Preparation method and application of colon-targeted chlorella vulgaris / paeoniflorin hydrogel

Abstract

The invention discloses a preparation method and application of colon-targeted chlorella vulgaris / paeoniflorin hydrogel. The colon-targeted chlorella vulgaris / paeoniflorin hydrogel can deliver chlorella vulgaris and paeoniflorin to colon in a targeted manner, and by lowering the level of intestinal proinflammatory cytokines, the intestinal epithelial barrier function is maintained, and intestinal inflammation is relieved. Besides, part of released paeoniflorin enters a blood brain barrier, and by adjusting an inflammation signal channel, abnormal activation of microglial cells is inhibited, brain inflammation is relieved, anxiety and depressive emotion are relieved, and brain cognition is recovered. Finally, in the aspect of regulating the diversity and composition of microbiota of a host, paeoniflorin and chlorella form an organic interaction mode, the richness and diversity of intestinal flora and metabolites are improved, and ulcerative colitis, depressive symptoms induced by intestinal dysfunction, cognitive impairment and other mental disorders are improved.

Description

Technical Field

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

[0002] Ulcerative colitis (UC) is a chronic intestinal inflammatory disease characterized by repeated relapses and remissions, heterogeneous clinical phenotypes, and unpredictable course. An increasing number of studies have reported that the prevalence of depression and anxiety in UC patients is relatively high. In particular, 41.3% of patients with active ulcerative colitis suffer from depression, while 70.8% suffer from anxiety disorders. In addition, cognitive dysfunction is now widely recognized as part of the symptoms of depression, which is also common in patients with ulcerative colitis. In this patient population, the presence of depression or anxiety can lead to a decrease in 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 functions in subjects with ulcerative colitis. However, the unknown mechanisms behind the signaling changes have hindered drug development.

[0003] Paeoniflorin (PA) is a Chinese medicine monomer isolated from the traditional Chinese medicine 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 regulation. At the same time, many 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 the disadvantages of poor stability, poor gastrointestinal absorption, and low permeability, resulting in low bioavailability and significant limitations in clinical application.

[0004] Chlorella vulgaris (CV) is a unicellular algae that is high in protein, high in polysaccharides, low in fat, rich in multiple vitamins and minerals. It has multiple health functions and can be used in the food industry as a functional food and nutritional enhancer. Studies have shown that the active ingredients of Chlorella vulgaris have functions such as regulating gastrointestinal absorption and promoting toxin excretion. In view of the high nutritional and pharmacological value and low cultivation cost of Chlorella, developing Chlorella into an oral therapeutic drug for ulcerative colitis and associated mental illness has broad development prospects. Summary of the invention

[0005] The purpose of the present invention is to provide a natural, environmentally friendly, simple, feasible and easy-to-scale colon-targeted Chlorella vulgaris / peonidin hydrogel preparation method and its application in the treatment of ulcerative colitis and associated mental illness, etc. The colon-targeted Chlorella vulgaris / peonidin hydrogel prepared by the method can improve the stability of Chlorella vulgaris and paeonidin in the gastrointestinal tract after oral administration, avoid the destruction of drugs by gastric acid and certain enzymes, reduce the physical and chemical degradation of drugs, and target the intestinal inflammation site. The stability, adhesion and mechanical properties of the hydrogel help to improve the retention time and tissue distribution of the drug in the intestine, and achieve sustained release of the drug, significantly improve the oral utilization of the drug, thereby improving the therapeutic effect of ulcerative colitis and depression induced by intestinal inflammation, and cognitive impairment, and avoiding systemic adverse reactions.

[0006] The technical solution adopted by the present invention is specifically as follows:

[0007] A colon-targeted Chlorella vulgaris / peonidin hydrogel, which consists of Chlorella vulgaris and paeonidin.

[0008] A method for preparing a colon-targeted Chlorella vulgaris / peonidin 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 crosslinking agent was added. The mixture was crosslinked 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 the added genipin ranges from 0.01 g / L to 0.10 g / L.

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

[0014] (1) Preparing an oral drug 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 beneficial effect of the present invention is that common Chlorella and paeoniflorin are used as oral active ingredients of medicine, which has great potential for commercialization and clinical transformation. In the present invention, carboxymethyl chitosan and sodium alginate are used as the matrix to co-coat Chlorella and paeoniflorin, and a colon-targeted common Chlorella / peoniflorin hydrogel is synthesized by a one-step chemical crosslinking method. The raw materials are easily available, green and environmentally friendly, and the preparation method is simple. The acid resistance and colon targeting properties of the hydrogel can improve the stability of the drug in the gastrointestinal tract, improve the retention time and tissue distribution of the drug in the intestine, and significantly improve the oral bioavailability of the drug.

[0018] In terms of oral therapeutic applications, the colon-targeted Chlorella vulgaris / peonidin hydrogel described in the present invention can relieve intestinal inflammation by downregulating the expression of intestinal proinflammatory cytokines in ulcerative colitis mice and maintaining intestinal epithelial function after oral administration; the paeonidin 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 microbiota, and further compound Chlorella vulgaris and paeonidin can form an organic interaction pattern. Experimental results show that the composite Chlorella vulgaris and paeonidin 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 / peonidin hydrogel CV@PA-gel.

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

[0021] Figure 3 Biodistribution diagram of CV and CV@PA-gel in vivo at different time periods after oral administration.

[0022] Figure 4 The results of anal bleeding (A), body weight (B), rectal bleeding, colon length (C), and spleen size after treatment of DSS-induced ulcerative colitis with Chlorella vulgaris, paeoniflorin, and CV@PA-gel represent the changes in inflammation levels (D). (*, p value < 0.05; **, p value < 0.01; ***, p value < 0.001; ****, p value < 0.0001).

[0023] Figure 5This is a comparison of the intestinal barrier 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). In the figure, A is an electron microscopic image of the changes in intestinal tight junctions and microvilli under an electron microscope, and B, C, D, and E are the results of the expression of intestinal barrier-related proteins.

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

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

[0026] Figure 8 The results of the protective effects of Chlorella vulgaris, paeoniflorin, and CV@PA-gel on hippocampal neural plasticity and adult neurogenesis in the hippocampus. (*, p value < 0.05; **, p value < 0.01; ***, p value < 0.001; ****, p value < 0.0001). In the figure, AC are the results of the number of surviving neurons and newborn neurons, respectively. DG are the results of the protein and mRNA levels of hippocampal synaptic-related proteins PSD95 and synaptophysin, respectively.

[0027] Fig. 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 transferase; AST, aspartate transferase; BUN, 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 will be further described below in conjunction with the following figures and examples, but the present invention is not limited to the following examples. In the following examples, w / v is a weight-to-volume ratio, and the specific unit is g / L. The common Chlorella in the present invention was purchased from Guangyu Company.

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

[0030] 2% (w / v) carboxymethyl chitosan (CMCS) and 2% (w / v) sodium alginate (SA) were mixed in 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 3 times with PBS. 1 mL of 14×10 7 Cells / mL of CV sample and 1mL, 20mg / mL of paeoniflorin (PA) were added to the above 3mL CMCS / SA mixed solution, and genipin was added to make the final concentration 0.02% (w / v). Cross-linking was carried out at 37℃ for 12h to obtain colon-targeted Chlorella vulgaris / peoniflorin hydrogel (CV@PA-gel). Results refer to Figure 1 After adding genipin for cross-linking, the color of the mixture of CMCS / SA, CV and PA changed from green to dark green, indicating that CV@PA-gel gelation was successful. The prepared CV@PA-gel showed a dense and porous network structure, which provided sufficient space 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 by UV-2600 spectrophotometer, and the cumulative drug release rate of peony inoside at different time points was calculated in combination with the PBS standard curve of peony inoside. The results are referred to Figure 2 , CV@PA-gel showed a slow and steady PA release curve under simulated gastric environment (pH=1.8), and the drug release rate in 72 hours was only 35.81%. In simulated intestinal environment (pH=7.4), CV@PA-gel rapidly released PA, and the drug release rate was as high as 74.38% within 72 hours. The pH-responsive drug release characteristics of CV@PA-gel help to reduce the gastric loss of CV and PA, achieve intestinal targeted release, and thus improve the bioavailability of oral CV and PA.

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

[0034] The whole-body fluorescence imaging of mice after intragastric 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 , 0.5h after administration, obvious fluorescence signals were observed in the abdomen of mice, and the fluorescence signals gradually moved downward and weakened over time. The fluorescence signal of the CV@PA-gel group was stronger and more persistent than that of the CV group, indicating that CMCS / SA hydrogel can effectively protect the loaded drugs from being degraded in the stomach, play an adhesion role, and prolong the retention time of drugs in the gastrointestinal tract.

[0035] Example 4. Anti-colitis properties

[0036] The mice were divided into 5 groups: control group CTR, DSS, DSS+CV, DSS+PA, and DSS+CV@PA-gel groups; the control group CTR was fed normally, and the DSS, DSS+CV, DSS+PA, and DSS+CV@PA-gel groups were fed C57BL / 6J mice alternately with 2wt% DSS solution (1 week) and water (1 week) for 9 weeks to construct a DSS-induced ulcerative colitis mouse model. Starting from the 5th week, the mice in the DSS+CV, DSS+PA, and DSS+CV@PA-gel groups were fed 300μL 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 The 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, which was significantly better than that of the other treatment groups. This indicates that CV@PA-gel has good anti-inflammatory ability 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 observed using a transmission electron microscope to evaluate the damage to the intestinal structure. Figure 5 The damage of intestinal tight junction structure in mice treated with CV@PA-gel was improved, and the colonic microvilli were denser. In addition, immunohistofluorescence analysis and qPCR results showed that CV@PA-gel could more significantly improve the expression of intestinal barrier-related proteins (ZO-1, OCLN, Claudin-1), indicating that CV@PA-gel can alleviate the 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 for 16S and non-targeted metabolomics analysis. Figure 6 , CV@PA-gel can restore the intestinal flora structure of colitis mice, increase bacterial diversity, and improve the abundance of beneficial bacteria. The results of non-targeted metabolomics analysis showed that CV@PA-gel can restore the abnormal metabolites and key pathway disorders caused by DSS. This shows that CV@PA-gel can change the intestinal flora and metabolism and maintain the homeostasis of intestinal bacterial microorganisms.

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

[0042] The DSS-induced ulcerative colitis mice in each group of Example 4 were subjected to open field test, elevated plus maze test, forced swimming 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 can more significantly increase the movement time in the central area and open arms, indicating that CV@PA-gel has the effect of improving anxiety. In the tail suspension test and forced swimming 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 colitis mice. In the Y maze test and novel object recognition test, CV@PA-gel can increase the exploration time of mice in new arms and new 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 of the mice were obtained. After fixation with 4% paraformaldehyde, the slices were sliced ​​and Nissl staining and immunofluorescence staining were performed to evaluate the neuroplasticity of the mice and the damage of adult neurogenesis in the hippocampus. 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 the decrease in the number of DCX+ cells caused by DSS, indicating that CV@PA-gel had a beneficial effect on the regenerative capacity of the hippocampus. 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 protective properties for adult neurogenesis in the hippocampus.

[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=4mg / mL) was injected into C57BL / 6J mice by gavage. After 30 days of administration, blood samples were collected from mice for routine blood tests and blood biochemical tests. The results were referred to Fig. 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 / peonidin 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 a genipin crosslinking agent was added and cross-linked to prepare a colon-targeted Chlorella vulgaris / peoniflorin hydrogel.

2. The preparation method according to claim 1, characterized in that: 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.

3. The preparation method according to claim 1, characterized in that: 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.

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

5. A colon-targeted Chlorella vulgaris / peonidin hydrogel prepared by the preparation method according to any one of claims 1 to 4, wherein the hydrogel is composed of Chlorella vulgaris and paeoniflorin loaded in the hydrogel.

6. An application of the colon-targeted Chlorella vulgaris / peonidin hydrogel according to claim 5, characterized in that: Includes one or more of the following: (1) Preparing an oral drug for treating ulcerative colitis; (2) Preparation of oral medications for the treatment of depression and cognitive impairment; (3) Prepare oral medication for treating ulcerative colitis associated with mental disorders.

Images