Application of hemerocallis citrina source exosome in preparation of anti-inflammatory drugs

Abstract

The invention provides a hemerocallis citrina derived exosome and an application thereof in preparation of an anti-inflammatory (especially anti-neuroinflammation) medicine. In-vitro and in-vivo verification on the hemerocallis citrina source exosome shows that the hemerocallis citrina source exosome not only has remarkable behavioral pharmacological activity, but also can effectively regulate and control the expression of related biochemical proteins, can be used as a potential alzheimer disease treatment preparation, is expected to reduce the medication cost of patients, and has a wide application prospect. The application of the plant exosome in the fields of behavioral pharmacology and neuroinflammation regulation and control is expanded, and the plant exosome has remarkable medical and economic values.

Description

Technical Field

[0001] This application belongs to the field of plant exosome preparation technology, specifically relating to an exosome derived from daylily and its application in the preparation of anti-inflammatory (especially anti-neuroinflammatory) drugs. Background Technology

[0002] Current technologies have significant limitations in the treatment of many neurological diseases. For example, in the treatment of Alzheimer's Disease (AD), existing drugs, such as cholinesterase inhibitors, can only slightly alleviate some cognitive symptoms and are unlikely to stop disease progression, with significant individual differences among patients. Furthermore, most existing drugs are chemically synthesized, and long-term use can easily cause side effects such as gastrointestinal discomfort and drowsiness, thus limiting their safety.

[0003] From a pathological perspective, inflammation is closely related to the development of various diseases. Existing research data has confirmed the presence of microglial overactivation and abnormally high expression of pro-inflammatory cytokines in Alzheimer's disease patients. Simultaneously, enhanced NF-κB nuclear translocation further exacerbates the inflammatory cascade, ultimately leading to cognitive decline and behavioral abnormalities. This pathological phenomenon is also significantly observed in other neurodegenerative diseases. Therefore, developing novel therapeutic drugs from an anti-inflammatory perspective provides a new approach for the research and development of neuroprotective drugs.

[0004] Plant exosomes, as a type of natural extract, possess good biocompatibility and high safety. However, research on their behavioral pharmacological activities and biochemical protein regulation related to Alzheimer's disease remains relatively scarce. Daylily, as a plant used both as food and medicine, has been consumed in China for thousands of years, and its safety has been verified through long-term consumption. Modern pharmacological studies have shown that the ethanol extract of daylily has certain neuroprotective effects, but its activity in combating Alzheimer's disease has not been systematically studied. In particular, the potential role of daylily-derived exosomes in improving memory impairment and anxiety behavior in Alzheimer's disease models, as well as regulating biochemical indicators such as Iba-1 and NF-κB, still requires further exploration and development. Summary of the Invention

[0005] In view of the problems existing in the prior art, the purpose of this application is to provide an exosome derived from daylily and its application in the preparation of anti-inflammatory (especially anti-neuroinflammatory) drugs.

[0006] Specifically, this application relates to the following aspects: 1. Application of daylily-derived exosomes in the preparation of anti-inflammatory drugs; Preferably, the inflammation includes peripheral inflammation or neuroinflammation; More preferably, the inflammation is neuroinflammation.

[0007] 2. Application of daylily-derived exosomes in the preparation of drugs that regulate the intestinal environment; Preferably, the exosomes derived from daylily regulate the intestinal environment by improving the release of short-chain fatty acids from the intestinal flora.

[0008] 3. The application according to item 1 or 2, wherein the exosomes derived from daylily have a particle size of 100-300 nm.

[0009] 4. The application according to any one of items 1-3, wherein the ZETA potential of the daylily-derived exosomes is (-35mV) to (-20mV).

[0010] 5. The application according to any one of items 1-4, wherein the method for preparing the exosomes derived from daylily comprises: Exosomes were obtained by extracting and separating them from daylilies; Preferably, the daylily is the flower bud of the daylily.

[0011] 6. According to the application described in item 5, the method for preparing the exosomes derived from daylily includes: Fresh daylily flowers are washed and crushed to obtain daylily juice. The daylily juice is then filtered to obtain a first solution. The first solution is centrifuged for the first time to extract the supernatant, thus obtaining the first supernatant; The first supernatant is centrifuged a second time to extract the supernatant and obtain the second supernatant. The second supernatant was centrifuged a third time to remove the supernatant, and the resulting precipitate was dissolved in PBS buffer to obtain the second solution. The second solution was centrifuged a fourth time to extract the supernatant, thus obtaining the third supernatant. The third supernatant was centrifuged for the fifth time, and the resulting precipitate was the exosomes derived from daylily.

[0012] 7. The application according to item 6, wherein the first centrifugation is performed at a speed of 2000-4000g for 10-30 minutes; the second centrifugation is performed at a speed of 9000-12000g for 20-60 minutes; the third centrifugation is performed at a speed of 100000-120000g for 70-120 minutes; the fourth centrifugation is performed at a speed of 9000-12000g for 20-60 minutes; and the fifth centrifugation is performed at a speed of 100000-120000g for 70-120 minutes.

[0013] 8. The application according to item 6 or 7, wherein the temperature of the first centrifugation, the second centrifugation, the third centrifugation, the fourth centrifugation and the fifth centrifugation is 3-5°C.

[0014] 9. A method for preparing exosomes derived from daylily, wherein the method comprises: Exosomes were obtained by extracting and separating them from daylilies; Preferably, the daylily is the flower bud of the daylily.

[0015] 10. The method according to item 9, wherein the method comprises: Fresh daylily flowers are washed and crushed to obtain daylily juice. The daylily juice is then filtered to obtain a first solution. The first solution is centrifuged for the first time to extract the supernatant, thus obtaining the first supernatant; The first supernatant is centrifuged a second time to extract the supernatant and obtain the second supernatant. The second supernatant was centrifuged a third time to remove the supernatant, and the resulting precipitate was dissolved in PBS buffer to obtain the second solution. The second solution was centrifuged a fourth time to extract the supernatant, thus obtaining the third supernatant. The third supernatant was centrifuged for the fifth time, and the resulting precipitate was the exosomes derived from daylily.

[0016] 11. The method according to item 10, wherein the first centrifugation is performed at a speed of 2000-4000g for 10-30 minutes; the second centrifugation is performed at a speed of 9000-12000g for 20-60 minutes; the third centrifugation is performed at a speed of 100000-120000g for 70-120 minutes; the fourth centrifugation is performed at a speed of 9000-12000g for 20-60 minutes; and the fifth centrifugation is performed at a speed of 100000-120000g for 70-120 minutes.

[0017] 12. The method according to item 10 or 11, wherein the temperature of the first centrifugation, the second centrifugation, the third centrifugation, the fourth centrifugation, and the fifth centrifugation is 3-5°C.

[0018] 13. Daylily-derived exosomes prepared according to any one of items 9-12.

[0019] 14. A composition comprising daylily-derived exosomes prepared according to any one of claims 9-12.

[0020] 15. The composition according to claim 14, wherein the composition is a pharmaceutical composition; Preferably, the composition further comprises a pharmaceutically acceptable carrier.

[0021] 16. The use of the composition according to item 14 or 15 in the preparation of an anti-inflammatory medicament; Preferably, the inflammation includes peripheral inflammation or neuroinflammation; More preferably, the inflammation is neuroinflammation.

[0022] 17. A drug carrier, wherein the drug carrier comprises exosomes derived from daylily; The exosomes derived from daylily are the daylily-derived exosomes used in the application described in item 1.

[0023] 18. The use of an exosome-loaded drug product in the preparation of an anti-inflammatory drug, wherein the exosome-loaded drug product uses daylily-derived exosomes as the drug carrier in the application described in item 1.

[0024] Beneficial effects: (1) The exosomes derived from daylily provided in this application have no toxic side effects of chemical drugs and are suitable for long-term use. Daylily resources are abundant and low in cost. The extraction process does not require special equipment and is conducive to large-scale production.

[0025] (2) The experimental data of this application show that the exosomes derived from daylily not only have significant behavioral pharmacological activities, but also effectively regulate the expression of related biochemical proteins. They can be used as a potential treatment agent for Alzheimer's disease, which is expected to reduce the cost of medication for patients and expand the application of plant exosomes in the fields of behavioral pharmacology and neuroinflammatory regulation, and have significant medical and economic value. Attached Figure Description

[0026] Figure 1 The images show the results of exosome detection from daylily using a transmission electron microscope. The scale bar for the top image is 100 nm, and the scale bar for the bottom image is 200 nm.

[0027] Figure 2 Results of nanoparticle tracking analysis of exosomes derived from daylily.

[0028] Figure 3 The results of ZETA potential detection for exosomes derived from daylily.

[0029] Figure 4The results of SDS-PAGE analysis of exosomes derived from daylily are shown. M represents the marker, and HHC-EXO represents exosomes derived from daylily.

[0030] Figure 5 The transcriptome analysis results are for exosomes derived from daylilies. Figure 5 A is a summary diagram of miRNA length distribution; Figure 5 B is a summary diagram of the top 20 miRNAs in exosomes derived from daylilies.

[0031] Figure 6 The results of CCK8 assay in BV2 cells after treatment with different concentrations of daylily-derived exosomes are shown. *p<0.05, **p<0.01.

[0032] Figure 7 The results show the NO release from BV2 cells after treatment with different concentrations of daylily-derived exosomes. *p<0.05, ***p<0.001 indicate a significant difference compared to the blank control group; #p<0.05, ###p<0.001 indicate a significant difference compared to the model group (i.e., stimulated only with 1 μg / mL LPS).

[0033] Figure 8 The results show the expression of Iba-1 in BV2 cells after treatment with different concentrations of daylily-derived exosomes. Green fluorescence represents positive Iba-1 expression, and blue fluorescence represents DAPI.

[0034] Figure 9 The results show the expression of NF-κB in BV2 cells after treatment with different concentrations of daylily-derived exosomes. Orange fluorescence represents positive NF-κB expression, and blue fluorescence represents DAPI.

[0035] Figure 10 The results show the expression of pro-inflammatory cytokines TNF-α, IL-1β, and IL-6 in BV2 cells after treatment with different concentrations of daylily-derived exosomes. Figure 10 A represents the detection results of TNF-α expression in BV2 cells; Figure 10 B represents the detection result of IL-1β expression in BV2 cells; Figure 10 C represents the detection results of IL-6 expression in BV2 cells. *p<0.05, **p<0.01, ***p<0.001 indicate a significant difference compared to the blank control group; #p<0.05, ##p<0.01, ###p<0.001 indicate a significant difference compared to the model group (i.e., stimulated only with 1 μg / mL LPS).

[0036] Figure 11The results show the spontaneous alternation rate of mice in each group during the Y-maze experiment. ***p<0.001 indicates a significant difference from the blank control group; #p<0.05 indicates a significant difference from the model group (i.e., injected only with 0.25 mg / kg LPS).

[0037] Figure 12 The results of the discrimination index test for each group of mice in the new object recognition experiment. **p<0.01 indicates a significant difference from the blank control group; #p<0.05 indicates a significant difference from the model group (i.e., injected only with 0.25 mg / kg LPS).

[0038] Figure 13 The results show the time mice spent in the central region during the open field test. ***p<0.001 indicates a significant difference from the blank control group; ##p<0.01 indicates a significant difference from the model group (i.e., injected with only 0.25 mg / kg LPS).

[0039] Figure 14 The results show the expression of Tau and its phosphorylated proteins in the brains of mice in each group. Figure 14 A shows the immunofluorescence staining results of phosphorylated Tau protein in the brains of mice in each group. Red fluorescence represents positive expression of phosphorylated Tau protein, and blue fluorescence represents DAPI. Figure 14 B represents the results of immunoblotting analysis of total Tau protein in the brains of mice in each group.

[0040] Figure 15 The results show the expression of Iba-1 in microglia in the brains of mice in each group. Red fluorescence represents microglia, and blue fluorescence represents DAPI.

[0041] Figure 16 The results show the expression levels of pro-inflammatory cytokines TNF-α, IL-1β, and IL-6 in the serum of mice in each group. Figure 16 A represents the detection result of TNF-α expression; Figure 16 B represents the detection result of IL-1β expression; Figure 16 C represents the detection result of IL-6 expression. *p<0.05, **p<0.01 indicate a significant difference from the blank control group; #p<0.05 indicates a significant difference from the model group (i.e., only injected with 0.25mg / kg LPS).

[0042] Figure 17 The results show the expression levels of pro-inflammatory cytokines TNF-α, IL-1β, and IL-6 in the brains of mice in each group. Figure 17 A represents the detection result of TNF-α expression; Figure 17B represents the detection result of IL-1β expression; Figure 17 C represents the detection result of IL-6 expression. *p<0.05, **p<0.01 indicate a significant difference from the blank control group; #p<0.05 indicates a significant difference from the model group (i.e., only injected with 0.25mg / kg LPS).

[0043] Figure 18 The results show the expression of short-chain fatty acids in the intestinal contents of mice in each group. Detailed Implementation

[0044] The present application is further illustrated below with reference to embodiments. It should be understood that the embodiments are only used to further illustrate and explain the present application and are not intended to limit the present application.

[0045] Unless otherwise defined, technical and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art. While similar or identical methods and materials may be applied in experimental or practical applications, materials and methods are described herein. In case of conflict, the definitions included herein shall prevail. Furthermore, materials, methods, and examples are for illustrative purposes only and are not intended to be limiting. The present application is further described below with reference to specific embodiments, but is not intended to limit the scope of the application.

[0046] definition As used herein, the term "inflammation" refers to acute and chronic inflammation of tissues, including neuroinflammation, which is acute and chronic inflammation of neural tissue, the main tissue component of the two parts of the nervous system: the brain and spinal cord of the central nervous system (CNS) and the peripheral nerves, branches of the peripheral nervous system (PNS). Chronic neuroinflammation is associated with neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, and multiple sclerosis. Acute neuroinflammation usually occurs immediately after injury to the central nervous system, for example, caused by traumatic brain injury (TBI).

[0047] As used in this article, the term “traumatic brain injury” (“TBI”, also known as “intracranial injury”) refers to brain damage caused by external mechanical forces such as rapid acceleration or deceleration, impact, shock wave, or projectile penetration.

[0048] As used herein, the term "neurodegenerative disease" refers to diseases associated with the progressive loss of structure or function of neurons, including neuronal death. Examples of neurodegenerative diseases include, but are not limited to, multiple sclerosis, Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis (ALS).

[0049] As used in this article, the term "Alzheimer's Disease (AD)" generally refers to early-onset dementia or senile dementia, a slowly progressing neurodegenerative disease that worsens over time. The most common early symptom is loss of short-term memory (difficulty remembering recent events). As the disease progresses, at least one of the following symptoms may gradually appear: language impairment, disorientation (e.g., getting lost), mood instability, loss of motivation, inability to care for oneself, and behavioral problems. The exact cause of Alzheimer's disease remains unknown, but its progression may be related to the deposition of amyloid plaques in the brain and fibrillary tangles caused by excessive phosphorylation of tau protein. Currently, there are no treatments that can stop or reverse the progression of the disease; only a few methods may temporarily alleviate or improve symptoms.

[0050] As used in this article, the terms "exosome," "daylily-derived exosome," and "daylily exosome" are used interchangeably and refer to small membrane vesicles containing complex RNA and proteins, a type of extracellular vesicle.

[0051] The terms “pharmaceuticalally acceptable carrier,” “pharmaceuticalally acceptable excipient,” “physiologically acceptable carrier,” or “physiologically acceptable excipient” refer to pharmaceutically acceptable materials, compositions, or carriers, such as liquid or solid fillers, diluents, solvents, or encapsulating materials. In some embodiments, each component is “pharmaceuticalally acceptable” in the sense of compatibility with other components of the pharmaceutical formulation and is suitable for use in contact with the tissues or organs of a subject (e.g., a human) without excessive toxicity, irritation, allergic reactions, immunogenicity, or other problems or complications, and is proportionate to a reasonable benefit / risk ratio. See, for example, Remington: The Science and Practice of Pharmacy, 23rd ed.; AdejareEd.; Academic Press, 2020; Handbook of Pharmaceutical Excipients, 9thed.; Sheskey et al., Eds.; Pharmaceutical Press, 2020; Handbook ofPharmaceutical Additives, 3rd ed.; Ash and Ash Eds.; Formulation,1st ed.;Gibson Ed.; CRC Press, 2015. In one aspect, this application provides the use of daylily-derived exosomes in the preparation of anti-inflammatory drugs.

[0052] In some embodiments, the inflammation includes peripheral inflammation or neuroinflammation.

[0053] In some implementations, the inflammation is peripheral inflammation.

[0054] In this application, the applicant established an LPS-induced AD mouse model via intraperitoneal injection of LPS. Previous studies have shown that, due to the blood-brain barrier, the neuroinflammatory response induced by intraperitoneal LPS injection is believed to primarily result from peripheral inflammation, thereby leading to the delivery of various inflammatory cytokines and chemokines to the central nervous system. Furthermore, other studies have found that in LPS-based neuroinflammatory models, intraperitoneal LPS injection can disrupt the blood-brain barrier, ultimately resulting in a large amount of LPS and inflammatory cytokines being transported to the brain via the circulatory system, thus exacerbating the neuroinflammatory response. Therefore, intraperitoneal LPS injection not only causes neuroinflammatory events but also leads to peripheral inflammation.

[0055] To investigate whether the daylily-derived exosomes of this application can improve peripheral inflammation, the applicant established an LPS-induced AD mouse model and measured the levels of pro-inflammatory cytokines in the mouse serum. The results showed that the exosomes could reduce the levels of TNF-α, IL-1β, and IL-6 in mouse serum (3.7 in Example 3). These results indicate that the daylily-derived exosomes of this application can effectively alleviate peripheral inflammation and, by reducing the peripheral inflammatory response, thereby improve neuroinflammation.

[0056] In some embodiments, the inflammation is neuroinflammation.

[0057] The exosomes derived from daylily in this application can indirectly improve neuroinflammation by alleviating peripheral inflammation; at the same time, given that plant-derived exosomes have the potential to cross the blood-brain barrier, they can also directly act on the central nervous system to exert anti-neuroinflammation effects.

[0058] In this application, after establishing an LPS-induced AD mouse model, the applicant conducted the following tests on the mice: (1) In the Y-maze test, the spontaneous alternation rate of the mice increased by 15%; (2) In the new object recognition test, the discrimination index of the mice increased by 21%; (3) In the open field test, the time the mice spent in the central region increased by 20%; (4) The number of Iba-1 positive cells in the mouse hippocampus decreased by 25%; (5) The levels of Tau protein and its phosphorylated proteins in the mouse brain decreased; (6) The levels of TNF-α, IL-1β, and IL-6 in the mouse brain decreased. These results indicate that exosomes can improve neuroinflammation.

[0059] In some embodiments, the neuroinflammation includes central nervous system inflammation caused by neurodegenerative diseases, trauma, or stroke. In some embodiments, the neurodegenerative diseases include Alzheimer's disease, Parkinson's disease, Huntington's disease, or amyotrophic lateral sclerosis (ALS).

[0060] In some embodiments, the exosomes derived from daylilies have a particle size of 100-300 nm, for example, 100 nm, 150 nm, 200 nm, 250 nm, 300 nm, or any value between the two aforementioned values.

[0061] Particle size determination is a standard procedure in the art. Specifically, in this application, an NTA nanoparticle size analyzer was used to analyze the particle size of the exosomes derived from daylily. More specifically, the average particle size of the exosomes derived from daylily was 129.9 nm.

[0062] In some embodiments, the ZETA potential of the exosomes derived from daylilies is (-35mv) to (-20mv), for example, -35mv, -30mv, -25mv, -20mv, -15mv, -10mv, -5mv, 5mv, 10mv, 15mv, 20mv, or any value between the two aforementioned values.

[0063] The ZETA potential measurement is standard practice in this field. Specifically, in this application, dynamic light scattering is used to analyze the ZETA potential of the exosomes derived from daylilies using a nanoparticle size and potential analyzer. More specifically, the ZETA potential of the exosomes derived from daylilies is -26.9 mV.

[0064] In some embodiments, the particle size of the exosomes derived from daylilies is 10. 9 ~10 13 Particle count / mL. Specifically, the particle size of the exosomes derived from daylily is 10. 11 Particle count / mL.

[0065] In some embodiments, the protein content of the exosomes derived from daylilies is 5 mg / mL to 20 mg / mL, for example, 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL, 10 mg / mL, 11 mg / mL, 12 mg / mL, 13 mg / mL, 14 mg / mL, 15 mg / mL, 16 mg / mL, 17 mg / mL, 18 mg / mL, 19 mg / mL, 20 mg / mL, or any value between the two aforementioned values.

[0066] The protein content determination is a routine procedure in the art. Specifically, in this application, the BCA method is used to analyze the protein content of the exosomes derived from daylilies. More specifically, the protein content of the exosomes derived from daylilies is 10 mg / mL.

[0067] The method for preparing exosomes derived from daylilies includes: extracting and separating daylilies to obtain exosomes.

[0068] In this application, the daylily can be the leaves, roots, and / or flowers (buds) of the daylily. In some embodiments, the daylily is the flower bud of the daylily.

[0069] This application does not limit the specific method for preparing the exosomes derived from daylily. Those skilled in the art can use any method known in the art to extract and separate daylilies to obtain exosomes.

[0070] In some embodiments, the preparation method includes: Fresh daylily flowers are washed and crushed to obtain daylily juice. The daylily juice is then filtered to obtain a first solution. The first solution is centrifuged for the first time to extract the supernatant, thus obtaining the first supernatant; The first supernatant is centrifuged a second time to extract the supernatant and obtain the second supernatant. The second supernatant was centrifuged a third time to remove the supernatant, and the resulting precipitate was dissolved in PBS buffer to obtain the second solution. The second solution was centrifuged a fourth time to extract the supernatant, thus obtaining the third supernatant. The third supernatant was centrifuged for the fifth time, and the resulting precipitate was the exosomes derived from daylily.

[0071] The daylily can be pulverized using conventional methods in the art, such as juicing it. In some embodiments, the daylily is placed in PBS buffer before juicing. In some embodiments, the mass ratio of daylily to PBS buffer is 1:1 to 10, for example, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, etc., and the PBS density is approximately 1 g / mL. In some embodiments, the mass ratio of daylily to PBS buffer is 1:2.

[0072] The daylily juice can be filtered using conventional methods in the art, such as filtering the daylily juice with gauze.

[0073] In some embodiments, the first centrifugation treatment is performed at a speed of 2000-4000g for 10-30 minutes. For example, the speed can be 2000g, 3000g, 4000g, or any value between these two; the time can be 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, or any value between these two. In some embodiments, the first centrifugation treatment is performed at a speed of 3000g for 20 minutes.

[0074] In some embodiments, the second centrifugation is performed at a speed of 9000-12000g for 20-60 minutes. For example, the speed can be 9000g, 10000g, 11000g, 12000g, or any value between these two; the time can be 20 minutes, 30 minutes, 40 minutes, 50 minutes, 60 minutes, or any value between these two. In some embodiments, the second centrifugation is performed at a speed of 10000g for 30 minutes.

[0075] In some embodiments, the third centrifugation is performed at a speed of 100,000-120,000 g for 70-120 minutes. For example, the speed can be 100,000 g, 110,000 g, 120,000 g, or any value between these two; the time can be 70 minutes, 80 minutes, 90 minutes, 100 minutes, 110 minutes, 120 minutes, or any value between these two. In some embodiments, the third centrifugation is performed at a speed of 120,000 g for 90 minutes.

[0076] In some embodiments, the fourth centrifugation is performed at a speed of 9000-12000g for 20-60 minutes. For example, the speed can be 9000g, 10000g, 11000g, 12000g, or any value between these two; the time can be 20 minutes, 30 minutes, 40 minutes, 50 minutes, 60 minutes, or any value between these two. In some embodiments, the fourth centrifugation is performed at a speed of 10000g for 30 minutes.

[0077] In some embodiments, the fifth centrifugation is performed at a speed of 100,000-120,000 g for 70-120 minutes. For example, the speed can be 100,000 g, 110,000 g, 120,000 g, or any value between these two; the time can be 70 minutes, 80 minutes, 90 minutes, 100 minutes, 110 minutes, 120 minutes, or any value between these two. In some embodiments, the fifth centrifugation is performed at a speed of 120,000 g for 90 minutes.

[0078] In some embodiments, the temperature of the first centrifugation, the second centrifugation, the third centrifugation, the fourth centrifugation, and the fifth centrifugation is 3-5°C, for example, 4°C.

[0079] In a preferred embodiment, the method for preparing the exosomes derived from daylilies includes: washing and crushing fresh daylilies to obtain daylily juice; filtering the daylily juice to obtain a first solution; centrifuging the first solution for the first time and extracting the supernatant to obtain a first supernatant; centrifuging the first supernatant for the second time and extracting the supernatant to obtain a second supernatant; centrifuging the second supernatant for the third time and removing the supernatant; dissolving the resulting precipitate in PBS buffer to obtain a second solution; centrifuging the second solution for the fourth time and extracting the supernatant to obtain a third supernatant; and centrifuging the third supernatant for the fifth time, the resulting precipitate being the exosomes derived from daylilies. The first centrifugation was performed at a speed of 3000g for 20 minutes at a temperature of 4°C; the second centrifugation was performed at a speed of 10000g for 30 minutes at a temperature of 4°C; the third centrifugation was performed at a speed of 120000g for 90 minutes at a temperature of 4°C; the fourth centrifugation was performed at a speed of 10000g for 30 minutes at a temperature of 4°C; and the fifth centrifugation was performed at a speed of 120000g for 90 minutes at a temperature of 4°C.

[0080] In a second aspect, this application provides a composition comprising exosomes derived from daylily as described in the first aspect of this application.

[0081] In some embodiments, the composition is a pharmaceutical composition. In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

[0082] Thirdly, this application provides the use of the composition described in the second aspect of this application in the preparation of an anti-inflammatory medicament.

[0083] In some embodiments, the inflammation includes peripheral inflammation or neuroinflammation.

[0084] In some embodiments, the inflammation is peripheral inflammation. In some embodiments, the inflammation is neuroinflammation.

[0085] In some embodiments, the neuroinflammation includes central nervous system inflammation caused by neurodegenerative diseases, trauma, or stroke. In some embodiments, the neurodegenerative diseases include Alzheimer's disease, Parkinson's disease, Huntington's disease, or amyotrophic lateral sclerosis (ALS).

[0086] Fourthly, this application provides a drug carrier, wherein the drug carrier comprises daylily-derived exosomes as described in the first aspect of this application.

[0087] In some embodiments, the exosomes derived from daylilies can serve as drug carriers to load active substances, such as nucleic acids, proteins, or small molecule compounds.

[0088] Fifthly, this application provides the use of the drug carrier described in the fourth aspect of this application in the preparation of anti-inflammatory drugs.

[0089] In some embodiments, the inflammation includes peripheral inflammation or neuroinflammation.

[0090] In some embodiments, the inflammation is peripheral inflammation. In some embodiments, the inflammation is neuroinflammation.

[0091] In some embodiments, the neuroinflammation includes central nervous system inflammation caused by neurodegenerative diseases, trauma, or stroke. In some embodiments, the neurodegenerative diseases include Alzheimer's disease, Parkinson's disease, Huntington's disease, or amyotrophic lateral sclerosis (ALS).

[0092] Sixthly, this application provides the use of an exosome-loaded drug product in the preparation of an anti-inflammatory drug, wherein the exosome-loaded drug product uses daylily-derived exosomes as the drug carrier as described in the first aspect of this application.

[0093] In some embodiments, the inflammation includes peripheral inflammation or neuroinflammation.

[0094] In some embodiments, the inflammation is peripheral inflammation. In some embodiments, the inflammation is neuroinflammation.

[0095] In some embodiments, the neuroinflammation includes central nervous system inflammation caused by neurodegenerative diseases, trauma, or stroke. In some embodiments, the neurodegenerative diseases include Alzheimer's disease, Parkinson's disease, Huntington's disease, or amyotrophic lateral sclerosis (ALS).

[0096] Seventhly, this application also provides the use of daylily-derived exosomes in the preparation of drugs that regulate the intestinal environment.

[0097] Specifically, the exosomes derived from daylily regulate the intestinal environment by improving the release of short-chain fatty acids from the gut microbiota.

[0098] The brain-gut axis is a bidirectional communication network between the brain and the gut, composed of four pathways: neural interaction pathways, neuroendocrine pathways, neuroimmune pathways, and microbial metabolic pathways. Studies have found that gut microbiota play a crucial role in various neurological disorders such as Alzheimer's disease, depression, and anxiety, and that gut microbiota can participate in the development and progression of these diseases by influencing neurodevelopment and neurotransmitter transmission. Furthermore, some studies have proposed that short-chain fatty acids released by gut microbiota can regulate the NLRP3 inflammasome, influencing neuroinflammation.

[0099] In this application, after establishing an LPS-induced AD mouse model, the applicant detected short-chain fatty acids released by the mouse gut microbiota (3.9 in Example 3). The results showed that exosomes could improve the expression of short-chain fatty acids such as butyric acid, isobutyric acid, valeric acid, and acetic acid, which represents the restoration of the "microbe-brain-gut" axis function. This indicates that the daylily-derived exosomes of this application can regulate the intestinal environment by improving the release of short-chain fatty acids from the gut microbiota, thereby improving neuroinflammation.

[0100] Compared with the prior art, the exosomes derived from daylily provided in this application have the following advantages: (1) Significant behavioral pharmacological activity: In addition to improving the behavioral abnormalities (including improving memory) of Alzheimer's disease mouse models, it can also alleviate anxiety-like symptoms; (2) Clear effect on regulating biochemical proteins: It can reduce microglial cell activation and NF-κB nuclear translocation, and can improve the pathological state from the level of neuroinflammation; (3) High safety: Daylily is a plant with both medicinal and edible properties, and its exosomes are a natural source without the toxic side effects of chemical drugs, making it suitable for long-term application; (4) Stable extraction process: The gradient ultracentrifugation method can be repeated, which is conducive to standardized production.

[0101] Specifically, the embodiments of this application, through in vitro verification of exosomes derived from daylilies, found that they could reduce Iba-1 expression and decrease NF-κB nuclear translocation in BV2 cells. Furthermore, in vivo verification revealed that after using daylily-derived exosomes in an Alzheimer's disease mouse model, the spontaneous alternation rate in the Y maze increased by 15%, the discrimination index increased by 21%, and the central dwell time in the open field test was prolonged by 20%. Simultaneously, the number of Iba-1 positive cells in the hippocampus decreased by 25%, the levels of Tau protein and its phosphorylated proteins decreased, and NF-κB nuclear translocation was significantly inhibited. These results confirm that the daylily-derived exosomes provided in this application have good anti-neuroinflammatory effects and can serve as a potential therapeutic agent for Alzheimer's disease, expanding the application of plant exosomes in behavioral pharmacology and neuroinflammatory regulation, and possessing significant medical and economic value.

[0102] Example The following description, in conjunction with specific embodiments, illustrates the content of this application, but the scope of this application is not limited thereto. Unless otherwise specified, the reagents and instruments used in the following embodiments are all conventional reagents and instruments in the art and can be obtained commercially. The methods used are all conventional experimental methods, and those skilled in the art can undoubtedly implement the described schemes and obtain corresponding results based on the embodiments.

[0103] Example 1: Preparation and Characterization of Exosomes from Daylily 1.1 Extraction and purification of exosomes from daylily High-purity exosomes were extracted using gradient ultracentrifugation, and the steps are as follows: (1) After washing 500g of daylily (flower buds), add 1000mL of pre-cooled 1×PBS and juice it using a juicer for 10 minutes. (2) Filter the residue with gauze and collect the filtered juice in a reagent bottle; (3) Centrifuge the filtered juice at 4°C and 3000g for 20 minutes to remove large impurities and take the supernatant; (4) Centrifuge the supernatant at 4°C and 10,000g for 30 minutes to further remove impurities, and take the supernatant again; (5) Centrifuge the supernatant at 4°C and 120,000g for 90 minutes, discard the supernatant, resuspend the precipitate with an appropriate amount of PBS, centrifuge at 4°C and 10,000g for 30 minutes, and take the supernatant again (i.e., obtain the exosome supernatant). (6) Dilute the obtained exosome supernatant with PBS to 25 mL, centrifuge at 4°C and 120,000 g for 90 minutes, discard the supernatant, and resuspend the precipitate with an appropriate amount of PBS to obtain the final exosomes.

[0104] 1.2 Characterization of exosome structure from daylily—Transmission electron microscopy analysis The exosomes obtained from daylily were slowly dropped onto the surface of a copper mesh covered with an ultrathin carbon film and allowed to stand at room temperature to dry naturally. Then, negative staining was performed, and after staining, the exosomes were dried naturally again. The exosomes were then placed in a transmission electron microscope to obtain ultrastructure images.

[0105] The results are as follows Figure 1 As shown, the exosomes derived from daylily exhibit a typical spherical nanoparticle morphology, consistent with the typical characteristics of plant exosomes.

[0106] 1.3 Characterization of exosome structure from daylily—Nanoparticle tracking analysis (NTA) The exosomes derived from daylily were analyzed using an NTA nanoparticle size analyzer (Particle Metrix, Zetaview-PMX120-Z) to obtain information on particle concentration and size of the exosomes.

[0107] The results are as follows Figure 2 As shown, the particle size of exosomes derived from daylilies was determined to be 3.2 × 10⁻⁶. 11 Particle count / mL, particle size range 100-300 nm, average particle size 129.9 nm.

[0108] 1.4 Characterization of exosome structure from daylily—ZETA potential detection The surface ZETA potential of the exosomes derived from daylily was determined using dynamic light scattering and a nanoparticle size and potential analyzer (Marvin Panaco Zetasizer Ultra (DLS) 2-105).

[0109] The results are as follows Figure 3 As shown, the potential measurement result is -26.9mV.

[0110] 1.5 Characterization of exosome structure from daylily—proteomics analysis Take 5 μL of the prepared daylily-derived exosomes and transfer them to a grinding tube. Add an appropriate amount of protein lysis buffer (8M urea + 1% SDS, containing protease inhibitors), and vortex three times (180 seconds each time) using a cryogenic grinder. Perform non-contact low-temperature sonication for 30 minutes, followed by centrifugation at 8°C and 14000g for 15 minutes. Collect the supernatant and determine the total protein content using a BCA protein assay kit (Hubei Milian Biotechnology Co., Ltd. Exosome BCA Kit E0025). Perform SDS-PAGE electrophoresis. The final protein concentration was 10 mg / mL. The SDS-PAGE analysis results are as follows: Figure 4 As shown.

[0111] The exosome protein solution was treated as follows: 100 μg of protein solution was added to 200 mM dithiothreitol solution and incubated at 37°C for 1 hour; then, 25 mM ammonium bicarbonate buffer was added to dilute the sample 4-fold, and trypsin was added at an enzyme:protein ratio of 1:50 (mass ratio). The enzyme was digested overnight at 37°C, and the next day, 50 μL of 0.1% formic acid was added to terminate the digestion; the digestion products were gradient eluted using a C18 column (EV01-06-01, C18 nano-liquid chromatography column (including packing material), 75 μm × 20 cm) and analyzed by mass spectrometry. Finally, 32 proteins were identified, as shown in Table 1 below.

[0112] Table 1. 32 proteins identified

[0113]

[0114] 1.6 Characterization of exosome structure from daylily—transcriptomic analysis Transcriptome analysis was performed on the exosomes obtained from daylily using high-throughput sequencing (contracted to Mylink Biotechnology (Hubei) Co., Ltd.) to assess the distribution of exosomal miRNAs. The specific experimental steps are as follows: RNA was extracted from exosomes derived from daylilies using an RNA purification and extraction kit (Qiagen, 217004). After determining the RNA concentration, miRNA libraries were constructed, and the constructed libraries were sequenced using the PE150 sequencing protocol for high-throughput sequencing. FastP was used to remove N-bases from both ends of the sequences, q20 filtering, and adapter removal. The cleaned sequences were then aligned to the Rfam library using the Bowtie short sequence alignment tool to remove rRNA, tRNA, and other ncRNAs, before being aligned to the genome using Bowtie. For non-model species, the miRbase library did not record their small RNA sequences; therefore, small RNAs from all plants in the miRbase library were used as a reference for quantitative analysis of possible small RNAs in the samples. A literature review was also conducted; if the literature provided small RNA sequences for the species, they were identified. miRNAs identified in the miRbase database were merged and retained as a single record based on complete sequence and expression level consistency. Small RNAs were then numbered sequentially from highest to lowest expression level based on sequence uniqueness. The corresponding families of the miRNAs were then searched using the miRbase database.

[0115] The results are as follows Figure 5 As shown, in exosomes derived from daylilies, miRNA readout fragments are mainly concentrated in the 17-25 nt range, consistent with the typical length distribution of plant exosomes.

[0116] Example 2: In vitro activity verification of exosomes derived from daylily 2.1 Detection of drug toxicity Mouse glial cells (BV2, Hefei Wanwu Biotechnology Co., Ltd., Delf-10370) were seeded at 8000 cells / well in 96-well plates and cultured overnight at 37℃ and 5% CO2 in DMEM medium (containing 10% FBS, 1% GlutaMAX, 1% HEPES buffer and 1% penicillin-streptomycin). Then, different concentrations of exosomes derived from daylily (400, 200, 100, 50, 25, 12.5, 6.25, 0 μg / mL) were added. After 2 hours of incubation, except for the blank control group, all other groups were stimulated with 1 μg / mL lipopolysaccharide (LPS, Wuhan Saiweier Biotechnology Co., Ltd., GC205009) for another 24 hours. The optimal concentration of exosomes was determined by a CCK8 assay (using the CCK-8 high-sensitivity rapid detection kit (G1613) (Hubei Wuhan Saiweier Biotechnology Co., Ltd.)).

[0117] The results are as follows Figure 6 As shown, the three optimal concentrations for the exosomes derived from daylily to exert their effects were determined to be 100, 50, and 25 μg / mL, respectively.

[0118] 2.2 Detection of NO levels BV2 cells were loaded at 2×10 5 Cells / well were seeded in 24-well plates and cultured overnight at 37°C and 5% CO2. Then, different concentrations of daylily-derived exosomes (100, 50, 25, and 0 μg / mL) were added. After 2 hours of incubation, except for the blank control group, 1 μg / mL LPS was added to all other groups for further stimulation for 24 hours. Afterward, the cell culture medium from each well was collected. Griess' reagent (Reagent A: p-aminobenzenesulfonic acid phosphate solution; Reagent B: naphthylethylenediamine hydrochloride solution) and sodium nitrate standard solution were prepared. Reagents A and B were added, and absorbance was measured to plot a standard curve. The collected cell culture medium was then used to add reagents A and B, and absorbance was measured and incorporated into the curve to detect the NO concentration in the cell culture medium.

[0119] The results are as follows Figure 7 As shown, exosomes derived from daylily have a certain inhibitory effect on the release of NO levels in LPS-induced BV2 cells, and this effect is dose-dependent.

[0120] 2.3 Activation detection of BV2 cells Place the smear at the bottom of the 24-well plate and add BV2 cells at a rate of 5 × 10⁻⁶. 4Cells / well were seeded in 24-well plates and cultured overnight at 37°C and 5% CO2. Then, different concentrations of daylily-derived exosomes (100, 50, 25, and 0 μg / mL) were added. After 2 hours of incubation, except for the blank control group, 1 μg / mL LPS was added to all other groups for further stimulation for 24 hours. After the incubation, the smears from each well were collected and incubated sequentially with primary antibody (Iba-1) (Hangzhou Huaan Biotechnology Co., Ltd., RT1316) and secondary fluorescent antibody (Alexa Fluor 488, Wuhan Sewell Biotechnology Co., Ltd., GB25301). After adding DAPI, the fluorescence level of Iba-1 under the same field of view was detected by fluorescence microscopy.

[0121] The results are as follows Figure 8 As shown, in the model group (i.e., stimulated only by 1 μg / mL LPS), the fluorescence level of Iba-1 was significantly higher than that of the blank control group. However, after treatment with daylily-derived exosomes, the fluorescence level decreased in a dose-dependent manner.

[0122] 2.4 Detection of NF-κB nuclear translocation Place the smear at the bottom of the 24-well plate and add BV2 cells at a rate of 5 × 10⁻⁶. 4 Cells / well were seeded in 24-well plates and cultured overnight at 37°C and 5% CO2. Then, different concentrations of daylily-derived exosomes (100, 50, 25, and 0 μg / mL) were added. After 2 hours of incubation, except for the blank control group, 1 μg / mL LPS was added to all other groups for further stimulation for 24 hours. After the incubation, the smears from each well were collected and incubated sequentially with primary antibody (NF-κB) (Hangzhou Huaan Biotechnology Co., Ltd., SP07-00) and fluorescent secondary antibody (Wuhan Sewell Biotechnology Co., Ltd., GB21303). After the addition of DAPI, the expression of NF-κB in the cytoplasm and nucleus was detected under the same field of view of a fluorescence microscope.

[0123] The results are as follows Figure 9 As shown, in the model group (i.e., stimulated only by 1 μg / mL LPS), NF-κB nuclear translocation was obvious, while after treatment with daylily-derived exosomes, both NF-κB expression and nuclear translocation were improved.

[0124] 2.5 Detection of pro-inflammatory cytokine secretion BV2 cells were seeded at a dose of 5 × 10⁻⁶. 4Cells / well were cultured overnight at 37°C and 5% CO2 in 24-well plates. Then, different concentrations of daylily-derived exosomes (100, 50, 25, and 0 μg / mL) were added. After 2 hours of incubation, except for the blank control group, 1 μg / mL LPS was added to all other groups for further stimulation for 24 hours. After the incubation, the cell culture medium of each well was collected, and the expression of TNF-α, IL-1β, and IL-6 in the culture medium was measured according to the EILSA enzyme-linked immunosorbent assay kit (TNF-α (MM-0132M1), IL-1β (MM-0040M2), IL-6 (MM-0163M2), Jiangsu Enzyme Immunosorbent Assay Co., Ltd.).

[0125] The results are as follows Figure 10 As shown, exosomes derived from daylily have a certain inhibitory effect on the release of TNF-α, IL-1β and IL-6 levels in LPS-stimulated BV2 cells, and this effect is dose-dependent.

[0126] Example 3: In vivo activity verification of exosomes derived from daylily 3.1 Establishing an LPS-induced AD mouse model The experimental environment was controlled under standard conditions of 12-hour light-dark cycle, temperature 24±2℃, and humidity 50±5%. The animals used in this example were SPF-grade male C57BL / 6J mice (6 weeks old), weighing 18-22g, totaling 32 mice. All mice underwent a 7-day pre-acclimatization process before the start of the experiment, during which they were provided with sufficient food and water. After the 7-day acclimatization period, the mice were divided into 4 groups: blank control group (blank group), model group (injected with LPS only), exosome administration group (exosomes derived from daylily), and donepezil positive control group. The blank control group consisted of 8 mice, and the other groups each consisted of 8 mice.

[0127] Except for the blank control group, all other groups of mice underwent modeling surgery. LPS was used as the modeling reagent and dissolved in physiological saline. After a 7-day adaptation period, the exosome administration group (exosome protein concentration of 10 mg / mL, 0.2 mL per mouse) and the donepezil (1.5 mg / kg) positive control group began 21 days of gavage administration. At the same time, the blank control group and the model group were gavaged with an equal volume of 0.5% sodium carboxymethyl cellulose (CMC-Na). Except for the blank control group, all other groups were intraperitoneally injected with LPS (0.25 mg / kg) for 7 consecutive days starting from the 14th day of gavage administration. The interval between the intraperitoneal injection of LPS and the daily gavage administration was 2 hours.

[0128] 3.2 Daylily-derived exosomes can improve spatial memory in mice. After 21 days of continuous drug administration, all mice in each group underwent the Y-maze test on day 22 to assess the improvement in their spatial memory ability. The specific experimental procedures are as follows: A Y-shaped maze was constructed using three identical arms (the three arms are at 120° angles and are labeled as arms A, B, and C). Each arm was evenly distributed on the same plane, and the experimental environment and lighting were kept stable. Mice were allowed to freely explore the center of the maze for 5-10 minutes to adapt to the environment. Then, the mice were placed into the maze from the center, and the order in which the mice entered each arm within 5 minutes was recorded. Entering an arm with all four limbs in one arm was counted as one entry. The spontaneous alternation rate of mice in each group was calculated (spontaneous alternation rate = (actual number of alternations / (total number of arm entries - 2)) × 100%).

[0129] The results are as follows Figure 11 As shown, when the concentration of exosomal protein from daylily was 10 mg / mL, continuous administration for 21 days could improve LPS-induced short-term spatial memory impairment in the Y maze in mice, manifested as an increased rate of spontaneous alternation.

[0130] 3.3 Daylily-derived exosomes can improve object recognition and memory abilities in mice. After 21 days of continuous drug administration, all mice in each group underwent a novel object recognition test on day 23 to assess the improvement in their object recognition memory ability. The specific experimental procedures are as follows: Familiarization phase: Place two identical objects in the experimental box and allow mice to explore freely for 5-10 minutes; Testing phase: After a 1-hour interval, replace one of the two identical objects with a different object and place it in the experimental box, allowing the mice to explore for another 5 minutes. Record the exploration time of each group of mice for the old and new objects, and calculate the discrimination index (DI = ...). (×100%), to assess object recognition and memory ability.

[0131] The results are as follows Figure 12 As shown, when the concentration of exosomal protein from daylily was 10 mg / mL, continuous administration for 21 days could improve the short- and medium-term object recognition memory impairment in LPS-induced mice in the new object recognition experiment, as evidenced by a significant increase in the exploration index of new objects. This result proves that exosomal protein from daylily has a certain improving effect on short-term object recognition memory.

[0132] 3.4 Daylily-derived exosomes can improve anxiety behavior in mice. After 21 days of continuous drug administration, all mice in each group underwent an open field test on day 24 to assess the improvement in anxiety behavior during the pathological progression of Alzheimer's disease. The specific experimental procedures are as follows: The open field box is a rectangular open box without a top, measuring 25cm×25cm×30cm (length×width×height). The bottom of the box is divided into a central area and an outer area. Twelve hours before the experiment, all mice in each group were transferred to the laboratory where the equipment was located to acclimatize to the environment. After the experiment started, the mice were placed in the box and allowed to explore freely for 5 minutes. The time spent in the central area and the movement trajectory of each group of mice were recorded.

[0133] The results are as follows Figure 13 As shown, exosomes derived from daylily significantly prolonged the time mice spent in the central region, indicating that they have a certain ameliorative effect on anxiety-like behavior.

[0134] 3.5 Detection of Tau and its phosphorylated protein levels in mouse brains Mice that had been administered the drug for 21 consecutive days were selected, and on day 25, immunofluorescence staining and Western blot analysis were performed to detect the levels of Tau and its phosphorylated proteins in the mouse brain in order to assess the impact of mice on the pathological progression of Alzheimer's disease.

[0135] The immunofluorescence staining procedure was as follows: Three mice were randomly selected from each group, and brain tissue (whole brain) was collected after sacrifice on day 25. The brain tissue was fixed, dehydrated, embedded in paraffin, and sectioned continuously along the coronal plane (5 μm). The sections were baked in a 60℃ oven for 2 hours, then soaked in xylene for 60 minutes, blotted dry, and then soaked in 100% ethanol, 95% ethanol, 85% ethanol, and 75% ethanol for 5 minutes in sequence, and rinsed with distilled water. The sections were placed in sodium citrate buffer (pH 6.0), heated to above 95℃ in a water bath and maintained for 10 minutes, and then naturally cooled to room temperature. The sections were washed three times with PBS for 5 minutes each time. After blotting dry, 100 μL of 3% PBS was added. Incubate with H2O2 at room temperature for 10 minutes, wash three times with PBS for 5 minutes each time; add sufficient 5% bovine serum albumin (BSA, Wuhan Sewell Biotechnology Co., Ltd., GC305010) blocking solution, block at room temperature for 30 minutes, add p-Tau primary antibody diluted with 5% BSA (Hangzhou Huaan Biotechnology Co., Ltd., ET1610-31), and incubate overnight at 4℃; after incubation, wash three times with PBS for 5 minutes each time, add fluorescently labeled secondary antibody diluted with 5% BSA (Wuhan Sewell Biotechnology Co., Ltd., GB21303), and incubate at room temperature in the dark for 1 hour; after incubation, add a solution containing DAPI, mount the slide, and observe and acquire images under a fluorescence microscope.

[0136] The steps for Western blot analysis of proteins are as follows: Three mice were randomly selected from each group. After sacrifice on day 25, hippocampal tissue was collected. The hippocampal tissue was added to 100-500 μL of RIPA solution containing 1% PMSF (Wuhan Sewell Biotechnology Co., Ltd., G2002), and treated with an ultrasonic homogenizer in an ice-water bath for 1 minute. The tissue was then allowed to stand for half an hour (4℃) to ensure complete lysis. The total protein concentration of each tissue was detected using a BCA protein assay kit (Hubei Milian Biotechnology Co., Ltd. Exosome BCA Kit E0025). Subsequently, the total protein concentration of each tissue was diluted to the same level with RIPA solution. After sample preparation, SDS-PAGE electrophoresis (Wuhan Sewell Biotechnology Co., Ltd., G2037) separation, membrane transfer (NC membrane), and blocking (5% BSA), anti-Tau antibody (Stressmarq, SPR-327) was added for incubation. After washing, the corresponding secondary antibody (Hangzhou Huaan Biotechnology Co., Ltd., HA710357) was added for incubation. ECL development, exposure, and protein band analysis were then performed.

[0137] The results are as follows Figure 14 As shown, exosomes derived from daylily have a certain inhibitory effect on the total Tau protein level in the mouse brain and can significantly reduce the level of inflammation-induced Tau protein phosphorylation.

[0138] 3.6 Detection of microglia activation in mouse brain Mice were selected after 21 consecutive days of drug administration. On day 25, mice in each group were sacrificed and hippocampal tissue was collected for immunofluorescence staining to detect the number of Iba-1 positive cells, in order to verify the regulatory effect of daylily-derived exosomes on neuroinflammatory-related proteins in mice.

[0139] Immunofluorescence staining was performed according to the method described in 3.5 of Example 3. Three mice were randomly selected from each group. The primary antibody used in immunofluorescence staining was anti-Iba-1 antibody (Hangzhou Huaan Biotechnology Co., Ltd., RT1316), and the secondary antibody was Cy5-labeled goat anti-rabbit IgG (Wuhan Sewell Biotechnology Co., Ltd., GB27303).

[0140] The results are as follows Figure 15 As shown, exosomes derived from daylily can improve LPS-induced activation of neuroinflammation in mice.

[0141] 3.7 Detection of pro-inflammatory cytokine secretion in mouse serum Mice that had undergone continuous drug administration for 21 days were selected. On day 25, the changes in serum pro-inflammatory cytokine levels in each group of mice were analyzed to assess the anti-peripheral and neuroinflammatory activities of the mice. The specific experimental procedures are as follows: Six mice were randomly selected from each group. Peripheral blood was collected from the mice via orbital blood sampling on day 25. Serum was obtained by centrifugation and stored at low temperature. The corresponding EILSA enzyme-linked immunosorbent assay (ELISA) kits (TNF-α (MM-0132M1), IL-1β (MM-0040M2), IL-6 (MM-0163M2, Jiangsu Enzyme Immunoassay Co., Ltd.) were used, and the levels of pro-inflammatory cytokines TNF-α, IL-1β, and IL-6 in the serum were measured according to the kit instructions. In short, the standards were diluted and a standard curve was prepared. After diluting the serum samples, they were added to the ELISA plate, incubated according to the kit steps, enzyme conjugates were added, and color development was performed. The absorbance of each well at 450 nm was measured using an ELISA reader, and the concentrations of pro-inflammatory cytokines were calculated based on the standard curve.

[0142] The results are as follows Figure 16 As shown, exosomes derived from daylily can reduce the levels of pro-inflammatory cytokines TNF-α, IL-1β, and IL-6 in mouse serum.

[0143] 3.8 Detection of pro-inflammatory cytokine secretion in mouse brain Mice were selected after 21 consecutive days of drug administration. On day 25, mice from each group were sacrificed, and hippocampal tissue was collected. Changes in the levels of pro-inflammatory cytokines in the brain were analyzed to assess the anti-neuroinflammatory activity of the mice. The specific experimental steps are as follows: Six mice were selected from each group. Hippocampal tissue was collected after sacrifice on day 25. The tissue samples were weighed and added to 100-300 μL of PBS solution. The tissue samples were then homogenized using a cell sonicator, centrifuged, and the supernatant was collected. The levels of pro-inflammatory cytokines TNF-α, IL-1β, and IL-6 in the brain were measured using the EILSA enzyme-linked immunosorbent assay kit (TNF-α (MM-0132M1), IL-1β (MM-0040M2), IL-6 (MM-0163M2), Jiangsu Enzyme Immunosorbent Assay Co., Ltd.). In short, standards were diluted and a standard curve was prepared. Tissue samples were diluted and added to an ELISA plate. Incubation, enzyme conjugate addition, and color development were performed according to the kit instructions. The absorbance of each well at 450 nm was measured using an ELISA reader, and the concentrations of pro-inflammatory cytokines were calculated based on the standard curve.

[0144] The results are as follows Figure 17 As shown, exosomes derived from daylily can reduce the levels of pro-inflammatory cytokines TNF-α, IL-1β, and IL-6 in the mouse brain.

[0145] 3.9 Daylily-derived exosomes can improve the expression of short-chain fatty acids in mice. Mice were selected after 21 consecutive days of drug administration. Feces were collected from each group on day 25. Gas chromatography-mass spectrometry (GC-MS) was used to evaluate the effects of daylily-derived exosomes on the levels of propionic acid, butyric acid, isobutyric acid, valeric acid, hexanoic acid, and acetic acid. The specific experimental procedures are as follows: (1) Six mice were selected from each group. On day 25, feces were collected and placed in a 2mL centrifuge tube. 1mL of pure water was added to the centrifuge tube and vortexed for 10 seconds. (2) Add steel balls, process with a 40Hz grinder for 4 minutes, then sonicate for 5 minutes (ice water bath), and repeat 3 times; (3) Centrifuge the fecal sample at 4°C and 5000 rpm for 20 minutes, and take 0.8 mL of the supernatant and place it in a 2 mL centrifuge tube; (4) Add 0.1 mL of 50% H2SO4 to the centrifuge tube, followed by 0.8 mL of extraction buffer (containing internal standard 2-methylvaleric acid, 25 mg / L, solvent is methyl tert-butyl ether), vortex for 10 seconds, shake for 10 minutes, and then sonicate for 5 minutes (ice water bath). After the process, let stand at -20℃ for 30 minutes. (5) Centrifuge at 4℃ and 10,000 rpm for 15 minutes, take the supernatant and transfer it to a sample vial for GC-MS analysis.

[0146] The analysis was performed using a Shimadzu GC2030-QP2020 NX gas chromatograph-mass spectrometer equipped with an Agilent HP-FFAP capillary column (30m×250μm×0.25μm, J&W Scientific, Folsom). The specific GC-MS analytical conditions are shown in Table 2 below.

[0147] Table 2 GC-MS Analysis Conditions

[0148] The results are as follows Figure 18 As shown, exosomes derived from daylilies can improve the expression of some short-chain fatty acids, such as butyric acid, isobutyric acid, valeric acid, and acetic acid, which represents the restoration of the function of the "microbe-brain-gut" axis.

[0149] The above description is merely a preferred embodiment of this application and is not intended to limit the application in any other way. Any person skilled in the art may make changes or modifications to the disclosed technical content to create equivalent embodiments. However, any simple modifications, equivalent changes, and modifications made to the above embodiments based on the technical essence of this application without departing from the scope of the technical solution of this application shall still fall within the protection scope of this application.

Claims

1. Application of daylily-derived exosomes in the preparation of anti-inflammatory drugs; Preferably, the inflammation includes peripheral inflammation or neuroinflammation; More preferably, the inflammation is neuroinflammation.

2. Application of daylily-derived exosomes in the preparation of drugs that regulate the intestinal environment; Preferably, the exosomes derived from daylily regulate the intestinal environment by improving the release of short-chain fatty acids from the intestinal flora.

3. The application according to claim 1 or 2, wherein the exosomes derived from daylily have a particle size of 100-300 nm.

4. The application according to any one of claims 1-3, wherein the ZETA potential of the exosomes derived from daylily is (-35mv) to (-20mv).

5. The application according to any one of claims 1-4, wherein the method for preparing the exosomes derived from daylily comprises: Exosomes were obtained by extracting and separating them from daylilies; Preferably, the daylily is the flower bud of the daylily.

6. The application according to claim 5, wherein the method for preparing the exosomes derived from daylily comprises: Fresh daylily flowers are washed and crushed to obtain daylily juice. The daylily juice is then filtered to obtain a first solution. The first solution is centrifuged for the first time to extract the supernatant, thus obtaining the first supernatant; The first supernatant is centrifuged a second time to extract the supernatant and obtain the second supernatant. The second supernatant was centrifuged a third time to remove the supernatant, and the resulting precipitate was dissolved in PBS buffer to obtain the second solution. The second solution was centrifuged a fourth time to extract the supernatant, thus obtaining the third supernatant. The third supernatant was centrifuged for the fifth time, and the resulting precipitate was the exosomes derived from daylily.

7. The application according to claim 6, wherein the first centrifugation treatment is performed at a speed of 2000-4000g for 10-30 minutes; the second centrifugation treatment is performed at a speed of 9000-12000g for 20-60 minutes; the third centrifugation treatment is performed at a speed of 100000-120000g for 70-120 minutes; the fourth centrifugation treatment is performed at a speed of 9000-12000g for 20-60 minutes; and the fifth centrifugation treatment is performed at a speed of 100000-120000g for 70-120 minutes.

8. The application according to claim 6 or 7, wherein the temperature of the first centrifugation, the second centrifugation, the third centrifugation, the fourth centrifugation, and the fifth centrifugation is 3-5°C.

9. A drug carrier, wherein the drug carrier comprises exosomes derived from daylily.

Images