Preparation and application research of nano nutrition carrier based on brown algae polyphenol

Abstract

The invention discloses preparation and application research of a nano nutrition carrier based on brown algae polyphenol, and belongs to the technical field of preparation of nano materials. The preparation method specifically comprises the following steps: dissolving arginine in water, and then adding brown algae polyphenol to obtain a mixed solution; pEG is dissolved in PBS, and a PEG solution is obtained; adding a PEG (Polyethylene Glycol) solution into the mixed solution, stirring, adding hydrogen peroxide for reaction, adding acetic acid for continuous reaction, dialyzing with flowing water, and drying in vacuum to obtain the brown algae polyphenol nanoparticles. According to the invention, the limitation of the natural polyphenol such as brown algae polyphenol in the aspects of solubility and stability is realized, and a powerful technical support is provided for the practical application of the natural polyphenol; the synthesized nanoparticles show excellent anti-inflammatory characteristics in lipopolysaccharide induced macrophage inflammatory response, show excellent anti-oxidation and anti-inflammatory capabilities in DSS induced mouse ulcerative colitis, and provide a certain theoretical basis for effective prevention of ulcerative colitis.

Description

Technical Field

[0001] The invention belongs to the technical field of nano material preparation, and specifically relates to a preparation and application research of a brown algae polyphenol-based nano nutrition carrier. Background Art

[0002] Ulcerative colitis (UC) is a chronic, multifactorial inflammatory disease of the colon. Studies have shown that the younger age group of the population is susceptible to the disease, with the highest incidence in the 30-40 age group. Mucosal inflammation can develop to varying degrees, from mild symptoms such as congestion to mucosal fragility, erosion, and ulceration. In UC, the rectum is usually involved, and the inflammation can spread proximally and present characteristic continuous lesions throughout the colon. The most common symptoms of UC include bloody diarrhea, abdominal pain, rectal urgency, and fecal incontinence; depending on the degree and severity of inflammation, systemic symptoms may also be commonly manifested as malaise, fatigue, fever, and weight loss. The quality of life of UC patients is often affected, especially during disease activity. Ulcerative colitis is a well-known risk factor for colorectal cancer and often leads to colectomy. The most commonly used method for UC is oral medication, and common therapeutic drugs include 5-aminosalicylic acid, immunomodulators, and corticosteroids. Oral medications can achieve therapeutic effects, but they cannot cure the disease, and long-term drug treatment can cause toxic side effects in the body. Therefore, it is urgent to explore effective methods for treating UC.

[0003] Research shows that the development of nano-delivery systems has become a hot topic in current research. Compared with traditional treatment methods, nano-delivery systems have better stability. They can not only provide a longer time period and reduce toxic side effects, but more importantly, they can improve the efficacy of active substances.

[0004] Polyphenols are a variety of compounds naturally present in plants, and have attracted much attention due to their excellent antioxidant and anti-inflammatory properties. Among them, brown algae polyphenols (PT), as a natural marine plant polyphenol with a wide range of sources, show great potential in antioxidant and anti-inflammatory aspects. However, as a natural polyphenol, brown algae polyphenols have certain shortcomings in their application areas. The high content of phenolic hydroxyl groups in the structure makes PT easy to oxidize and degrade, resulting in its poor stability in the biological environment. In addition, the rapid metabolism and excretion of PT in the body will also affect the efficient utilization of brown algae polyphenols, so its development potential needs to be further explored and improved.

[0005] In previous inventions, polyphenols were loaded onto proteins to form nanoparticles, aiming to improve the stability of polyphenols and ensure their release in the colon, thereby exerting antioxidant and anti-inflammatory effects and effectively alleviating colitis symptoms. Although using protein as a carrier for synthesizing polyphenol nanoparticles has its advantages, it also has some potential disadvantages: proteins may cause immune or allergic reactions, especially for certain individuals who are sensitive to specific proteins. This may lead to reduced tolerance to treatment or serious side effects. Secondly, proteins may be unstable under specific environmental conditions (such as pH, temperature, and ionic strength) and are prone to denaturation or degradation, which may affect the stability of nanoparticles and the release characteristics of polyphenols. More importantly, the degradation rate of proteins in the body may be difficult to control, which may cause polyphenols to be released prematurely before reaching the colon, reducing the therapeutic effect. Summary of the invention

[0006] In order to solve the above problems, the present invention provides a preparation method of brown algae polyphenol nano-nutrient carrier, through the Schiff base reaction between brown algae polyphenol and arginine, that is, through the oxidation of the phenolic hydroxyl group of brown algae polyphenol to a carbonyl-containing ketone and the reaction with the amino group of arginine, polyphenol nanoparticles with Schiff base bonds are synthesized. The brown algae polyphenol nanoparticles not only retain the inherent antioxidant properties of brown algae polyphenol intact, but also significantly improve the stability of polyphenol; more importantly, the antioxidant efficacy of arginine itself complements the polyphenol, and together enhances the antioxidant function of the synthesized nanoparticles; in addition, the immunomodulatory and repair effects of arginine combined with the antioxidant and anti-inflammatory effects of polyphenols may be more effective in treating colitis.

[0007] The present invention provides a method for preparing a brown algae polyphenol nanonutrient carrier, which specifically comprises:

[0008] (1) dissolving arginine in water to obtain an arginine solution, and then adding brown algae polyphenols to obtain a mixed solution;

[0009] (2) dissolving polyethylene glycol (PEG) in PBS to obtain a polyethylene glycol solution;

[0010] (3) Adding the polyethylene glycol solution to the mixed solution and stirring, and adding hydrogen peroxide, after a period of reaction, adding acetic acid to continue the reaction, and finally dialyzing with running water and vacuum drying to obtain brown algae polyphenol nanoparticles PT NPs.

[0011] In one embodiment of the present invention, in step (1), the concentration of the arginine solution is 0.5-2 mg / mL.

[0012] In one embodiment of the present invention, in step (1), the concentration of brown algae polyphenols in the mixed solution is 0.5-2 mg / mL.

[0013] In one embodiment of the present invention, in step (2), the relative molecular weight of PEG is 1000, and the concentration of the polyethylene glycol solution is 1-2.5 mg / mL.

[0014] In one embodiment of the present invention, in step (3), the ratio of polyethylene glycol to the sum of the mass of arginine and brown algae polyphenols is 1:10-20.

[0015] In one embodiment of the present invention, in step (3), the volume concentration of hydrogen peroxide is 30%, and 5 mL of hydrogen peroxide is added every 5 minutes to promote the oxidation of brown algae polyphenols, and the addition is performed four times in total.

[0016] In one embodiment of the present invention, in step (3), 75 μL of acetic acid is added, with a volume concentration of 36%.

[0017] In one embodiment of the present invention, in step (3), hydrogen peroxide is added and the reaction is continued for 18-24 hours, and acetic acid is added and the reaction is continued for 6 hours.

[0018] The present invention provides a brown algae polyphenol-based nano-nutrient carrier prepared by the method described above.

[0019] The present invention also provides the use of the above-mentioned brown algae polyphenol-based nano-nutrient carrier in the preparation of a drug for treating ulcerative colitis.

[0020] Compared with the prior art, the present invention has the following advantages:

[0021] (1) The polyphenol nanoparticles of the present invention exhibit significant antioxidant and anti-inflammatory properties in the treatment of ulcerative colitis. In this process, the use of arginine plays a key role. Arginine, as an amino acid, has multiple biological activities, including antioxidant properties. Arginine can enhance the antioxidant capacity of polyphenol nanoparticles. Studies have shown that the guanidine group contained in arginine can capture free radicals, reduce oxidative stress, and thus protect cells from oxidative damage. In the present invention, by introducing arginine during the synthesis of polyphenol nanoparticles, the antioxidant properties of the nanoparticles are further improved, making it more effective in reducing inflammation and oxidative damage when treating ulcerative colitis.

[0022] (2) In the treatment of ulcerative colitis, the addition of arginine to the polyphenol nanoparticles of the present invention not only enhances the stability of the nanoparticles, but also improves their antioxidant capacity, thereby better maintaining the integrity of the active substances and exerting an excellent therapeutic effect. This discovery is of great significance for the treatment of inflammation, especially for diseases related to oxidative stress.

[0023] (3) Arginine is a naturally occurring amino acid with good biocompatibility. Compared with protein, arginine may be more easily accepted by the human body, reducing potential allergic reactions or side effects. Secondly, arginine can enhance the stability and efficiency of drug delivery systems. It can promote the cellular uptake of nanoparticles by forming ion pairs or interacting with intestinal cell membranes using its positive charge. In addition, arginine can increase the solubility of polyphenols in water, thereby increasing their absorption in the intestine. In contrast, protein may affect the solubility and release characteristics of polyphenols under certain conditions. More importantly, the combination of arginine and polyphenols may reduce the degradation of polyphenols by intestinal digestive enzymes, ensuring that the polyphenols remain stable before reaching the colon, thereby improving their bioavailability.

[0024] (4) The polyphenol nanoparticles successfully prepared by the present invention showed good therapeutic effects in the treatment of ulcerative colitis, and their therapeutic effects were similar to those of the commonly used clinical drug 5-aminosalicylic acid (5-ASA). This shows that polyphenol nanoparticles have potential application value in the field of inflammation treatment and are expected to become a new and effective means of treating ulcerative colitis. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is the infrared spectrum of the brown algae polyphenol nanoparticles in Example 1.

[0026] Figure 2 This is a SEM image of the brown algae polyphenol nanoparticles in Example 1.

[0027] Figure 3 This is a TEM image of brown algae polyphenol nanoparticles in Example 1.

[0028] Figure 4 This is a graph showing the storage time stability of brown algae polyphenol nanoparticles in Example 1.

[0029] Figure 5 This is a graph showing the salt solution concentration stability of brown algae polyphenol nanoparticles in Example 1.

[0030] Figure 6 This is a graph showing the in vitro cytotoxicity of brown algae polyphenol nanoparticles in Example 1.

[0031] Figure 7 This is a graph of the nanoparticle hemolysis experiment in Example 1.

[0032] Figure 8 This is a diagram of the uptake of brown algae multi-nanoparticles of Example 1 by macrophages.

[0033] Fig. 9 This is a fluorescence quantitative graph of the uptake of brown algae multi-nanoparticles in Example 1 by macrophages.

[0034] Fig.10This is a diagram showing that brown algae polyphenol nanoparticles regulate oxidative stress levels in Example 1.

[0035] Fig.11 This is a graph showing the effects of different PT sample treatments on SOD activity.

[0036] Fig.12 This is a graph showing the effects of different PT sample treatments on GSH content.

[0037] Fig.13 Figure 2 is the colon diagram of mice after treatment with different groups of PT.

[0038] Fig.14 This is the body weight change curve of mice after being treated with different PT groups.

[0039] Fig.15 This is a graph showing the effects of different PT sample treatments on the level of the inflammatory factor TNF-α.

[0040] Fig.16 This is a graph showing the changes in NO content after treatment with different PT groups.

[0041] Fig.17 This is a graph showing changes in iNOS activity after treatment with different groups of PT.

[0042] Fig.18 This is a graph showing the changes in SOD activity after treatment with different PT groups.

[0043] Fig.19 This is a graph showing changes in MPO content after treatment with different PT groups.

[0044] Fig. 20 This is a graph showing changes in MDA content after treatment with different PT groups.

[0045] Fig.21 These are H&E staining images of the main organs after different PT groups were treated. DETAILED DESCRIPTION

[0046] Brown algae polyphenols were purchased from Xi'an Kangnuo Chemical Co., Ltd.; polyethylene glycol with a relative molecular weight of 1000 was purchased from Shanghai MacLean Biochemical Technology Co., Ltd.

[0047] The room temperature of the present invention is 20-25°C

[0048] The following examples serve to illustrate the invention.

[0049] Example 1

[0050] A preparation method of brown algae polyphenol nano nutrition carrier, the steps are as follows:

[0051] (1) Weigh 40 mg of arginine and dissolve it in water to keep the concentration at 0.5 mg / mL, and weigh 60 mg of brown algae polyphenols and add them to the water to keep the concentration at 1 mg / mL to obtain a mixed solution;

[0052] (2) Weigh 5 mg of PEG and dissolve it in 2 mL of PBS to obtain a PEG solution;

[0053] (3) The PEG solution was added to the mixture and stirred at room temperature. Then, 20 mL of 30 vol% hydrogen peroxide solution was added, and 5 mL of hydrogen peroxide was added every 5 minutes to promote the oxidation of brown algae polyphenols. The mixture was added in four portions. After reacting for 24 hours, 75 μL of 36 vol% acetic acid was added to promote the reaction. After reacting for 6 hours, the mixture was dialyzed in running water and freeze-dried to obtain polyphenol nanoparticles.

[0054] This embodiment also includes pre-treatment steps such as dialysis bag activation:

[0055] Dialysis bag activation: 0.2 g EDTA-2Na and 10 g NaHCO 3 10H 2 After mixing, add deionized water to 500 mL, boil for 10 to 15 minutes, wash twice with deionized water, and then boil with distilled water for 10 to 15 minutes.

[0056] like Figure 1 As shown in Figure 2, the infrared spectra of brown algae polyphenols and their nanoparticles show their basic structural features, such as aromatic rings and hydroxyl groups (C=C aromatic stretching vibration (1600-1700cm -1 ) and OH stretching vibration (3300-3700cm -1 )). The newly added characteristic peak in the nanoparticle spectrum (1400cm -1 ) indicates that the Schiff base reaction (N=C) has occurred, confirming the successful synthesis of the nanoparticles. The synthesized brown algae polyphenol nanoparticles are relatively uniform spherical ( Figure 2 , Figure 3 ).

[0057] Example 2

[0058] A preparation method of brown algae polyphenol nano nutrition carrier, the steps are as follows:

[0059] (1) Weigh 40 mg of arginine and dissolve it in water to keep the concentration at 1 mg / mL, and weigh 60 mg of brown algae polyphenols and add them to the water to keep the concentration at 1 mg / mL to obtain a mixed solution;

[0060] (2) Weigh 5 mg of PEG and dissolve it in 2 mL of PBS to obtain a PEG solution;

[0061] (3) The PEG solution was added to the mixture and stirred at room temperature. Then, 20 mL of 30 vol% hydrogen peroxide solution was added, and 5 mL of hydrogen peroxide was added every 5 minutes to promote the oxidation of brown algae polyphenols. The mixture was added in four portions. After reacting for 24 hours, 75 μL of 36 vol% acetic acid was added to promote the reaction. After reacting for 6 hours, the mixture was dialyzed in running water and freeze-dried to obtain polyphenol nanoparticles.

[0062] This embodiment also includes pre-treatment steps such as dialysis bag activation:

[0063] Dialysis bag activation: 0.2 g EDTA-2Na and 10 g NaHCO 3 10H 2 After mixing, add deionized water to 500 mL, boil for 10 to 15 minutes, wash twice with deionized water, and then boil with distilled water for 10 to 15 minutes.

[0064] Example 3

[0065] A preparation method of brown algae polyphenol nano nutrition carrier, the steps are as follows:

[0066] (1) Weigh 40 mg of arginine and dissolve it in water to keep the concentration at 1.5 mg / mL, and weigh 60 mg of brown algae polyphenols and add them to the water to keep the concentration at 1 mg / mL to obtain a mixed solution;

[0067] (2) Weigh 5 mg of PEG and dissolve it in 2 mL of PBS to obtain a PEG solution;

[0068] (3) The PEG solution was added to the mixture and stirred at room temperature. Then, 20 mL of 30 vol% hydrogen peroxide solution was added, and 5 mL of hydrogen peroxide was added every 5 minutes to promote the oxidation of brown algae polyphenols. The mixture was added in four portions. After reacting for 24 hours, 75 μL of 36 vol% acetic acid was added to promote the reaction. After reacting for 6 hours, the mixture was dialyzed in running water and freeze-dried to obtain polyphenol nanoparticles.

[0069] This embodiment also includes pre-treatment steps such as dialysis bag activation:

[0070] Dialysis bag activation: 0.2 g EDTA-2Na and 10 g NaHCO 3 10H 2 After mixing, add deionized water to 500 mL, boil for 10 to 15 minutes, wash twice with deionized water, and then boil with distilled water for 10 to 15 minutes.

[0071] Study on the stability of brown algae polyphenol nanoparticles

[0072] This study comprehensively tested the stability of the nanoparticles of Example 1 under different environmental conditions, including storage time and salt solution concentration.

[0073] The specific detection methods are as follows:

[0074] Storage time stability: 0 days, 2 days, 4 days, 6 days and 8 days were selected as time nodes to measure the particle size and polydispersity index (PDI) of the nanoparticles. Effect of salt solution concentration: The nanoparticles were dispersed in 0M, 0.1M, 0.2M, 0.3M and 0.4M NaCl solutions, and after being placed at room temperature for 3 hours, their particle size and PDI were detected. Through these detailed stability tests, the stability performance of nanoparticles under various conditions can be accurately evaluated.

[0075] like Figure 4 As shown in the figure, the particle size and polydispersity index of polyphenol nanoparticles did not change significantly over 8 days, proving that they have good stability. In salt solutions of different concentrations, polyphenol nanoparticles can remain relatively stable ( Figure 5 ).

[0076] In vitro cytotoxicity assay of brown algae polyphenol nanoparticles

[0077] The 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay is an experimental method used to assess cytotoxicity.

[0078] Macrophages were cultured in DMEM medium containing 10% premium fetal bovine serum (Sangon Biotechnology Co., Ltd., Shanghai) and 1% antibiotics (100 U / mL penicillin and 100 μg / mL streptomycin) in an incubator at 37°C with 5% CO. 2 Then, the cells were cultured at 10 5 The cells were seeded into 96-well plates at a density of 1 μL / well and incubated at 37°C in 5% CO 2 The cells were cultured for 24 h in an atmosphere to allow the cells to adhere to the wall, with 100 μL of cell suspension per well. Then, the brown algae polyphenol nanoparticles of Example 1 were added to the culture medium to a final concentration of 0.3125, 0.625, 1.25, 2.5, 5 and 10 μg / mL, respectively, and the culture medium was incubated at 37°C in an incubator with 5% CO 2 Next, 20 μL of MTT (5 mg / mL) was added to each well and the cells were incubated at 37°C in an incubator with 5% CO 2The culture was continued for 4 hours under atmosphere. The culture medium was then removed and 150 μL DMSO (dimethyl sulfoxide) was added to each well to dissolve the purple crystals at the bottom of the well plate. The plate was shaken for 5 minutes and then the absorbance was measured at 570 nm using a microplate reader immediately. By comparing the absorbance of the treatment group with that of the control group, the relative percentage of cell proliferation or cytotoxicity can be calculated.

[0079] The culture medium is DMEM containing 10% by volume of premium fetal bovine serum (Sangon Biotechnology Co., Ltd., Shanghai) and 1% by volume of antibiotics (100 U / mL penicillin and 100 μg / mL streptomycin).

[0080] like Figure 6 As shown, at all concentrations, the cell viability was greater than 100, and the cell viability was maximum when the concentration was 5 μg / mL, and this concentration was selected for subsequent experiments.

[0081] To further explore the in vitro safety of nanoparticles, a hemolysis experiment was also conducted. Blood was drawn from mice, and the plasma and other components were removed by centrifugation to obtain pure red blood cells. The red blood cells were then washed with saline several times until the supernatant no longer appeared yellow. The washed red blood cells were prepared into a 1 mg / mL suspension with saline. The polyphenol nanoparticle solution was added at a concentration gradient ( Figure 7 ), 0.5mL of each group was added to a test tube containing a red blood cell suspension, and a negative control group and a positive control group (Tween-80) were set up at the same time, that is, the same volume of PBS and Tween-80 solution was added to the suspension instead of the nanoparticle solution. The test tube was placed in a water bath or incubator, usually incubated at 37°C for 30 minutes. After incubation, the test tube was placed in a centrifuge and centrifuged to precipitate the non-hemolyzed red blood cells. Carefully aspirate the supernatant and measure the absorbance at 540nm. The hemolysis rate was calculated by comparing the absorbance of the sample group to be tested with the negative control group and the positive control group. The hemolysis rate can be calculated by the following formula:

[0082] Hemolysis rate (%) = (absorbance of sample group - absorbance of negative control group) / (absorbance of positive control group - absorbance of negative control group) × 100%

[0083] Generally, a substance is considered hemolytic if the hemolysis rate exceeds 5%.

[0084] like Figure 7 As shown, no hemolysis occurred in the entire concentration range, further proving that the synthesized brown algae polyphenol nanoparticles have excellent biocompatibility.

[0085] Macrophage uptake of brown algae polyphenol nanoparticles

[0086] First, prepare FITC-labeled brown algae polyphenol nanoparticles: weigh 1 mg FITC and fully dissolve it with 1 mL DMSO. Add 10 μL FITC solution to the brown algae polyphenol nanoparticle solution (5 mL, 5 mg / mL). Stir at 600 rpm for 12 hours. The FITC-labeled brown algae polyphenol nanoparticle solution was dialyzed with 200 mL of distilled water for 48 hours under light-proof conditions, and the water was changed every 2 hours. A dialysis bag with a molecular weight cutoff of 500 Da was used. The solution was lyophilized to obtain the final product (FITC-PTNPs). The samples were divided into two groups: FITC-PT and FITC-PTNPs. Macrophages were cultured at 10 5 The cells were seeded into 12-well plates at a density of 1 mL per well and incubated at 37°C with 5% CO 2 incubator for 24 hours. The above two groups of samples were added separately: FITC-PT group: FITC-PT was added to the culture medium and cultured for 3 hours. FITC-PTNPs group: FITC-PTNPs were added to the culture medium and cultured for 3 hours. The cells were washed 3 times with PBS, then fixed with 1 mL of 4% paraformaldehyde for 15 minutes and washed again with PBS. The nuclei were stained with 1 mL of 2 μg / mL DAPI solution for 15 minutes, and then washed 3 times with PBS. The cells were sealed with a mixture of VPBS:Vglycerol=1:9. The cells were observed and images were taken using a confocal microscope.

[0087] The absorption of nanoparticles by cells was evaluated by observing the blue cell nuclei stained with DAPI and the green brown algae polyphenol nanoparticles labeled with FITC. The cellular absorption behavior of brown algae polyphenol nanoparticles was analyzed by comparing the fluorescence intensity of different treatment groups. Figure 8 As shown, using DAPI stain, the cell nucleus exhibited blue fluorescence, while the FITC-labeled sample showed strong green fluorescence around the cell nucleus, indicating that the nanoparticles were effectively internalized into the cell. The experimental results showed that the fluorescence intensity of the nanoparticle group was higher than that of the free polyphenol subgroup, indicating that the stability was increased after assembly into nanoparticles. Quantitative analysis further confirmed this trend ( Fig. 9 ).

[0088] In summary, this study shows that the effective absorption of nanoparticles into cells is the key to their anti-inflammatory effect. Nanoparticles showed higher cellular internalization efficiency and fluorescence intensity. These findings provide important experimental basis for the application of nanoparticles in anti-inflammatory treatment.

[0089] The culture medium is DMEM containing 10% by volume of premium fetal bovine serum (Sangon Biotechnology Co., Ltd., Shanghai) and 1% by volume of antibiotics (100 U / mL penicillin and 100 μg / mL streptomycin).

[0090] Determination of oxidative stress level

[0091] The fluorescent probe DCFH-DA was used to evaluate the reactive oxygen species (ROS) production capacity of PT and PTNPs in macrophage cells. The PTNPs were prepared in Example 1 of the present invention. The level of ROS was measured by a fluorescent microplate reader.

[0092] The samples were divided into two groups: free PT and PTNPs. 5 The cells were seeded into 12-well plates at a density of 1 mL per well and incubated at 37 °C with 5% CO 2 The two groups of samples (free PT ， For the free PT group, PT was added to the culture medium to a final concentration of 5 μg / mL; the cells were incubated at 37°C in a 5% CO 2 For the PTNPs group, PTNPs were added to the culture medium to make the final PT concentration of 5 μg / mL, and then the cells were incubated at 37°C in a constant temperature incubator with 5% CO 2 The untreated cells were used as blank controls. 30 μM H 2 O 2 , and incubated at 37 ° C for 30 min. Subsequently, the liquid in the 12-well plate was removed, and 1 mL of DCFH-DA solution dispersed in PBS was added to each well and incubated for 30 min. The cells were digested with trypsin, centrifuged at 1000 rpm for 5 min, and then collected from each well. Then, the cells were washed with 1 mL of PBS and resuspended in 1 mL of PBS. Finally, the cells were transferred to a 96-well plate, and the ROS content was detected by a fluorescent microplate reader at an excitation wavelength of 485 nm and an emission wavelength of 525 nm. The levels of intracellular superoxide dismutase (SOD) and glutathione (GSH) were evaluated using a commercial kit (Nanjing Jiancheng Bioengineering Institute, Nanjing).

[0093] The culture medium is DMEM containing 10% by volume of premium fetal bovine serum (Sangon Biotechnology Co., Ltd., Shanghai) and 1% by volume of antibiotics (100 U / mL penicillin and 100 μg / mL streptomycin).

[0094] like Fig.10 As shown in Figure 2, the ROS level induced by PTNPs increased significantly (p < 0.05), 23.11% higher than that of the control group. Fig.11 , Fig.12As shown, compared with the control group, the PTNPs group could significantly reduce (p<0.05) the activities of SOD and GSH; the SOD level was reduced by 45.26, and the GSH level was reduced by 28.37%, respectively.

[0095] Mouse ulcerative colitis (UC) model, changes in mouse colon length

[0096] The DSS (dextran sulfate sodium)-induced mouse ulcerative colitis animal model is a commonly used model for the pathological mechanism and treatment of colitis. The following are the steps to establish a DSS-induced mouse ulcerative colitis model:

[0097] 1. Experimental Animal Preparation

[0098] Healthy B / c male mice (such as C57B / c6 mice) were selected. Before the experiment, the mice were adapted to be raised in the laboratory environment for 7 days and provided with sufficient drinking water and food.

[0099] 2. Preparation of DSS

[0100] Dissolve DSS powder in distilled water to a DSS concentration of 3%.

[0101] 3. Induction of Colitis

[0102] Provide DSS solution as the only drinking water for mice for 7 days (adjustable according to experimental needs).

[0103] The water intake of mice was monitored daily, and body weight and general condition were recorded.

[0104] 4. Colitis Assessment

[0105] Before inducing colitis, mice were given free PT and PTNPs samples by intragastric administration of 200 μL of samples and 5-ASA at the same dose every day for 21 days. The mice were killed after the experiment. The colon of the mice was taken and its length was measured.

[0106] Fig.13 As shown in the figure, the colon length of mice was significantly shortened after DSS induction. However, after treatment with PT and PTNPs, the colon length increased significantly and was closer to the 5-ASA group. The colon length of the PT group increased by 2.1 cm, and the colon length of the PTNPs group increased by 3.4 cm. This result shows that PT and PTNPs can effectively improve the shortening of the colon in enteritis.

[0107] In addition, colitis also caused mice to lose weight. Fig.14As shown in the figure, the body weight of mice was significantly reduced after DSS induction (p < 0.05). The weight loss in the PT and PTNPs groups was alleviated, and the effect was more prominent in the PTNPs group. This further proves that PT and PTNPs can effectively improve the inflammatory response of colitis.

[0108] A group of mice without any heat treatment was used as blank control and compared with the DSS-induced group and the PT and PTNPs groups.

[0109] The steps for preparing mice in the blank group are as follows:

[0110] After the mice entered the laboratory, a one-week adaptation observation was carried out to ensure that the mice were in good health. The experimental group was treated with drugs; the blank group was not treated and was only used as a control. During the experiment, the growth status, behavioral activities, diet and drinking water of the mice were regularly observed and recorded. At the end of the experiment, the blank group mice were tested for corresponding indicators, and samples of blood, tissues and organs were collected. The data of the blank group were compared and analyzed with the experimental group to evaluate the effect of the experimental treatment.

[0111] Study on the preventive effect of brown algae polyphenol nanoparticles on inflammation in UC model

[0112] The colon tissue collected from the above-mentioned mouse ulcerative colitis (UC) model was accurately weighed, and then physiological saline (g / mL=1 / 9) was added for ice bath homogenization. After centrifugation at 1000rpm for 10min, the supernatant was discarded and the precipitate was retained. The content of TNF-α, a key inflammatory factor in colon tissue, was measured using an ELISA kit. Subsequently, a dedicated detection kit was used to evaluate the content of nitric oxide (NO), nitric oxide synthase (iNOS) and myeloperoxidase (MPO) activity levels in colon tissue. In addition, blood samples from mice were collected and centrifuged to obtain serum. The levels of superoxide dismutase (SOD) and malondialdehyde (MDA) in serum were determined using a dedicated analysis kit. The results prove that PTNPs can effectively prevent ulcerative colitis and relieve inflammatory responses.

[0113] like Fig.15 As shown in the figure, DSS induction led to a significant increase in the content of TNF-α. In contrast, the content of pro-inflammatory factors in the PTNPs group was significantly downregulated after treatment (p < 0.05). And after PTNPs treatment, the content of NO ( Fig.16 ) and iNOS activity ( Fig.17 ) were significantly decreased (p < 0.05). This indicates that PTNPs have excellent anti-inflammatory activity. In addition, PTNPs treatment can significantly improve the decrease in SOD activity induced by DSS ( Fig.18) and MDA content increased (p < 0.05). These data prove that PTNPs have excellent anti-inflammatory and anti-inflammatory abilities.

[0114] In the above mouse ulcerative colitis (UC) model, the heart, liver, spleen, lung and kidney were taken for H&E (hematoxylin-eosin) staining and in vivo safety evaluation

[0115] The harvested organs are fixed in an appropriate fixative, usually formalin, followed by dehydration, clearing, and paraffin embedding. After sectioning, the paraffin sections are mounted on slides in preparation for H&E staining. The paraffin sections are stained with hematoxylin to stain the nuclei, followed by eosin staining to stain the cytoplasm. Through this process, the structure and morphology of the cells, including details of the nucleus and cytoplasm, can be observed. The stained sections are then examined using an optical microscope to observe the tissue structure of the organ. The results of H&E staining are used to evaluate the potential toxicity or side effects of the nanoparticles on organs such as the heart, liver, spleen, lungs, and kidneys.

[0116] like Fig.21 As shown, H&E staining of major organs showed that the organ structures were normal without obvious pathological changes, indicating that PTNPs have good in vivo safety.

[0117] The embodiments provided above are not intended to limit the scope of the present invention, and the steps described are not intended to limit the execution order thereof. Those skilled in the art may make obvious improvements to the present invention in combination with existing common knowledge, which also fall within the scope of protection defined by the claims of the present invention.

Claims

1. A method for preparing brown algae polyphenol nanoparticles, characterized in that: The steps include: (1) dissolving arginine in water to obtain an arginine solution, and then adding brown algae polyphenols to obtain a mixed solution; (2) dissolving polyethylene glycol in PBS to obtain a polyethylene glycol solution; (3) adding the polyethylene glycol solution to the mixed solution and stirring, and adding hydrogen peroxide. After a period of reaction, adding acetic acid to continue the reaction. Finally, dialyzing with running water and vacuum drying are performed to obtain brown algae polyphenol nanoparticles.

2. The method for preparing brown algae polyphenol nanoparticles according to claim 1, characterized in that: In step (1), the concentration of the arginine solution is 0.5-2 mg / mL.

3. The method for preparing brown algae polyphenol nanoparticles according to claim 1, characterized in that: In step (1), the concentration of brown algae polyphenols in the mixed solution is 0.5-2 mg / mL.

4. The method for preparing brown algae polyphenol nanoparticles according to claim 1, characterized in that: In step (2), the relative molecular weight of polyethylene glycol is 1000, and the concentration of the polyethylene glycol solution is 1-2.5 mg / mL.

5. The method for preparing brown algae polyphenol nanoparticles according to claim 1, characterized in that: In step (3), the ratio of polyethylene glycol to the sum of the mass of arginine and brown algae polyphenols is 1:10-20.

6. The method for preparing brown algae polyphenol nanoparticles according to claim 1, characterized in that: In step (3), the volume concentration of hydrogen peroxide is 30%, the added amount is 20 mL, and 5 mL of hydrogen peroxide is added dropwise every 5 minutes to promote the oxidation of brown algae polyphenols.

7. The method for preparing brown algae polyphenol nanoparticles according to claim 1, characterized in that: In step (3), 75 μL of acetic acid is added, and the volume concentration of acetic acid is 36%.

8. The method for preparing brown algae polyphenol nanoparticles according to claim 1, characterized in that: In step (3), hydrogen peroxide is added and the reaction is continued for 18-24 hours, and acetic acid is added and the reaction is continued for 6 hours.

9. Brown algae polyphenol nanoparticles prepared by the method according to any one of claims 1 to 8.

10. Use of the brown algae polyphenol nanoparticles according to claim 9 in preparing a drug for treating ulcerative colitis.

Images