitate is washed with deionized water, and freeze-dried to obtain Fe3O4 nanoparticles coated with Panax notoginseng polysaccharide.

[0013] The application of the Panax notoginseng polysaccharide iron nanoparticles prepared by the method of this invention in the preparation of drugs for treating iron deficiency anemia, inflammatory bowel disease-related anemia, iron deficiency anemia-related colitis, inflammatory anemia, or inflammatory bowel disease.

[0014] The aforementioned Panax notoginseng polysaccharide iron nanoparticle preparation is an oral formulation.

[0015] The above description is merely a preferred embodiment of the present invention and is not intended to limit the scope of protection of the present invention. For those skilled in the art, various improvements and modifications can be made to the specific embodiments without departing from the principles of the present invention, and these improvements and modifications should also be considered to fall within the scope of protection of the present invention. The scope of protection claimed by the present invention is determined by the claims and their equivalents.

[0016] The preparation method of this invention is simple, uses abundant raw materials, and has low production costs. The prepared Panax notoginseng polysaccharide iron nanoparticles can significantly improve the stability and absorption efficiency of iron nanoparticles in the intestine, reduce their irritation to the gastrointestinal tract, not only help improve the oral bioavailability of iron nanoparticles, but also effectively reduce the level of inflammation in the body, and have both anti-inflammatory and iron-supplementing functions. It can synergistically enhance the therapeutic effect on inflammatory bowel disease-related anemia. Specific experimental data are as follows (taking Example 1 as an example): Experiment 1: Macrophage Polarization RAW264.7 macrophages were induced to differentiate into M1 type using lipopolysaccharide (LPS), and then co-cultured with iron nanoparticles and Panax notoginseng polysaccharide-iron nanoparticles, respectively. The levels of the pro-inflammatory cytokine interleukin-12 (M1 marker) and the anti-inflammatory cytokine interleukin-10 (M2 marker) in the supernatant were detected using enzyme-linked immunosorbent assay (ELISA). The results showed that compared with the saline-negative control group, treatment with iron nanoparticles and Panax notoginseng polysaccharide-iron nanoparticles reduced interleukin-12 levels by approximately 10% and 60%, respectively, while increasing interleukin-10 levels by approximately 4-fold and 6-fold, respectively. These results indicate that both iron nanoparticles and Panax notoginseng polysaccharide-iron nanoparticles can induce macrophages from M1 to M2 type in a LPS-induced inflammatory environment, with the Panax notoginseng polysaccharide-iron nanoparticles showing a significantly higher induction effect than iron nanoparticles.

[0017] Experiment 2: Mouse model of inflammatory bowel disease-associated anemia A model of iron deficiency anemia associated with inflammatory bowel disease was established using BALB / c female mice. Mice were randomly divided into 6 groups (n=10 per group): a saline control group, a disease model group, a FeSO4 positive control group, an iron nanoparticle group, a Panax notoginseng polysaccharide group, and a Panax notoginseng polysaccharide iron nanoparticle group. Mice in the saline control group had free access to sterile water and were fed a normal diet throughout the treatment. The other groups alternated between drinking dextran sulfate sodium solution and sterile water, and were fed a low-iron diet. Hemoglobin levels were measured periodically. Treatment was initiated when hemoglobin levels fell below 80 g / L. The dosage was 2 mg / kg of iron and 600 mg / kg of Panax notoginseng polysaccharide, administered once daily for 41 days.

[0018] The results showed that, compared with the saline control group, the mice in the disease model group exhibited a periodic and significant decrease in body weight, a periodic and significant increase in disease activity index, a significant shortening of colon length, and hemoglobin levels within the range of anemia. Compared with the model group, treatment with Panax notoginseng polysaccharide iron nanoparticles significantly restored the body weight loss in colitis mice, reduced the disease activity index, and alleviated colon shortening, demonstrating significant therapeutic effects on inflammatory bowel disease and anemia.

[0019] Experiment 3: In vivo anti-anemia effect Blood was collected from mice in each group using the orbital blood sampling method, and hemoglobin levels were measured using a fully automated hematology analyzer. Results showed that after treatment with different agents, the hemoglobin levels in all groups were increased to varying degrees compared to the model group. Specifically, the hemoglobin levels in the FeSO4 group and the iron nanoparticle group increased to (95.8±4.9) g / L and (92.1±5.3) g / L, respectively, which were significantly higher than those in the model group. P < 0.05, but still significantly lower than the normal control group ( P < 0.01). The hemoglobin content of mice in the Panax notoginseng polysaccharide iron nanoparticle group increased to (121.6±5.1) g / L, which was not statistically different from that of the normal control group. P > 0.05), and significantly higher than the FeSO4 group and the iron nanoparticle group ( P < 0.01). Experimental results show that the anti-anemia effect of Panax notoginseng polysaccharide iron nanoparticles is significantly higher than that of FeSO4 and iron nanoparticles.

[0020] Experiment 4: Mouse colon length The colons of mice in each group were dissected and their lengths measured. The results showed that the colon length of mice in the disease model group was significantly shorter compared to the normal control group. Compared to the model group, treatment with FeSO4, iron nanoparticles, Panax notoginseng polysaccharide, and Panax notoginseng polysaccharide-iron nanoparticles all resulted in varying degrees of increase in colon length. The Panax notoginseng polysaccharide-iron nanoparticle group had the longest colon, indicating that the preparation had the lowest gastrointestinal irritation effect.

[0021] While the above experiments were performed on Example 1, the Panax notoginseng polysaccharide iron nanoparticles prepared in other examples of the present invention were also verified in the same way. The results were basically the same as or similar to those of Example 1, so they will not be described in detail here.

[0022] In summary, the present invention has the following beneficial effects: 1. The preparation method is simple, the raw materials are abundant, and it is easy to carry out industrial production; 2. The prepared Panax notoginseng polysaccharide iron nanoparticles can improve the bioavailability of iron nanoparticles in the intestine, and have both good anti-inflammatory and iron-supplementing effects, while reducing gastrointestinal side effects; 3. This formulation has good biocompatibility and safety, and a wide range of applications. It can be used to prepare oral formulations for the treatment of iron deficiency anemia, inflammatory bowel disease-related anemia, iron deficiency anemia-related colitis, inflammatory anemia, and other diseases, and has clear clinical application prospects and promotional value.

Claims

1. A method for preparing Panax notoginseng polysaccharide iron nanoparticles, the specific steps of which are: purifying the crude polysaccharide solution of Panax notoginseng using conventional purification methods to obtain purified Panax notoginseng polysaccharide, dissolving it in ultrapure water, adding an iron source under the protection of an inert gas, adjusting the pH value, loading iron nanoparticles onto the Panax notoginseng polysaccharide, and obtaining iron nanoparticles coated with Panax notoginseng polysaccharide. The conventional purification methods mentioned refer to the CTAB method, the Sevag method, or the trichloroacetic acid method; The inert gas is nitrogen or argon; The iron source is FeCl3·6H2O, FeSO4·7H2O, FeSO4, or a combination thereof; The iron nanoparticles are one or more of iron oxide nanoparticles, iron tetroxide nanoparticles, or multivalent iron nanoparticles, with a particle size of 40-200 nm and a mass ratio of 1-3:10 with Panax notoginseng polysaccharide.

2. The method for preparing the Panax notoginseng polysaccharide iron nanoparticles according to claim 1, characterized in that, The specific steps are as follows: Mix the crude polysaccharide solution of Panax notoginseng with an equal volume of hexadecyltrimethylammonium bromide solution, let it stand at 4°C, centrifuge, discard the supernatant, take the precipitate, and obtain purified Panax notoginseng polysaccharide. Dissolve it in ultrapure water, and under nitrogen protection, add FeCl3·6H2O and FeSO4·7H2O at a molar ratio of 1-1.2:

1. After stirring the reaction at room temperature, adjust the pH of the solution to 9-10, continue stirring the reaction, and let it stand. The precipitate is separated by magnetic separation, washed, and freeze-dried to obtain Fe3O4 magnetic nanoparticles coated with Panax notoginseng polysaccharide.

3. The method for preparing the Panax notoginseng polysaccharide iron nanoparticle formulation according to claim 1, characterized in that, The specific steps are: adding equal volume of Sevag reagent into the solution of notoginseng polysaccharide, the Sevag reagent is made by mixing chloroform and n-butanol with a volume ratio of 4:1, standing overnight, centrifuging, taking supernatant, adding 5 times volume of anhydrous ethanol, standing overnight; centrifuging, discarding supernatant, taking the precipitate, obtaining purified notoginseng polysaccharide, dissolving in ultrapure water, under argon protection, adding FeSO4 and NaBH4 into the above solution with a molar ratio of 1:0.8-1.0, reacting until no gas bubble is generated, centrifuging, discarding supernatant, resuspending the precipitate with ultrapure water, stirring at room temperature; passing in argon, adding the same mass of FeSO4 again, adjusting pH to 10-12, stirring and reacting, standing, centrifuging, discarding supernatant, washing the precipitate with deionized water, freeze-drying, obtaining notoginseng polysaccharide coated Fe 0 @Fe3O4 core-shell nanoparticles.

4. The method for preparing the Panax notoginseng polysaccharide iron nanoparticles according to claim 1, characterized in that, The specific steps are as follows: Trichloroacetic acid is added to the Panax notoginseng polysaccharide solution to adjust the pH to 2-3, the solution is allowed to stand overnight, centrifuged, the supernatant is collected, 4 times the volume of anhydrous ethanol is added, the solution is allowed to stand overnight, centrifuged, the supernatant is discarded, the precipitate is collected, the purified Panax notoginseng polysaccharide is obtained, it is dissolved in ultrapure water, FeSO4 is added under argon protection, the pH is adjusted to 10-12, the solution is heated to 80°C and stirred to react, the solution is allowed to stand, centrifuged, the precipitate is washed with deionized water, and the solution is freeze-dried to obtain Fe3O4 nanoparticles coated with Panax notoginseng polysaccharide.

5. The use of the Panax notoginseng polysaccharide iron nanoparticles prepared by the method according to any one of claims 1-4 in the preparation of drugs for treating iron deficiency anemia, inflammatory bowel disease-related anemia, iron deficiency anemia-related colitis, inflammatory anemia, or inflammatory bowel disease.

6. The application according to claim 5, characterized in that, The aforementioned Panax notoginseng polysaccharide iron nanoparticle preparation is an oral formulation.