Preparation method and application of colon-targeted Chlorella vulgaris / peoniflorin hydrogel

Abstract

The present invention discloses a preparation method and application of a colon-targeted Chlorella vulgaris / peoniflorin hydrogel. The colon-targeted Chlorella vulgaris / peoniflorin hydrogel can deliver Chlorella and paeoniflorin to the colon in a targeted manner, thereby alleviating intestinal inflammation by downregulating the level of intestinal pro-inflammatory cytokines and maintaining intestinal epithelial barrier function. In addition, partially released paeoniflorin enters the blood-brain barrier and, by regulating inflammatory signaling pathways, inhibits abnormal activation of microglia, reduces brain inflammation, relieves anxiety and depression, and restores brain cognition. Finally, in terms of regulating the diversity and composition of the host's microbiota, paeoniflorin forms an organic interaction pattern with Chlorella, improves the richness and diversity of intestinal flora and metabolites, and improves ulcerative colitis and mental disorders such as depressive symptoms and cognitive impairment induced by intestinal dysfunction.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to the preparation of a colon-targeted Chlorella vulgaris / peoniflorin hydrogel and its oral application. Background Art

[0002] Ulcerative colitis (UC) is a chronic inflammatory intestinal disease characterized by repeated relapses and remissions, heterogeneous clinical phenotypes, and an unpredictable course. An increasing number of studies have reported a relatively high prevalence of depression and anxiety in UC patients. In particular, 41.3% of patients with active UC suffer from depression, while 70.8% suffer from anxiety disorders. Furthermore, cognitive dysfunction, now widely recognized as part of the depressive symptom spectrum, is also common in patients with UC. In this patient population, the presence of depression or anxiety can lead to decreased quality of life, complicate the clinical management of UC, and even increase the frequency of active disease. The brain-gut axis hypothesis explains the correlation between biobehavioral, emotional, and cognitive function in subjects with UC. However, the unknown mechanisms underlying these signaling alterations have hindered drug development.

[0003] Paeoniflorin (PA) is a traditional Chinese medicine monomer isolated from the traditional Chinese medicines red peony root and white peony root. Functional studies have shown that paeoniflorin has multiple pharmacological activities, such as anti-inflammatory, antidepressant, anti-tumor, neuroprotective, and immune-modulating. At the same time, multiple studies have shown that different forms of paeoniflorin can enter the brain, suggesting that paeoniflorin may be a potential drug for improving colitis-associated depression and cognitive decline. However, paeoniflorin has disadvantages such as poor stability, poor gastrointestinal absorption, and low permeability, resulting in low bioavailability and significant limitations in its clinical application.

[0004] Chlorella vulgaris (CV) is a single-celled algae that is high in protein, polysaccharides, low in fat, and rich in various vitamins and minerals. It possesses numerous health benefits and can be used in the food industry as a functional food and nutritional supplement. Studies have shown that the active ingredients in Chlorella vulgaris can regulate gastrointestinal absorption and promote toxin excretion. Given Chlorella's high nutritional and pharmacological value and low cultivation cost, developing Chlorella into an oral treatment for ulcerative colitis and associated psychiatric disorders holds great promise. Summary of the Invention

[0005] The present invention aims to provide a natural, environmentally friendly, simple, feasible, and easily scalable method for preparing a colon-targeted Chlorella vulgaris / peoniflorin hydrogel, and its application in the treatment of ulcerative colitis and associated mental illnesses. The colon-targeted Chlorella vulgaris / peoniflorin hydrogel prepared by this method, after oral administration, can improve the stability of Chlorella vulgaris and paeoniflorin in the gastrointestinal tract, prevent drug damage by gastric acid and certain enzymes, reduce physical and chemical degradation of the drug, and target the drug to the site of intestinal inflammation. The hydrogel's stability, adhesion, and mechanical properties help improve the drug's residence time and tissue distribution in the intestine, achieve sustained drug release, and significantly increase the drug's oral availability, thereby enhancing the therapeutic efficacy of ulcerative colitis and depression and cognitive impairment induced by intestinal inflammation, while avoiding systemic adverse reactions.

[0006] The technical solutions adopted in the present invention are as follows:

[0007] A colon-targeted Chlorella vulgaris / peoniflorin hydrogel, comprising Chlorella vulgaris and paeoniflorin.

[0008] A method for preparing a colon-targeted Chlorella vulgaris / peoniflorin hydrogel, specifically comprising:

[0009] Chlorella vulgaris and paeoniflorin were added to a carboxymethyl chitosan / sodium alginate mixture, incubated with stirring in the dark, and genipin crosslinker was added. The mixture was cross-linked at 37°C to prepare a colon-targeted Chlorella vulgaris / peoniflorin hydrogel.

[0010] Furthermore, the concentrations of Chlorella vulgaris and paeoniflorin added to the carboxymethyl chitosan / sodium alginate mixture were 1×10 5 -1×10 9 cells / mL, 200-10000μg / mL.

[0011] Furthermore, the final concentration ranges of carboxymethyl chitosan and sodium alginate are 0.5% g / L-5% g / L and 0.5% g / L-5% g / L, respectively.

[0012] Furthermore, the final concentration of genipin added ranges from 0.01 g / L to 0.10 g / L.

[0013] The application of the colon-targeted Chlorella vulgaris / peoniflorin hydrogel of the present invention includes one or more of the following:

[0014] (1) preparing an oral medication for treating ulcerative colitis;

[0015] (2) Preparation of oral medications for the treatment of depression and cognitive impairment;

[0016] (3) Prepare oral medication for treating ulcerative colitis associated with mental disorders.

[0017] The present invention has the beneficial effect of using Chlorella vulgaris and paeoniflorin as active ingredients for oral medications, demonstrating significant potential for commercialization and clinical transformation. In this invention, Chlorella vulgaris and paeoniflorin are co-encapsulated with carboxymethyl chitosan and sodium alginate as a matrix, and a colon-targeted Chlorella vulgaris / peoniflorin hydrogel is synthesized via a one-step chemical crosslinking process. The raw materials are readily available, environmentally friendly, and the preparation method is simple. The hydrogel's acid resistance and colon-targeting properties can enhance drug stability in the gastrointestinal tract, improve drug retention time and tissue distribution in the intestine, and significantly enhance the drug's oral bioavailability.

[0018] In terms of oral therapeutic applications, the colon-targeted Chlorella vulgaris / peoniflorin hydrogel described in the present invention can alleviate intestinal inflammation by downregulating the expression of intestinal proinflammatory cytokines in ulcerative colitis mice and maintaining intestinal epithelial function after oral administration; the paeoniflorin released in the hydrogel can further pass through the blood-brain barrier, inhibit the proinflammatory pathways in the hippocampus, and improve anxiety and depression-like symptoms by inhibiting neuroinflammation in ulcerative colitis mice; the active substances such as proteins and polysaccharides rich in Chlorella vulgaris can regulate the diversity and composition of the host's microbiome, and further compounding Chlorella vulgaris and paeoniflorin can form an organic interaction pattern. Experimental results show that the composite Chlorella vulgaris and paeoniflorin have a synergistic effect, greatly improving the therapeutic effect on ulcerative colitis and associated mental illnesses, and has unexpected technical effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Preparation flow chart (top) and scanning electron micrograph (bottom) of colon-targeted Chlorella vulgaris / paeoniflorin hydrogel CV@PA-gel.

[0020] Figure 2 Drug release curves of CV@PA-gel in different environments.

[0021] Figure 3 Biodistribution diagrams of CV and CV@PA-gel in the body at different times after oral administration.

[0022] Figure 4 Figures showing the effects of Chlorella vulgaris, paeoniflorin, and CV@PA-gel on anal bleeding (A), body weight (B), rectal bleeding, colon length (C), and spleen size in DSS-induced ulcerative colitis, representing changes in inflammation (D). (*, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001).

[0023] Figure 5Comparative images of the intestinal barrier effects of Chlorella vulgaris, paeoniflorin, and CV@PA-gel after treatment of DSS-induced ulcerative colitis (*, p-value < 0.05; **, p-value < 0.01; ***, p-value < 0.001; ****, p-value < 0.0001). Figure A shows electron microscopic images of changes in intestinal tight junctions and microvilli; Figures B, C, D, and E show the expression of intestinal barrier-related proteins.

[0024] Figure 6 Figure 1 shows the changes in the intestinal bacterial microbiome after treatment with Chlorella vulgaris, paeoniflorin, and CV@PA-gel in DSS-induced ulcerative colitis. (*, p-value < 0.05; **, p-value < 0.01; ***, p-value < 0.001; ****, p-value < 0.0001). In the figure, A and B represent α- and β-diversity, respectively. C represents the principal axis analysis. D and E represent changes at the phylum and genus levels, respectively. F and G represent differential metabolites between CV@PA-gel and DSS.

[0025] Figure 7 Figure 1 shows the effects of Chlorella vulgaris, paeoniflorin, and CV@PA-gel on DSS-induced depression, anxiety-like behaviors, and cognitive impairment. (*, p-value < 0.05; **, p-value < 0.01; ***, p-value < 0.001; ****, p-value < 0.0001). Figures A and B show the results of the open field test and elevated plus maze test, respectively, to measure anxiety-like behaviors. Figures C and D show the results of the tail suspension test and forced swim test, respectively, to measure depression-like behaviors. Figures E and F show the results of the Y-maze test and novel object test, respectively, to measure cognitive function.

[0026] Figure 8 Figure 1 shows the protective effects of Chlorella vulgaris, paeoniflorin, and CV@PA-gel on hippocampal neuroplasticity and adult neurogenesis (*, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001). Figures A and B show the number of surviving and newly born neurons, respectively. Figures D and G show the protein and mRNA levels of the hippocampal synaptic proteins PSD95 and synaptophysin, respectively.

[0027] Figure 9This is a comparison chart of the test results of blood routine (WBC, white blood cell; RBC, red blood cell; HGB, hemoglobin; MCH, mean corpuscular hemoglobin; MCHC, mean corpuscular hemoglobin concentration; MCV, mean cell volume; PLT, platelet; HCT, hematocrit) and blood biochemical indicators (ALT, alanine aminotransferase; AST, aspartate aminotransferase; BUN, blood urea nitrogen; CREA, blood creatinine) after continuous oral administration of Chlorella vulgaris, paeoniflorin, and CV@PA-gel for 30 days. DETAILED DESCRIPTION

[0028] The present invention is further described below with reference to the following figures and examples, but the present invention is not limited to the following examples. In the following examples, w / v is weight-to-volume ratio, specifically in g / L. Chlorella vulgaris in the present invention was purchased from Guangyu Company.

[0029] Example 1. Synthesis of colon-targeted Chlorella / peoniflorin hydrogel

[0030] 2% (w / v) carboxymethyl chitosan (CMCS) and 2% (w / v) sodium alginate (SA) were mixed at a volume ratio of 1:1 to obtain a CMCS / SA mixed solution. Chlorella vulgaris (CV) samples were collected by centrifugation (4500 rpm, 10 min) and washed three times with PBS. 1 mL of 14×10 7 1 mL of CV sample (100 cells / mL) and 1 mL of 20 mg / mL paeoniflorin (PA) were added to the above 3 mL of CMCS / SA mixed solution, and genipin was added to a final concentration of 0.02% (w / v). Cross-linking was carried out at 37°C for 12 h to obtain the colon-targeted Chlorella vulgaris / paeoniflorin hydrogel (CV@PA-gel). Figure 1 After cross-linking with genipin, the color of the CMCS / SA, CV, and PA mixture changed from green to dark green, indicating successful gelation of the CV@PA-gel. The prepared CV@PA-gel exhibited a dense and porous network structure, providing sufficient spatial support for the loading and adhesion of CV and PA. Figure 1 (Bottom) Scanning electron microscopy (SEM) images show that a large number of CV cells are attached to the hydrogel surface or distributed between the hydrogel pores.

[0031] Example 2. Drug release performance

[0032] 5mL CV@PA-gel was placed in a dialysis bag with a molecular weight cutoff of 3kDa. The dialysis bag was immersed in 200mL PBS with different pH values ​​(1.8 or 7.4) and stirred continuously at 37°C. At different time points (0.5, 1, 2, 4, 6, 8, 12, 24, 48, 72h), a certain volume of release medium was taken out to calculate the drug release rate, and an equal volume of fresh PBS was added at the same time. The absorbance at 413nm was measured using a UV-2600 spectrophotometer, and the cumulative drug release rate of paeoniflorin at different time points was calculated based on the PBS standard curve of paeoniflorin. The results were referred to Figure 2 CV@PA-gel exhibited a slow and steady PA release profile under a simulated gastric environment (pH = 1.8), with a 72-hour drug release rate of only 35.81%. Under a simulated intestinal environment (pH = 7.4), CV@PA-gel rapidly released PA, achieving a drug release rate of 74.38% within 72 hours. The pH-responsive drug release properties of CV@PA-gel help reduce gastric loss of CV and PA, achieve intestinal-targeted release, and thus improve the oral bioavailability of CV and PA.

[0033] Example 3. Fluorescence imaging capability and in vivo distribution

[0034] The whole-body fluorescence imaging of mice after oral administration of CV@PA-gel was tested using a small animal in vivo imaging system. 7 cells / mL) and CV@PA-gel (prepared in Example 1; CV = 2.8 × 10 7 cells / mL, PA = 4 mg / mL) and injected into Balb / c nude mice by gavage. After different time periods, the signal images of the whole body of the mice were obtained using a small animal in vivo imaging device. The results refer to Figure 3 A clear fluorescent signal was observed in the mouse abdomen 0.5 hours after administration. Over time, the signal gradually shifted downward and weakened. The fluorescent signal in the CV@PA-gel group was stronger and more persistent than that in the CV group, indicating that the CMCS / SA hydrogel effectively protects the loaded drug from degradation in the stomach and acts as an adhesive, prolonging its retention in the gastrointestinal tract.

[0035] Example 4. Anti-colitis properties

[0036] Mice were divided into five groups: control group (CTR), DSS group, DSS+CV group, DSS+PA group, and DSS+CV@PA-gel group. The control group (CTR) was fed a normal diet, while the DSS group, DSS+CV group, DSS+PA group, and DSS+CV@PA-gel group were fed with 2 wt% DSS solution (1 week) and water (1 week) alternately for 9 weeks to establish a DSS-induced ulcerative colitis mouse model. Starting from the 5th week, mice in the DSS+CV group, DSS+PA group, and DSS+CV@PA-gel group were given 300 μL of CV (CV = 2.8 × 10 7 cells / mL), PA (PA = 4 mg / mL), CV@PA-gel (prepared in Example 1; CV = 2.8 × 10 7 cells / mL, PA = 4 mg / mL) were injected into C57BL / 6J mice by gavage. Figure 4 Mice treated with CV@PA-gel showed significant improvement in weight loss and spleen weight, mild rectal bleeding, and colon length similar to that of the normal group, significantly outperforming the other treatment groups. This suggests that CV@PA-gel has excellent anti-inflammatory properties and can effectively alleviate the inflammatory response of colitis.

[0037] Example 5. Intestinal barrier protection performance

[0038] The intestines of the mice with DSS-induced ulcerative colitis in each group of Example 4 were fixed with glutaraldehyde and then observed using a transmission electron microscope to assess the damage to the intestinal structure. Figure 5 In mice treated with CV@PA-gel, damage to the intestinal tight junction structure was alleviated, and the colonic microvilli became denser. Furthermore, immunohistofluorescence analysis and qPCR results showed that CV@PA-gel significantly improved the expression of intestinal barrier-related proteins (ZO-1, OCLN, and Claudin-1), indicating that CV@PA-gel can alleviate intestinal barrier damage caused by colitis.

[0039] Example 6. Intestinal bacterial microbial homeostasis protection performance

[0040] After euthanasia, the mice with DSS-induced ulcerative colitis in each group of Example 4 were immediately collected for fecal samples to be analyzed by 16S and non-targeted metabolomics. Figure 6 CV@PA-gel was able to restore the intestinal microbiome structure of colitis mice, increase bacterial diversity, and improve the abundance of beneficial bacteria. Non-targeted metabolomics analysis showed that CV@PA-gel could reverse DSS-induced metabolite abnormalities and key pathway disturbances. This suggests that CV@PA-gel can alter intestinal microbiome and metabolism, maintaining intestinal bacterial microbial homeostasis.

[0041] Example 7. Anti-colitis-induced depression, anxiety and cognitive impairment performance

[0042] The mice with DSS-induced ulcerative colitis in each group of Example 4 were subjected to open field test, elevated plus maze test, forced swim test, tail suspension test, Y maze and novel object recognition test to evaluate the depression, anxiety-like behavior and cognitive impairment of the mice. Figure 7 In the open field test and elevated plus maze test, CV@PA-gel significantly increased the time spent locomotion in the central area and open arms, demonstrating its anxiety-relieving effect. In the tail suspension test and forced swim test, the immobility time of mice in the CV@PA-gel group was reduced, indicating that CV@PA-gel improved the depressive-like behavior of mice with colitis. In the Y-maze test and novel object recognition test, CV@PA-gel increased the time mice spent exploring novel arms and objects, indicating that CV@PA-gel treatment can improve cognitive impairment.

[0043] Example 8. Neuroplasticity and hippocampal adult neurogenesis protective properties

[0044] The DSS-induced ulcerative colitis mice in each group of Example 4 were euthanized and the brain tissues were taken out. They were fixed with 4% paraformaldehyde and then sliced. Nissl staining and immunofluorescence staining were performed to evaluate the neuroplasticity and hippocampal adult neurogenesis damage of the mice. Figure 8 . The number and morphological structural integrity of Nissl bodies can reflect the functional activity of neurons. The results showed that CV@PA-gel was able to reverse the decrease in the number of Nissl-positive cells in the mouse hippocampus caused by DSS; in addition, immature neurons were labeled with doublecortin (DCX), and it was found that CV@PA-gel reversed the trend of decreased number of DCX+ cells caused by DSS, indicating that CV@PA-gel had a beneficial effect on hippocampal regeneration ability. In addition, CV@PA-gel reversed the DSS-induced decrease in the expression levels of synaptic-related proteins (synaptophysin, SYP; postsynaptic density 95, PSD-95) in the hippocampus. The above results indicate that CV@PA-gel has neuroplasticity and hippocampal adult neurogenesis protective properties.

[0045] Example 9. Good oral safety

[0046] 300 μL CV (CV = 2.8 × 10 7 cells / mL), PA (PA = 4 mg / mL), CV@PA-gel (prepared in Example 1; CV = 2.8 × 10 7cells / mL, PA = 4 mg / mL) were injected into C57BL / 6J mice by gavage. After 30 days of administration, blood samples were collected from the mice for routine blood tests and blood biochemistry tests. The results were referred to Figure 9 After administration of CV@PA-gel, the main blood routine and blood biochemical indicators of mice were within the normal range, indicating that CV@PA-gel has good oral safety.

Claims

1. A method for preparing a colon-targeted Chlorella vulgaris / peoniflorin hydrogel, characterized in that: Specifically: Chlorella vulgaris and paeoniflorin were added to a carboxymethyl chitosan / sodium alginate mixture, incubated with stirring in the dark, and genipin crosslinking agent was added to prepare a colon-targeted Chlorella vulgaris / peoniflorin hydrogel through crosslinking. The concentrations of Chlorella vulgaris and paeoniflorin added to the carboxymethyl chitosan / sodium alginate mixture were 1×10 5 -1×10 9 cells / mL, 200-10000 μg / mL; The final concentration ranges of carboxymethyl chitosan and sodium alginate were 0.5% g / L-5% g / L and 0.5% g / L-5% g / L, respectively.

2. The preparation method according to claim 1, characterized in that The final concentration of genipin added ranged from 0.01 g / L to 0.10 g / L.

3. A colon-targeted Chlorella vulgaris / peoniflorin hydrogel prepared by the preparation method according to any one of claims 1-2, wherein the hydrogel is composed of Chlorella vulgaris and paeoniflorin loaded in the hydrogel.

4. A use of the colon-targeted Chlorella vulgaris / peoniflorin hydrogel according to claim 3, characterized in that: Include one or more of the following: (1) Preparation of oral medication for the treatment of ulcerative colitis; (2) Prepare oral medications for the treatment of depression and cognitive impairment.

5. The use according to claim 4, characterized in that The oral medication for treating ulcerative colitis is specifically an oral medication for treating ulcerative colitis associated with mental disorders.

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