Preparation method of blueberry honeysuckle exosome and application thereof

Abstract

The application discloses a preparation method of blueberry honeysuckle exosome and application thereof, and belongs to the field of biological medicines. The blueberry honeysuckle exosome is prepared by a four-step method of differential centrifugation combined with ultrahigh-speed centrifugation, and it is proved that the blueberry honeysuckle exosome can effectively improve the pathological symptoms of ulcerative colitis model mice, significantly reverses the weight loss of the model mice, reduces the hematochezia index and restores the colon length, and shows a clear intervention potential for ulcerative colitis. Meanwhile, the exosome also shows significant anti-depression activity. The application provides a new research and development direction for the prevention and treatment of colitis and anti-depression.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, and in particular relates to a method for preparing Lonicera japonica exosomes and their application in the preparation of anti-colitis active drugs and antidepressant products. Background Technology

[0002] Extracellular vesicles are naturally occurring nanoscale vesicles that are actively produced and released by various cells. In the past, these structures were often mistaken for metabolic waste, but current research has confirmed that they are important mediators of intercellular communication. These vesicles contain various functional molecules such as proteins and nucleic acids, which can be recognized and absorbed by target cells, thereby regulating a variety of physiological and pathological processes.

[0003] Based on their biogenetic pathways, exosomes can be mainly divided into two categories: exosomes and microvesicles. They participate in numerous processes such as immunity, coagulation, and disease development, and their specific contents can serve as disease biomarkers. Simultaneously, their natural penetrating ability makes them highly promising drug delivery carriers.

[0004] Similar structures also exist in plants, namely plant-derived exosome nanoparticles, which also have anti-inflammatory and gut microbiota-regulating biological activities, showing broad application prospects in the treatment and drug delivery.

[0005] CN119662517A discloses the use of Schisandra chinensis-like exosome nanovesicles in the preparation of antidepressant products. Exosome-like nanovesicles (SCDENs) with anti-inflammatory and antidepressant activities are prepared from Schisandra chinensis, which effectively improve depressive-like behavior and hippocampal tissue damage. However, the extraction and preparation process of this patent application is complicated, and a five-step centrifugation method is used to obtain better exosomes.

[0006] Blue honeysuckle (scientific name: *Lonicera caerulea* L.), also known as blue honeysuckle or blue honeysuckle, is a deciduous plant belonging to the genus *Lonicera* in the family Caprifoliaceae (see *Liaoning Plants*, edited by Zhang Shumei, p. 1391). The fruit of *Lonicera caerulea* can be used medicinally, possessing heat-clearing and detoxifying properties. It can be used to treat symptoms such as abdominal distension and gastric ulcers, and also has anti-cancer and lipid-lowering effects. The fruit is also edible, sweet and juicy, and is a nutritious berry containing vitamins, minerals, and other bioactive substances.

[0007] This invention is the first to prepare Lonicera japonica exosome nanovesicles (LCDENs) from Lonicera japonica and discover that they have therapeutic effects on inflammatory bowel disease, providing a new approach for the clinical relief of colitis. Summary of the Invention

[0008] The purpose of this invention is to provide a method for preparing Lonicera japonica exosomes and their application in relieving colitis and antidepressant effects. The LCDENs prepared by this method have intact structures, good stability, good biocompatibility, and can effectively exert anti-inflammatory and antidepressant activities.

[0009] To achieve the above-mentioned objectives, the following technical solution is adopted:

[0010] This invention discloses a method for preparing exosomes from *Lonicera japonica*. The method employs a four-step centrifugation process to prepare *Lonicera japonica* exosomes, comprising the following steps:

[0011] 1) Juice the blue honeysuckle fruit and filter it with gauze to obtain filtrate a; centrifuge filtrate a at 1500 g ultra-low speed to remove the bottom precipitate and obtain supernatant b;

[0012] 2) Centrifuge the supernatant b at a low speed of 4000 g to 5000 g using an ultrafiltration tube to obtain the supernatant concentrate c;

[0013] 3) Centrifuge the supernatant concentrate c at 10000 g to obtain supernatant d;

[0014] 4) Take the supernatant d and filter it through a 0.45 μm filter membrane to obtain filtrate e; the precipitate obtained by ultra-high speed centrifugation of filtrate e at 100,000 g to 120,000 g is the exosome of Lonicera japonica.

[0015] Preferably, the juicing and centrifugation temperature is 1°C to 16°C; more preferably, it is 4°C.

[0016] Preferably, the high-speed centrifugation conditions are 10,000 g × 30 min, and the ultra-high-speed centrifugation conditions are 120,000 g × 60 min.

[0017] Preferably, the average particle size of the *Lonicera japonica* exosomes is 150 nm to 300 nm, and the Zeta potential is -18 mV ± 5 mV; preferably, the average particle size of the *Lonicera japonica* exosomes is 196.1 nm, and the Zeta potential is -17.8 mV.

[0018] The present invention also provides a blue honeysuckle exosome prepared by the above preparation method.

[0019] This invention also provides the application of Lonicera japonica exosomes in the preparation of antidepressant products, wherein the products are preferably food or pharmaceuticals, wherein the food is preferably functional food, health product, or special dietary food, and wherein the food form is preferably solid, semi-solid, liquid, or fluid other than semi-solid and liquid.

[0020] This invention also provides the application of Lonicera japonica exosomes in the preparation of pharmaceuticals for improving intestinal inflammation.

[0021] This invention also provides the application of Lonicera japonica exosomes in the preparation of functional foods, health products, and special dietary foods for improving enteritis, wherein the preferred form is solid, semi-solid, liquid, or fluid other than semi-solid and liquid.

[0022] The present invention also provides a medicament for treating colitis, the medicament comprising honeysuckle exosomes and a pharmaceutically acceptable carrier.

[0023] The dosage form of the drug is tablets, capsules, pills, powders, granules, suspensions, oral solutions, injections, or rectal preparations.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] First, this invention is the first to pioneer the study of the medical value of exosomes from *Lonicera japonica*, particularly in its anti-colitis and anti-depression effects. Unexpectedly, *Lonicera japonica* exosomes prepared from *Lonicera japonica* significantly improved disease symptoms in mice with ulcerative colitis, effectively reversing weight loss, reducing fecal bleeding index, and restoring colon length, demonstrating excellent therapeutic potential for ulcerative colitis. Furthermore, *Lonicera japonica* exosomes can alleviate pathological damage to hippocampal tissue and effectively improve depressive-like behaviors.

[0026] Secondly, this invention prepares Lonicera japonica exosomes by combining differential centrifugation with ultra-high speed centrifugation. In particular, it improves upon existing centrifugation methods by combining differential centrifugation and ultra-high speed centrifugation for Lonicera japonica fruit pulp. The steps and centrifugal force of differential centrifugation are adjusted, reducing the overall centrifugation process from the traditional five-step method to a four-step method. This simplifies the centrifugation process, improves preparation efficiency, and also enables the acquisition of stable Lonicera japonica exosomes. Attached Figure Description

[0027] Figure 1 For the morphological characterization of LCDENs provided in Example 1, A is a photograph of LCDENs precipitated at the bottom after ultra-high speed centrifugation; B is a transmission electron microscope image of LCDENs.

[0028] Figure 2 The particle size and zeta potential of the LCDENs provided in Example 1 are characterized, where A represents the particle size and B represents the zeta potential.

[0029] Figure 3 The particle size stability of the LCDENs provided in Example 1 after 24 hours of light irradiation;

[0030] Figure 4The fecal bleeding in mice with colitis after treatment with LCDENs provided in Example 3;

[0031] Figure 5 The colon length of mice with colitis treated with LCDENs as provided in Example 3;

[0032] Figure 6 Pathological analysis of colon tissue from mice with colitis after LCDENs treatment provided in Example 3, with a scale bar of 50 μm;

[0033] Figure 7 Y-maze experiment after establishing the LPS-induced depression model in C57 mice provided in Example 3. A is the number of times the mouse entered the arm; B is the spontaneous alternation rate of mice in each group. *** P<0.001, ## P<0.01, ns indicates no statistical difference;

[0034] Figure 8 After establishing the LPS-induced depression model in C57 mice provided in Example 3, an open field experiment was conducted. A represents the average speed of the mice; B represents the total distance traveled by the mice in each group; and C represents the number of times the mice in each group entered the central region. *** P<0.001, ## P<0.01, ### P < 0.001, ns indicates no statistical difference;

[0035] Figure 9 The pathological analysis of various brain regions of mice by LCDENs after the establishment of the LPS-induced depression model in C57 mice provided in Example 3 was performed using a scale bar of 200 μm, with a further magnified scale bar of 20 μm. Detailed Implementation

[0036] The present invention will be further described below with reference to specific embodiments. It should be understood that the specific embodiments described herein are for illustrative purposes only and are not intended to limit the scope of the invention.

[0037] Unless otherwise specified, the techniques or conditions described in the examples shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels. Unless otherwise specified, the experimental methods in the examples are conventional methods.

[0038] Example 1

[0039] In this embodiment, exosomes with vesicle structures from mature Lonicera blueae fruit were extracted and isolated.

[0040] The ultracentrifuge (manufactured by HITACHI, Japan) was provided by the Pharmacology Platform of Shenyang Pharmaceutical University, and the PBS buffer was provided by Wuhan Saiwei Biotechnology Co., Ltd. The BCA kit was provided by Beyotime Biotechnology Co., Ltd.

[0041] Juice was extracted from mature blue honeysuckle fruits, and the filtrate (a) was obtained by filtering through gauze. Filtrate (a) was centrifuged at 1500 g for 5 min at 4°C to obtain supernatant (b). Supernatant (b) was then centrifuged at 4000 g for 20 min using a 100 kDa ultrafiltration tube at 4°C to obtain concentrated supernatant (c). Concentrated supernatant (c) was centrifuged at 10000 g for 30 min at 4°C to obtain supernatant (d). Supernatant (d) was filtered through a 0.45 μm filter membrane to obtain filtrate (e). Filtrate (e) was then centrifuged at 120000 g for 60 min at 4°C to obtain a precipitate (…). Figure 1 Results A showed that precipitation was present at the bottom of the supernatant tube. The suspension was resuspended in 1 mL of 1×PBS (pH=7.4) to obtain the LCDENs suspension. The concentration of the extracted Lonicera japonica exosomes was then tested using a BCA kit.

[0042] Example 2

[0043] In this embodiment, the morphology of exosomes from Lonicera japonica was observed and their particle size, zeta potential, and photostability were detected.

[0044] The dynamic light scattering (DLS) particle size analyzer was provided by the instrument platform of the School of Functional Food and Wine, Shenyang Pharmaceutical University, and the transmission electron microscope was provided by Wuhan Milian Biotechnology Co., Ltd.

[0045] Observation morphology: Use a pipette to draw 10 μL of LCDENs suspension and drop it onto a disposable sealing film. Invert a 200-mesh copper mesh to ensure that the adsorption surface of the copper mesh is in full contact with the sample solution. Allow it to naturally adsorb for 20 min based on capillary action and the adsorption effect of the support film on the copper mesh surface. Then, use a pipette to draw 10 μL of 2% phosphotungstic acid solution to stain the Lonicera japonica exosomes and drop it onto the front side of the copper mesh. Let it stand for 90 s, then use a filter paper strip to remove excess droplets. Allow it to air dry at room temperature in the dark for 30 min and observe it using a transmission electron microscope. Figure 1 B shows that the LCDENs have a saucer-like structure.

[0046] Particle size determination: 20 μL of Lonicera japonica exosomes extracted by ultracentrifugation were taken, diluted with 980 μL of 1×PBS (pH=7.4), and the particle size was determined using a DLS particle size analyzer. The precipitate was resuspended in 1 mL of pure water and the zeta potential was determined. The particle size and zeta potential were detected at room temperature. Figure 2The results show that the exosomes of Lonicera japonica have a particle size range of about 150 nm to 300 nm, an average particle size of 196.1 nm, and a PDI index of 0.172, which is consistent with the particle size distribution of exosomes. This indicates that the LCDENs prepared in this invention have exosome-like nanovesicle morphology characteristics.

[0047] Potential detection: The higher the absolute value of the zeta potential, the more stable the system is and the less likely it is to aggregate. Figure 2 B shows that the Zeta potential of LCDENs is -17.8 mV, indicating that LCDENs are negatively charged and the solution system is relatively stable.

[0048] Photostability: 20 μL of Lonicera japonica exosomes extracted by ultracentrifugation were taken and diluted with 980 μL of 1×PBS (pH=7.4) to form solutions A and B. Solution A was left untreated as the blank group, and solution B was placed under natural light as the experimental group. The particle size of both solutions was measured after 24 h. Figure 3 The results showed that the particle size of LCDENs did not change significantly after 24 hours of light exposure, indicating that LCDENs are not easily decomposed by light and demonstrate a certain degree of stability.

[0049] Example 3

[0050] This embodiment demonstrates the construction of a DSS-induced acute colitis mouse model and the therapeutic effect of Lonicera japonica exosomes on acute colitis.

[0051] Twenty-five male C57BL / 6 mice, aged 6-8 weeks and weighing 23-25 ​​g, were provided by Liaoning Changsheng Biotechnology Co., Ltd. (Experimental Animal Production License No.: SCXK(Liaoning)2020-0001). All animal experimental procedures strictly adhered to relevant regulations on the management and use of experimental animals and were approved by the Animal Management Committee of Shenyang Pharmaceutical University (Animal Ethics Approval No.: SYPU-IACUC-S2024-0919-101). Acute colitis was induced in mice by feeding them drinking water containing 2.5% sodium dextran sulfate (DSS), thus establishing a mouse model of acute colitis. The 25 mice were randomly divided into five groups of five each: a normal control group (Ctrl group), a model group (DSS group), a low-dose LCDENs (25 mg / kg) group, a medium-dose LCDENs (50 mg / kg) group, and a high-dose LCDENs (100 mg / kg) group. The modeling period lasted from day 1 to day 7. The DSS group and the different doses of LCDENs groups had free access to 2.5% DSS aqueous solution, while the Ctrl group had free access to purified water. Drug administration was carried out from day 1 to day 7. A 10 mg / mL LCDENs suspension was prepared and administered by gavage according to the mice's body weight. The DSS group received an equal volume of purified water by gavage, and the control group received no treatment. On day 8, the presence of rectal bleeding was observed in each group of mice. The mice were dissected, and colon tissue was collected for length comparison. The colon tissue was fixed and subjected to H&E pathological staining.

[0052] The results show:

[0053] Blood in stool: Figure 4 The results showed that mice in the DSS group had rectal bleeding. Different doses of LCDENs showed signs of improvement after treatment, and the rectal bleeding was significantly improved after treatment with 100 mg / kg LCDENs.

[0054] Colon morphology: Figure 5 The results showed that the colon length of mice in the DSS group was significantly shorter, while the colon length of mice in the LCDENs groups with different doses was longer than that of mice in the DSS group, and similar to that of mice in the blank group. At the same time, it was clearly visible that the feces in the colon of mice in the DSS model group were unformed and there was blood in the stool, while the feces in the colon of mice in the LCDENs groups with different doses were normal in shape and there was no blood in the stool.

[0055] Colon status: Figure 6The results showed that, as indicated by the orange arrows, the colonic H&E stained sections of the DSS-affected mice exhibited significant intestinal epithelial cell shedding and crypt tortuosity (indicated by the yellow arrows). Simultaneously, as indicated by the blue arrows, significant colonic edema was observed in the DSS group. After treatment with LCDENs, the results showed that LCDENs improved the pathological features caused by colitis, including villus destruction, intestinal epithelial cell shedding, crypt tortuosity, and reduced goblet cells. After treatment with 100 mg / kg LCDENs, the colonic crypts significantly recovered, and goblet cell count increased, showing no significant difference from the colonic structure of normal mice.

[0056] Example 4

[0057] This embodiment investigates the construction of an LPS-induced depressed mouse model and the effect of Lonicera japonica exosomes on improving depressive-like behavior.

[0058] The experimental animals were 6-8 week old, 18-20 g male C57BL / 6 mice, provided by Liaoning Changsheng Biotechnology Co., Ltd. (Experimental Animal Production License No.: SCXK(Liaoning)2020-0001). All animal experimental procedures strictly adhered to relevant regulations on the management and use of experimental animals and were approved by the Animal Management Committee of Shenyang Pharmaceutical University (Animal Ethics Approval No.: SYPU-IACUC-S2024-1113-101). A lipopolysaccharide (LPS)-induced mouse depression model was used in the experiment. A total of 15 mice were randomly divided into 3 groups of 5 mice each: the Ctrl group, the LPS group, and the LCDENs (2.5 mg / kg) group. A 10 mg / mL LCDENs suspension was prepared. The LCDENs group was administered the drug by gavage according to the mice's body weight, the LPS group was administered an equal volume of purified water by gavage, and the Ctrl group received no treatment. The drug administration lasted for 7 days. Starting on day 8, mice in both the LPS and LCDENs groups were intraperitoneally injected with LPS (0.05 mg / kg) once daily for 7 days. Behavioral experiments (Y-maze and open field tests) began on day 14. The purpose of these experiments was to assess the successful establishment of the depression model and the therapeutic effect of LCDENs.

[0059] 1. Behavioral experiments:

[0060] (1) Y-maze experiment

[0061] The Y-maze test was used to assess the memory ability of mice.

[0062] A Y-shaped maze was constructed using three arms of equal length (30 cm), width (5 cm), and height (12 cm), with an angle of 120 degrees between any two adjacent arms. The three arms were randomly named "A," "B," and "C." All mice were tested individually, with each mouse given 8 minutes of testing time. First, a mouse was placed in one arm, and the name of that arm was recorded. Then, the mouse entered any other arm, and the name of that arm was quickly recorded. Before the test, the inside of each arm was wiped with an alcohol swab, and any excrement left by the mouse was cleaned. The number of times each mouse entered an arm within 8 minutes was counted.

[0063] Spontaneous alternation rate per mouse (%) = (Number of correct alternation responses / (Total responses - 2)) × 100%

[0064] The results are as follows Figure 7 As shown, there was no significant difference in the total number of arm advances among the groups, indicating that the mice in each group had good spontaneous activity ability and that it did not affect the experiment. Spontaneous alternation was used to assess the mice's learning and memory abilities. Compared with the Ctrl group, the spontaneous alternation rate in the LPS group was significantly lower, indicating that LPS induced a decrease in spatial memory ability in mice. Compared with the LPS group, the accuracy rate of spontaneous alternation behavior in the LCDENs-treated group was significantly increased, indicating that LCDENs improved the memory and spatial exploration abilities of LPS mice.

[0065] (2) Open field experiment

[0066] The depressive-like behavior of mice was evaluated using an open field test.

[0067] The open field experiment setup consisted of an open cardboard box, with the bottom of the box divided into 16 square grids. The four middle grids were designated as the central area, and the remaining 12 grids as the peripheral area. Mice were placed in the central area of ​​the box, ensuring there was no noise interference, and allowed to move and explore freely. The number of times the mouse crossed the central area and the time it spent in the central area were observed and recorded within 10 minutes, with the number of times the mouse's two paws crossed a grid as the criterion.

[0068] The results are as follows Figure 8 As shown, there was no significant difference in average movement speed among the Ctrl group, LPS group, and LCDENs-treated group, indicating that the mice's spontaneous activity ability was normal and had no impact on the experiment. Compared with the Ctrl group, the LPS group showed a decrease in total exploration distance and the number of times it entered the central region, indicating that LPS induced a decrease in the mice's spatial exploration ability and the occurrence of depressive-like behavior. Compared with the LPS group, the LCDENs-treated group showed a significant increase in total exploration distance and the number of times it entered the central region. These data demonstrate that LCDENs can improve the spatial learning ability, exploration ability, and depressive-like behavior in mice.

[0069] 2. H&E staining:

[0070] H&E staining was used to observe hippocampal cell tissue damage.

[0071] After dehydration, the whole brain tissue fixed in tissue fixative was embedded in paraffin and cut into 5 μm sections. The sections were then stained with hematoxylin and eosin sequentially. After staining, the sections were washed with water, dehydrated with ethanol, soaked in xylene, and mounted with resin of appropriate concentration. Finally, the morphology of the mouse brain tissue was observed and photographed using an optical microscope.

[0072] The results are as follows Figure 9 As shown, compared with the Ctrl group, the LPS group showed obvious nuclear condensation. After administering a certain concentration of LCDENs, the nuclear condensation in the DG, CA1, and CA3 regions of the hippocampus was significantly improved, indicating that LCDENs have the effect of improving hippocampal tissue damage.

[0073] The above description is only a partial embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing exosomes from Lonicera japonica, characterized in that, Includes the following steps: 1) Juice the blue honeysuckle fruit, filter it to obtain filtrate a; centrifuge filtrate a at 1500 g ultra-low speed to remove the bottom precipitate and obtain supernatant b; 2) Centrifuge the supernatant b at a low speed of 4000 g to 5000 g using an ultrafiltration tube to obtain the supernatant concentrate c; 3) Centrifuge the supernatant concentrate c at 10000 g to obtain supernatant d; 4) Take the supernatant d and filter it through a 0.45 μm filter membrane to obtain filtrate e; the precipitate obtained by ultra-high speed centrifugation of filtrate e at 100,000 g to 120,000 g is the exosome of Lonicera japonica.

2. The preparation method according to claim 1, characterized in that, The juicing and centrifugation temperatures are 1℃~16℃.

3. The preparation method according to claim 1, characterized in that, The high-speed centrifugation conditions were 10,000 g × 30 min, and the ultra-high-speed centrifugation conditions were 120,000 g × 60 min.

4. The preparation method according to claim 1, characterized in that, The average particle size of the *Lonicera japonica* exosomes is 150 nm to 300 nm, the Zeta potential is -18 mV ± 5 mV, and the PDI is 0.

172.

5. The application of a blue honeysuckle exosome in the preparation of antidepressant products, characterized in that, The products are functional foods, health products, special dietary foods, and medicines. The food forms are solid, semi-solid, liquid, or fluid forms other than semi-solid and liquid.

6. The application of a honeysuckle exosome in the preparation of a drug for improving enteritis.

7. The application of a blue honeysuckle exosome in the preparation of functional foods, health products, and special dietary foods for improving enteritis, characterized in that... The product can be in solid, semi-solid, liquid, or fluid form other than semi-solid and liquid.

8. A drug for treating colitis, characterized in that, The drug comprises honeysuckle exosomes and a pharmaceutically acceptable carrier.

9. The medicament according to claim 8, characterized in that, The dosage form of the drug is tablets, capsules, pills, powders, granules, suspensions, oral solutions, injections, or rectal preparations.

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