Extraction method and application of ananas comosus exosome

Abstract

The present application relates to a kind of anaphalis contorta exosome extraction method and its application.The exosome is obtained by ultra-high speed centrifugation method from fresh anaphalis contorta, particle size is 160-200 nm, Zeta potential is-21.63±1.34 mV, with double membrane structure, mainly containing protein (such as resveratrol O-methyltransferase etc.) and phospholipid, polysaccharide content is only 12.5% of anaphalis contorta fresh medicine, and does not contain traditional active ingredient anaphalis contorta glycoside.Experiments show that anaphalis contorta exosome can be targeted to enrich in colorectal tissue, significantly improve AOM / DSS induced colorectal cancer model mouse tumor microenvironment, reduce tumor number, regulate T cell and macrophage ratio, and relieve pathological damage, better than anaphalis contorta fresh medicine.The mechanism is related to protein component, not polysaccharide or small molecule component.The anaphalis contorta exosome provided in the present application has simple preparation process, high safety, and has significant anti-colorectal cancer application potential.

Description

Technical Field

[0001] This invention relates to the field of plant exosome technology, specifically to a method for extracting exosomes from *Anoectochilus roxburghii* and their application in the preparation of anti-colorectal cancer drugs. Background Technology

[0002] Related studies have shown that increased release of pro-inflammatory factors and alterations in inflammatory bowel disease-related signaling pathways play a crucial role in the development of colitis-associated colorectal cancer. Therefore, intervention during the intestinal inflammation and early tumor development stages is of paramount importance for effectively controlling the incidence and mortality of colorectal cancer.

[0003] With the development of traditional Chinese medicine, herbal medicines play an important role in improving inflammatory responses and the tumor microenvironment. *Anoectochilus roxburghii*, a perennial herb belonging to the genus *Anoectochilus* of the Orchidaceae family, thrives in moist environments. It is sweet and cool in nature, and enters the lung, liver, kidney, and bladder meridians. It has the effects of clearing heat and cooling blood, removing dampness and detoxifying, and is clinically used to treat lung heat cough with hemoptysis, infantile convulsions, nephritis edema, and rheumatic pain. Modern pharmacological studies have shown that *Anoectochilus roxburghii* contains abundant flavonoids, polysaccharides, steroids, and other bioactive components, possessing pharmacological effects such as hypoglycemic, anti-tumor, anti-inflammatory, and analgesic properties.

[0004] However, traditional Chinese medicine preparations and extracts suffer from problems such as unstable active ingredients, low bioavailability, and poor targeting in practical applications, limiting their modern application in tumor treatment. Current technologies lack effective natural products with strong therapeutic effects that can target and inhibit tumors. Exosomes, vesicles with a diameter of approximately 30-200 nm, possess natural targeted delivery characteristics and can carry various bioactive substances such as proteins and RNA, attracting widespread attention in drug delivery systems in recent years. Plant-derived exosomes, due to their safe origin, high scalability, and low immunogenicity, have become a research hotspot for novel drug delivery systems.

[0005] Therefore, developing a stable and highly targeted plant-derived exosome preparation and verifying its anti-tumor effects has great application potential and clinical value. Summary of the Invention

[0006] To overcome the problems of poor targeting and low stability of active ingredients in traditional Chinese medicine treatment, this invention provides *Anoectochilus roxburghii* exosomes, a method for preparing *Anoectochilus roxburghii* exosomes, and their applications.

[0007] On the one hand, the present invention provides the application of Anoectochilus roxburghii exosomes in the preparation of anti-colorectal cancer drugs, wherein the Anoectochilus roxburghii exosomes can directly target colorectal tissue to exert their effects.

[0008] Furthermore, the colorectal cancer is azomethane / dextran sulfate sodium (AOM / DSS) induced colorectal cancer.

[0009] On the other hand, the present invention provides the application of *Anoectochilus roxburghii* exosomes in the preparation of drugs for relieving colorectal inflammation, wherein the *Anoectochilus roxburghii* exosomes can directly target colorectal tissue to exert their effects.

[0010] Furthermore, the *Anoectochilus roxburghii* exosomes include proteins, including resveratrol O-methyltransferase, a protein containing the FAS1 domain, a pyrophosphate-excited vacuum membrane proton pump, a large subunit of ribulose-1,5-bisphosphate carboxylase / oxygenase, a protein containing the C-terminal domain of the large ribosomal subunit protein uL4, 60S ribosomal protein L3, allene oxide synthase, a catalytic subunit of vacuole ATP synthase, a large ribosomal subunit protein uL6 with an N-terminal domain, a small ribosomal subunit protein uS5, and a 40S ribosomal protein S2.

[0011] Furthermore, the size of the exosomes from *Anoectochilus roxburghii* is 150-200 nm;

[0012] Furthermore, the Zeta potential of the *Anoectochilus roxburghii* exosomes is -21.63 ± 1.34 mV, and they appear spherical under an electron microscope, exhibiting a double-membrane structure.

[0013] The method for preparing the *Anoectochilus roxburghii* exosomes includes the following steps:

[0014] (1) Crush fresh Anoectochilus roxburghii to obtain crushing liquid;

[0015] (2) After filtering the above-mentioned crushing liquid, the precipitate was separated and collected by ultra-high speed centrifugation.

[0016] (3) The precipitate was resuspended in pre-cooled PBS to obtain the exosomes of Anoectochilus roxburghii.

[0017] Further, the fresh Anoectochilus roxburghii described in step (1) needs to be washed with sterile water and mixed with pre-cooled PBS buffer before being crushed;

[0018] The mixing ratio is Anoectochilus roxburghii:PBS buffer = 1 g : (3-4) mL; the pH of PBS buffer is 7.4-7.6.

[0019] Further, the filtration in step (2) refers to filtration using a 0.45 µm filter membrane to obtain filtrate;

[0020] Furthermore, the ultra-high-speed centrifugation method in step (2) includes the following steps:

[0021] a) Centrifuge the filtrate at 4℃ and 1000-2000g for 10-15 min, and collect the supernatant I;

[0022] b) Centrifuge supernatant I at 4℃, 3000-4000g for 15-20 min, and collect supernatant II;

[0023] c) Centrifuge supernatant II at 4℃, 10000-15000 g for 40-60 min, and collect supernatant III;

[0024] d) Centrifuge the supernatant III at 100,000-150,000 g for 70-90 min at 4℃ and collect the precipitate;

[0025] e) Resuspend each precipitate in pre-cooled PBS, repeat 3-5 times, and then centrifuge the resuspended solution at 100,000-150,000 g for 70-90 min at 4°C, and collect the precipitate again.

[0026] Further, in step (3), the precipitate in the tube is resuspended with pre-cooled PBS and then filtered. The resulting filtrate is a solution containing Anoectochilus roxburghii exosomes. The filtrate is stored at 4°C for later use.

[0027] Furthermore, in step (3), filtration is performed using a 0.22 µm filter membrane;

[0028] Furthermore, in step (3), the precipitate in the tube is resuspended with 2-3 mL of pre-cooled PBS.

[0029] Furthermore, the *Anoectochilus roxburghii* exosomes are soluble in pre-cooled PBS buffer and stored at -80°C.

[0030] Compared with the prior art, the present invention has the following beneficial effects:

[0031] (1) The extraction process of Anoectochilus roxburghii exosomes in this invention is simple and controllable, safe and pollution-free; it has sufficient safety, stability of effective drug components, low toxicity, solubility and targeting, and has good application prospects.

[0032] (2) The *Anoectochilus roxburghii* exosomes in this invention improve AOM / DSS-induced colorectal shortening in mice, reduce the number of colorectal tumors, regulate T cell subsets and macrophage ratios, and alleviate intraepithelial neoplasia and inflammatory cell infiltration. The anti-colorectal cancer mechanism of *Anoectochilus roxburghii* exosomes is clear, the efficacy is reliable, and it has high potential for industrialization and clinical translation.

[0033] (3) The exosomes of Anoectochilus roxburghii in this invention can be enriched in normal colonic mucosal epithelial cells, fibroblasts, macrophages and colorectal tissue. In addition, the exosomes of Anoectochilus roxburghii can be stably enriched in mouse colorectal tissue within 24 h, thereby targeting colorectal tissue to exert anti-tumor effects.

[0034] (4) The *Anoectochilus roxburghii* exosomes of the present invention are not traditional Chinese herbal medicines. The polysaccharide content in the *Anoectochilus roxburghii* exosomes provided is only 12.5% ​​of that in fresh *Anoectochilus roxburghii*, and does not contain the traditional active ingredient *Anoectochilus roxburghii* glycoside.

[0035] (5) The main components of the Anoectochilus roxburghii exosomes provided by the present invention are proteins and phospholipids, wherein the protein Trans-resveratrol di-O-methyltransferase accounts for about 30% of the protein content of Anoectochilus roxburghii exosomes. Attached Figure Description

[0036] Figure 1 A schematic diagram illustrating the isolation process of exosomes from fresh Anoectochilus roxburghii.

[0037] Figure 2 To examine the morphology of exosomes from *Anoectochilus roxburghii* using transmission electron microscopy;

[0038] Figure 3 The results of particle size analysis of Anoectochilus roxburghii exosomes;

[0039] Figure 4 The results of Zeta potential analysis of Anoectochilus roxburghii exosomes;

[0040] Figure 5 The results of protein concentration analysis of *Anoectochilus roxburghii* exosomes;

[0041] Figure 6 TIC plot for LC-MS / MS protein identification of Anoectochilus roxburghii exosomes;

[0042] Figure 7 These are the top 10 proteins with the highest responsiveness to *Anoectochilus roxburghii* exosomes;

[0043] Figure 8 TIC chromatogram for LC-MS / MS chemical composition identification of Anoectochilus roxburghii exosomes;

[0044] Figure 9 The UV scan spectrum of glucose and sample solution in Example 2;

[0045] Figure 10 This is the glucose standard curve from Example 2;

[0046] Figure 11 The following is a list of the body weights of colorectal cancer mice in each group in Example 3;

[0047] Figure 12 These are visual images of the colorectal tissue of mice with colorectal cancer in each group in Example 3.

[0048] Figure 13 This is a statistical chart showing the number of colorectal tumors in each group of mice in Example 3;

[0049] Figure 14 This is a statistical diagram of the colorectal length of mice in each group in Example 3;

[0050] Figure 15 CD4+ T cells and CD8+ T cells in the colorectal tissue of Example 3 + T cell flow cytometry;

[0051] Figure 16 For each group of CD4 in Example 3 + T cell count statistics;

[0052] Figure 17 For each group of CD8 in Example 3 + T cell count statistics;

[0053] Figure 18 For each group of CD8 in Example 3 + T / CD4 + T cell percentage statistics;

[0054] Figure 19 This is a statistical chart showing the proportion of macrophages in each group in Example 3;

[0055] Figure 20 The results of colorectal histopathological tissue sections from each group of mice in Example 3;

[0056] Figure 21 These are in vivo imaging images at different time points after oral administration of DIR-labeled Anoectochilus roxburghii exosomes in Example 4;

[0057] Figure 22 These are imaging images of different organs and tissues at different time points in Example 4;

[0058] Figure 23 This is a statistical graph of the average fluorescence intensity in the colorectal tissue of Example 4;

[0059] Figure 24 Fluorescence microscopy shows the uptake of PKH26-labeled Anoectochilus roxburghii exosomes by NCM460 cells in Example 4;

[0060] Figure 25 Fluorescence microscopy shows the uptake of PKH26-labeled Anoectochilus roxburghii exosomes by L929 cells in Example 4;

[0061] Figure 26 Fluorescence microscopy shows the uptake of PKH26-labeled Anoectochilus roxburghii exosomes by RAW264.7 cells in Example 4. Detailed Implementation

[0062] To enable those skilled in the art to better understand the technical solutions of the present invention, the present invention will be described in detail below with reference to specific embodiments. It should be noted that the following embodiments will help those skilled in the art to further understand the present invention, but do not limit the present invention in any way. It should be pointed out that those skilled in the art can make several modifications and improvements without departing from the concept of the present invention. These all fall within the protection scope of the present invention.

[0063] All raw materials used in this invention are not particularly limited in their source; they can be purchased from the market or prepared using conventional methods known to those skilled in the art.

[0064] All instruments obtained must be sterilized before use.

[0065] Example 1: Preparation of Anoectochilus roxburghii exosomes

[0066] 1. Morphological characterization

[0067] Morphological characterization of *Anoectochilus roxburghii* exosomes under transmission electron microscopy, such as... Figure 2 As shown, the exosomes of *Anoectochilus roxburghii* exhibit a double-membrane structure.

[0068] 2. Particle size determination

[0069] The particle size of *Anoectochilus roxburghii* exosomes was determined using a Zetasizer Nano ZS (DLS) nanoparticle size potentiometer. The results are as follows: Figure 3 As shown, the results indicate that the size of the exosomes from *Anoectochilus roxburghii* is approximately 150-200 nm.

[0070] 3. Potential Measurement

[0071] The zeta potentials of *Anoectochilus roxburghii* exosomes were measured using a zetasizer Nano ZS (DLS) nanoparticle size potentiometer. The results are as follows: Figure 4 As shown, the results indicate that the Zeta potential of the Anoectochilus roxburghii exosomes is -21.63 ± 1.34 mV.

[0072] 4. Protein concentration of Anoectochilus roxburghii exosomes

[0073] like Figure 5 As shown, the protein concentration of *Anoectochilus roxburghii* exosomes was 3 mg / mL.

[0074] 5. Protein composition of Anoectochilus roxburghii exosomes

[0075] Processing of Anoectochilus roxburghii exosome samples:

[0076] Enzyme digestion steps: Transfer the excised protein bands into 1.5 mL imported centrifuge tubes and wash twice with ultrapure water; remove deionized water, add 50 μL of destaining solution to each tube (enough to cover the gel particles, the same below), and destain for 5 min; remove the destaining solution, add 200 μL of deionized water to wash the gel and repeat twice, and incubate overnight; remove deionized water, add dehydration solution 1 (50% acetonitrile solution), and dehydrate for 30 min; remove dehydration solution 1, add dehydration solution 2 (100% acetonitrile), and dehydrate for 30 min, then freeze-dry under vacuum; add 50 μL of reducing solution 1 (containing 10 mM DTT and 25 mM NH4HCO3 aqueous solution) to the freeze-dried gel and incubate at 55℃ for 1 h; remove the liquid, cool to room temperature, and add 50 μL of reducing solution 2 (containing 50 mM iodoacetamide and 25 mM acetonitrile solution)... (In an aqueous solution of NH4HCO3), incubate in the dark for 30 min; aspirate the liquid and add swelling buffer, incubate for 10 min; aspirate the swelling buffer and add dehydration buffer 1, dehydrate for 30 min; aspirate the dehydration buffer 1 and add dehydration buffer 2, dehydrate for 30 min; aspirate the dehydration buffer 2 and add 10 μL of enzymatic hydrolysis working solution, incubate for 30 min; add 20 μL of enzymatic hydrolysis covering buffer, incubate at 37℃ for 16 h; after enzymatic hydrolysis, transfer the supernatant to another new centrifuge tube; add 50 μL of peptide extraction solution to the remaining gel, incubate at 37℃ for 30 min, centrifuge at 5000g for 5 min, combine the supernatants, and repeat the above operation once more. After evaporation, prepare for desalting.

[0077] Peptide desalting steps:

[0078] Prepare a C18 membrane-packed column; redissolve the evaporated peptide sample in Nano-HPLC Buffer A; centrifuge once with 40 μL methanol, discarding the liquid at the bottom of the EP tube, repeat twice; centrifuge once with 40 μL of 0.1% formic acid-water solution, discarding the liquid at the bottom of the EP tube, repeat twice; centrifuge once with the peptide sample, collecting the liquid at the bottom of the EP tube and centrifuging again; centrifuge once with 40 μL of 0.1% formic acid-water solution, discarding the liquid at the bottom of the EP tube, repeat twice; replace with a new EP tube, centrifuge once with 40 μL of 0.1% formic acid-acetonitrile eluent, collecting the liquid at the bottom of the EP tube, repeat once. After desalting, evaporate 80 μL of the 0.1% formic acid-acetonitrile eluent containing the peptide sample to dryness.

[0079] Chromatographic conditions: Trap column (100 μm × 20 mm), Analysis column (75 μm × 250 mm); Mobile phase: 0.1% formic acid aqueous solution (A) - 0.1% formic acid-acetonitrile solution (B); Elution gradient: 0-110 min (5-38% B), 110-112 min (38-95% B), 112-120 min (95-95% B); Flow rate: 300 nL / min; Samples were washed once with blank solvent using the mobile phase gradient for 30 min; Mass spectrometry conditions: Ion source was electrospray ionization (ESI), carrier gas was nitrogen, positive and negative ion mode Full MS / dd-MS2 scan, high-energy collision dissociation HCD, NCE energy 28, 32, dynamic exclusion time: 30 s. The primary resolution is 60,000, the secondary resolution is 15,000, the AGC target is set to 8e5, the maximum injection time is 50ms, the MS2 resolution is set to 15,000, the AGC target is set to 5e4, the maximum injection time is 100ms, and the scan range is 350-1250 m / z.

[0080] LC-MS / MS proteomic analysis of *Anoectochilus roxburghii* exosomes yielded 7653 peptides, 7171 unique peptides, and 1581 proteins. Figures 6-7As shown. The Top 10 proteins include resveratrol di-O-methyltransferase, FAS1 domain-containing protein, pyrophosphate-energized vacuolar membrane proton pump-like protein, ribulose-1,5-bisphosphate carboxylase / oxygenase large subunit, large ribosomal subunit protein uL4 C-terminal domain-containing protein, 60S ribosomal protein L3, allene oxide synthase, vacuolar ATP synthase catalytic subunit, and large ribosomal subunit protein uL6 N-terminal domain-containing protein. The protein composition of *Anoectochilus roxburghii* exosomes includes ribosomal subunit protein uL6 (N-terminal domain-containing protein), small ribosomal subunit protein uS5, and 40S ribosomal protein S2. Trans-resveratrol di-O-methyltransferase accounts for approximately 30% of the protein content in these exosomes. The top 10 proteins in *Anoectochilus roxburghii* exosomes are likely key components in the efficacy of these exosomes against AOM / DSS-induced colorectal cancer in mice.

[0081] 5. Chemical composition analysis of exosomes from *Anoectochilus roxburghii*

[0082] Take 100 µL of the *Anoectochilus roxburghii* exosome solution (protein concentration 3 mg / mL) prepared in Example 1 into a 2 mL centrifuge tube; add 200 μL of pre-cooled methanol:acetonitrile (1:1, v / v), vortex for 30 s; freeze at -20℃ for 30 min; centrifuge at 12000 rpm and 4℃ for 10 min, take 200 μL of the supernatant and concentrate under vacuum to dryness; add 150 μL of 50% methanol to reconstitute, vortex for 30 s; centrifuge at 12000 rpm and 4℃ for 10 min, take the supernatant and filter through a 0.22 μm filter membrane to obtain the *Anoectochilus roxburghii* exosome test solution.

[0083] Chromatographic conditions: ACQUITY UPLC HSS T3 (100 mm × 2.1 mm, 1.8 μm); Mobile phase: 0.1% formic acid aqueous solution (A) - acetonitrile (containing 0.1% formic acid (B); Elution gradient: 0–1 min (95–95% B), 1–4.7 min (95–5% B), 4.7–6 min (5–5% B), 6–6.1 min (5–95% B), 6.1–8.1 min (95–95% B); Flow rate: 0.4 mL / min; Column temperature: 40 °C; Injection volume: 2 μL; Mass spectrometry conditions: Ion source: electrospray ionization (ESI); Carrier gas: nitrogen; Full MS / dd-MS2 scan in positive and negative ion modes; Sheath gas flow rate: 40 arb; Assist gas flow rate: 10 The arb capillary voltage is 3.5 kV and 3.0 kV in positive and negative ion modes, with a primary resolution of 60,000 and a secondary resolution of 15,000. The ion transmission tube temperature is 320°C, the vaporization temperature is 300°C, and the scanning range is 70-1000 m / z.

[0084] The results are as follows Figure 8 Analysis revealed that the chemical components mainly included organic acids, esters and benzene ring derivatives, as well as glycosides and sugars. Polysaccharides were the most abundant chemical component. No traditional active ingredient, anoside, was detected in the exosomes of *Anoectochilus roxburghii*. Furthermore, the abundance of polysaccharides was higher than that of other organic acids, esters, and benzene ring derivatives, such as ferulic acid.

[0085] Example 2: Determination of polysaccharide content in *Anoectochilus roxburghii* exosomes and fresh *Anoectochilus roxburghii* herbs

[0086] (1) Preparation of glucose standard solution:

[0087] Accurately weigh an appropriate amount of anhydrous glucose standard and dissolve it in distilled water to prepare a glucose standard stock solution with a concentration of 1.25 μg / mL. Accurately pipette 0, 31.25, 62.5, 125, 250, 500, and 1000 μL of the glucose standard stock solution and dilute to 1 mL with distilled water to prepare a series of glucose standard solutions with different mass concentrations.

[0088] (2) Preparation of the test solution of Anoectochilus roxburghii exosomes:

[0089] Accurately pipette 100 μL of the Anoectochilus roxburghii exosome solution (protein concentration 3 mg / mL) prepared in Example 1, dilute with distilled water to 300 μL to obtain the Anoectochilus roxburghii exosome sample solution (protein concentration 1 mg / mL, for pharmacodynamic evaluation of gavage volume and protein concentration), and then add distilled water to 600 μL to obtain the Anoectochilus roxburghii exosome test solution.

[0090] (3) Preparation of the test solution of fresh Anoectochilus roxburghii:

[0091] Take 60 g of fresh Anoectochilus roxburghii, wash thoroughly with sterile water, chop, add 100 mL of pre-cooled PBS buffer (4℃), and juice using a juicer. Collect the juice, filter it through a 0.45 µm filter membrane, and collect the filtrate. Place the filtrate in a 50 mL centrifuge tube, centrifuge at 1000 g for 10 min at 4℃, and collect the supernatant I. Place the supernatant I in a 50 mL centrifuge tube, centrifuge at 3000 g for 15 min at 4℃, and collect the supernatant. This yields the fresh Anoectochilus roxburghii herb (0.6 g fresh Anoectochilus roxburghii / mL, for pharmacodynamic evaluation of gavage volume and concentration). Accurately pipette 300 μL of the fresh Anoectochilus roxburghii herb and add distilled water to a final volume of 6000 μL to obtain the test solution of the fresh Anoectochilus roxburghii herb.

[0092] (4) Ultraviolet absorption spectroscopy measurement:

[0093] Accurately pipette 500 μL of the standard solution or test solution, add 250 μL of 5% phenol solution and 1.25 mL of concentrated sulfuric acid in sequence, and shake to mix thoroughly.

[0094] Using distilled water as a blank control, scanning was performed using a UV-Vis spectrophotometer in the wavelength range of 400-600 nm. Figure 9 The results showed that both the glucose standard solution and the Anoectochilus roxburghii exosome test solution had maximum absorption at around 488.80 nm. Therefore, 488.80 nm was selected as the detection wavelength for determining the polysaccharide content in the Anoectochilus roxburghii exosome test solution.

[0095] The absorbance was measured at a wavelength of 488.80 nm. A standard curve was plotted by performing linear regression with the mass concentration X of the glucose standard solution as the abscissa and the absorbance Y as the ordinate. Results Figure 10 As shown, the glucose standard exhibits a good linear relationship with its absorbance in the concentration range of 0.0391-1.250 μg / mL, with a linear regression equation of y=2.356x-0.1242 and R2=0.9989.

[0096] The absorbance was measured at a wavelength of 488.80 nm, and the sugar content in the *Anoectochilus roxburghii* exosome solution and the fresh *Anoectochilus roxburghii* herb solution was calculated. The results are shown in Table 1. The sugar content in 300 μL of *Anoectochilus roxburghii* exosome solution and fresh *Anoectochilus roxburghii* herb solution was 2.77 μg and 22.19 μg, respectively.

[0097] Table 1. Results of polysaccharide content determination in *Anoectochilus roxburghii* exosome solution and fresh AR herb solution.

[0098]

[0099] The volumes of the *Anoectochilus roxburghii* exosome test solution and the fresh *Anoectochilus roxburghii* herb test solution used in the detection were the same as those used in the gavage evaluation. The results showed that the sugar content in 300 μL of *Anoectochilus roxburghii* exosome solution and fresh *Anoectochilus roxburghii* herb solution was 2.77 μg and 22.19 μg, respectively. The polysaccharide content in the *Anoectochilus roxburghii* exosome solution was approximately 12.5% ​​of that in the fresh *Anoectochilus roxburghii* herb solution. Therefore, the polysaccharide concentration in the *Anoectochilus roxburghii* exosome solution was significantly lower than that in the fresh *Anoectochilus roxburghii* herb solution.

[0100] The above results indicate that the *Anoectochilus roxburghii* exosomes prepared in this invention contain very few small molecule active chemical components, far lower than those found in fresh *Anoectochilus roxburghii*, and even lower than those found in extracts of *Anoectochilus roxburghii*, such as alcohol extracts or petroleum ether extracts.

[0101] Example 3: Effects of Anoectochilus roxburghii exosomes on AOM / DSS-induced colorectal cancer

[0102] (I) Construction of an AOM / DSS-induced mouse model of colorectal cancer:

[0103] C57BL / 6 mice were randomly divided into five groups (n=6): blank control group, model control group, *Anoectochilus roxburghii* exosome group (10 mg protein / kg / d), *Anoectochilus roxburghii* fresh herb group (6 g fresh herb / kg / d), and positive control group (aspirin, 30 mg / kg / d). Blank control group: normal feeding, no intervention. Model control group: induced by AOM+DSS, administered by gavage with saline. *Anoectochilus roxburghii* exosome group: induced by AOM+DSS, administered by gavage with *Anoectochilus roxburghii* exosome solution (10 mg / kg / d). *Anoectochilus roxburghii* fresh herb group: induced by AOM+DSS, administered by gavage with *Anoectochilus roxburghii* fresh herb solution (600 mg / kg / d). Positive control group: induced by AOM+DSS, administered by gavage with aspirin (30 mg / kg / d).

[0104] Days 1-7: Adaptive feeding of mice;

[0105] Day 8: Except for the blank control group, the mice in the other 4 groups were injected intraperitoneally with AOM (10 mg / kg, calculated as 200 μL / 20g body weight), while the blank control group was injected with an equal volume of physiological saline (PBS).

[0106] Days 15-35: This is the first cycle. Days 15-21: The model group, the Anoectochilus roxburghii exosome group, the Anoectochilus roxburghii group, and the aspirin group were given 2.0% DSS drinking water (free access for 7 days), while the blank control group continued to drink ordinary water. Days 22-35: All groups resumed drinking ordinary drinking water (14 days), completing the first cycle.

[0107] Days 36-56: This is the second cycle; repeat the DSS intervention and water restoration procedures described above.

[0108] Days 57-77: This is the third cycle; repeat the DSS intervention and water restoration procedures described above.

[0109] Days 78-96: Continue to provide normal drinking water until the end of the experiment.

[0110] Starting from the second cycle (day 36), mice in each group were administered the drug by gavage until the end of the experiment (day 96).

[0111] (ii) Medicines

[0112] Drug administration of Anoectochilus roxburghii exosomes: The Anoectochilus roxburghii exosomes prepared in Example 1 were administered at a dose of 10 mg protein / kg / d.

[0113] Fresh Anoectochilus roxburghii (Golden Thread Orchid) treatment: Take 60 g of fresh Anoectochilus roxburghii, wash thoroughly with sterile water, chop, add 100 mL of pre-cooled PBS buffer (4℃), and juice using a juicer. Collect the juice, filter it through a 0.45 µm filter membrane, and collect the filtrate. Place the filtrate in a 50 mL centrifuge tube, centrifuge at 1000 g for 10 min at 4℃, and collect the supernatant I. Place the supernatant I in a 50 mL centrifuge tube, centrifuge at 3000 g for 15 min at 4℃ to obtain the fresh Anoectochilus roxburghii (0.6 g fresh drug / mL, pharmacodynamic evaluation of gavage volume and concentration). The dosage is 6 g fresh drug / kg / day.

[0114] Aspirin group: 150 mg of aspirin was dissolved in 50 mL of sterile saline to obtain a 3 mg / mL aspirin solution. The dosage was 30 mg / kg / day.

[0115] (III) Results

[0116] The results showed that, compared with the control group, the model group mice experienced a sharp decrease in body weight upon administration of DSS, while the mice with *Anoectochilus roxburghii* exosomes showed a slight decrease in body weight, though the reduction was less severe. Figure 11 As shown. Furthermore, compared to the control group, the colorectal length of the model group mice was significantly shortened, and tumor nodules of varying sizes and numbers were visible to the naked eye in the mouse colorectal region. *Anoectochilus roxburghii* exosomes could improve colon length and weight, such as... Figures 12-14 shown. (Note: Figure 13 and Figure 14 In comparison with the blank group: #p<0.05, ##p<0.01; compared with the model group: *p<0.05, **p<0.01, n=6)

[0117] Flow cytometry results showed that, compared with the control group, the number of CD8+ T cells in the colorectal tissue of mice in the model group was significantly reduced. There was no significant difference in CD8+ T cells between the *Anoectochilus roxburghii* exosome group and the control group, but the number of CD8+ T cells was significantly increased in the *Anoectochilus roxburghii* exosome group compared with the model group. Figures 15-18 As shown; furthermore, compared with the blank group, the number of macrophages in the colorectal tissue of mice in the model group was significantly increased, and the number of macrophages in the *Anoectochilus roxburghii* exosome group was significantly increased, such as Figure 19 As shown, *Anoectochilus roxburghii* exosomes therefore affect the tumor microenvironment of colonic tissue, thereby exerting an anti-tumor effect. (Note:) Figures 16-19 In comparison with the control group: #p<0.05, ##p<0.01; compared with the model group: *p<0.05, **p<0.01, n=3)

[0118] The results of histopathological sections of mouse colorectal tissue showed that, Figure 20 As shown, the colorectal tissue structure of mice in the blank control group was clear, with goblet cells, crypts, and intestinal glands arranged regularly and without related inflammatory cell infiltration. In the model group, the colorectal tissue of mice showed high-grade intraepithelial neoplasia, deformed intestinal crypt structure, and polyp protrusions. The polyps contained a large number of red blood cells and were accompanied by a large number of inflammatory cell infiltrations. Under the microscope, the colorectal tissue structure of mice in the *Anoectochilus roxburghii* exosome group still showed normal-shaped goblet cells, their crypts, and intestinal glands. Although there was a small amount of inflammatory cell infiltration in the submucosa, there were no obvious polyp protrusions. In the fresh *Anoectochilus roxburghii* herb group, the colorectal tissue of mice showed deformed crypt structure and a large number of inflammatory factors infiltration, and the number of polyp protrusions did not improve significantly.

[0119] The above results suggest that *Anoectochilus roxburghii* exosomes can effectively slow down AOM / DSS-induced colorectal cancer lesions in mice, and the effect is better than that of the fresh *Anoectochilus roxburghii* drug group.

[0120] Combining the results from Example 1, where the abundance of *Anoectochilus roxburghii* polysaccharides in the exosomes was higher than that of other organic acids, esters, and benzene ring derivatives such as ferulic acid, and the results from Example 2, the polysaccharide concentration in the *Anoectochilus roxburghii* exosome solution was significantly lower than that in the fresh *Anoectochilus roxburghii* herb. It can be inferred that the polysaccharide content in both the *Anoectochilus roxburghii* exosome solution and the fresh *Anoectochilus roxburghii* herb solution is not a major factor affecting the efficacy against AOM / DSS-induced colorectal cancer in mice. Furthermore, the polysaccharide content in the *Anoectochilus roxburghii* exosomes was significantly lower than the current therapeutic dose for cancer (800 mg / kg).

[0121] Example 4: Intake of Anoectochilus roxburghii exosomes

[0122] (a) Accumulation of Anoectochilus roxburghii exosomes in normal mice

[0123] (1) Staining of Anoectochilus roxburghii exosomes with DIR staining solution

[0124] Experimental steps:

[0125] a. Add 4 mL of Anoectochilus roxburghii exosomes to 1 mL of DIR dye, vortex mix for 1 min, and incubate for 30 min to allow the dye and Anoectochilus roxburghii exosomes to fully bind, thus obtaining DIR dye-labeled Anoectochilus roxburghii exosomes.

[0126] b. To remove free dye, add 15 mL of pre-chilled PBS buffer to the above-incubated mixed solution, place it in an ultracentrifuge tube, centrifuge at 100,000 g for 70 min at 4°C, collect the precipitate, dilute the labeled Anoectochilus roxburghii exosome precipitate in 4 mL of pre-chilled PBS buffer and use it for uptake experiments.

[0127] (2) Uptake of DIR-labeled Anoectochilus roxburghii exosomes by normal mice

[0128] Experimental group: DIR-labeled Anoectochilus roxburghii exosome solution was administered by gavage;

[0129] Control group: PBS solution was administered by gavage.

[0130] The experimental steps are as follows:

[0131] C57BL / 6 mice were randomly divided into 6 groups (n=3): blank control group, experimental group - 3h post-gavage (10 mg / kg / d), experimental group - 6h post-gavage (10 mg / kg / d), experimental group - 12h post-gavage (10 mg / kg / d), experimental group - 24h post-gavage (10 mg / kg / d), and experimental group - 48h post-gavage (10 mg / kg / d). In vivo imaging was performed at different time points, and organs were dissected and collected for in vivo imaging detection.

[0132] In vivo imaging results as follows Figures 21-23 As shown, no fluorescence enrichment was observed in the heart, liver, spleen, lung, and kidney tissues, indicating that *Anoectochilus roxburghii* exosomes possess biocompatibility. Furthermore, DIR-labeled *Anoectochilus roxburghii* exosomes exhibited varying degrees of absorption at 3h, 6h, 12h, and 24h time points, with the strongest fluorescence intensity at 6h, and no fluorescence intensity observed at 48h. These results suggest that *Anoectochilus roxburghii* exosomes can directly target colorectal tissue to exert an antitumor effect.

[0133] (II) Uptake of Anoectochilus roxburghii exosomes by NCM460 cells, L929 cells, and RAW264.7 cells

[0134] Colorectal tissue contains cell types such as epithelial cells, fibroblasts, and macrophages. Therefore, this invention selected normal human colonic mucosal epithelial cells (NCM460 cells), L929 cells, and RAW264.7 cells to demonstrate the uptake effect of these cells on *Anoectochilus roxburghii* exosomes.

[0135] The samples were labeled using PKH26 staining solution and observed using a fluorescence microscope. The specific operating procedures for the experimental group are as follows:

[0136] (1) Staining of Anoectochilus roxburghii exosomes with PKH26 staining solution

[0137] Experimental steps:

[0138] a. Add 1 µL of PKH26 dye to 9 µL of diluent C to obtain the PKH26 dye working solution;

[0139] b. Add 150 µL of Anoectochilus roxburghii exosomes to the PKH26 dye working solution, vortex mix for 1 min, and incubate for 10 min to allow the dye and Anoectochilus roxburghii exosomes to fully bind, thus obtaining Anoectochilus roxburghii exosomes labeled with PKH26 dye.

[0140] c. To remove free dye, 15 mL of pre-chilled PBS buffer was added to the above-incubated mixed solution, and the mixture was further placed in an ultracentrifuge tube and centrifuged at 100,000 g for 70 min at 4°C. The precipitate was collected, and the labeled Anoectochilus roxburghii exosome precipitate was diluted in 150 µL of pre-chilled PBS buffer and used for uptake experiments.

[0141] (2) Uptake of Anoectochilus roxburghii exosomes by NCM460 cells, L929 cells and RAW264.7 cells

[0142] Experimental treatment system: PKH26-labeled Anoectochilus roxburghii exosomes were added to NCM460 cells, L929 cells, and RAW264.7 cells, respectively;

[0143] Blank control group system: only NCM460 cells, L929 cells, and RAW264.7 cells, without exosome treatment.

[0144] The experimental steps are as follows:

[0145] a. After culturing the cells of the control group and the experimental group in 200 µL DMEM basal medium, PMI basal medium and RPMI 1640 basal medium for 12 h, respectively, the cells were washed twice with PBS.

[0146] b. Cover the cells with fixative containing 4% formaldehyde and fix at room temperature for 10 min;

[0147] c. Wash cells with PBS buffer 2-3 times, 0.5-1 min each time;

[0148] d. Cover the cells with a cell permeabilizing agent and permeabilize at room temperature for 10 min;

[0149] e. Wash cells 2-3 times with PBS buffer, 0.5-1 min each time;

[0150] f. Take 200 μL of freshly prepared FITC-labeled phalloidin staining working solution to completely cover the cells and incubate at room temperature in the dark for 30 min;

[0151] g. Wash cells 2-3 times with PBS buffer, 0.5-1 min each time;

[0152] h. Add 50 μL of DAPI staining solution (enough to cover the bottom of the dish), incubate at room temperature in the dark for 5 min to allow the staining agent to fully bind with the cells;

[0153] i. After incubation, wash with PBS 2-3 times to remove unbound dye, each time for 0.5-1 min, and finally observe under a microscope.

[0154] The results of fluorescence microscopy are as follows Figures 24-26 As shown in the figure, PKH26-stained exosomes appear red, FITC-labeled phalloidin-stained cytoskeleton appears green, and DAPI-stained cells appear blue. The positions of the red, green, and blue fluorescence in the figure are largely overlapping. Therefore, based on the position and color of the fluorescence in the figure, it can be determined that *Anoectochilus roxburghii* exosomes were successfully taken up and absorbed by NCM460 cells, L929 cells, and RAW264.7 cells. Furthermore, compared with the results for "NCM460 cells" and "L929 cells," RAW264.7 cells showed higher efficiency in taking up *Anoectochilus roxburghii* exosomes.

[0155] In summary, the *Anoectochilus roxburghii* exosomes provided by this invention can significantly inhibit the progression of inflammatory colon cancer, regulate T cell subsets and macrophage ratios, and alleviate pathological damage to colorectal tissue in an AOM / DSS-induced colorectal cancer mouse model. Furthermore, the *Anoectochilus roxburghii* exosomes are stably enriched in mouse colorectal tissue within 24 hours. In vitro studies show that the *Anoectochilus roxburghii* exosomes can be taken up by normal colonic mucosal epithelial cells, fibroblasts, and macrophages, thereby targeting colorectal tissue to exert anti-tumor effects. In addition, the *Anoectochilus roxburghii* exosomes contain 72 chemical components. The polysaccharide content in the *Anoectochilus roxburghii* exosome solution is only 12.5% ​​of that in the fresh *Anoectochilus roxburghii* solution, and the exosomes do not contain the traditional active ingredient *Anoectochilus roxburghii* glycosides. The content of glycosides and sugars in the *Anoectochilus roxburghii* exosomes is significantly lower than the polysaccharide content in the fresh *Anoectochilus roxburghii* solution and the dosage of existing *Anoectochilus roxburghii* polysaccharide anticancer drugs. Therefore, the efficacy of *Anoectochilus roxburghii* exosomes against AOM / DSS-induced colorectal cancer in mice is mainly due to the lack of significant correlation between the proteins, polysaccharides, and small molecule components in the exosomes. Thus, the treatment of colorectal cancer with *Anoectochilus roxburghii* exosomes is an innovation based on traditional Chinese medicine, possessing sufficient safety, stability of the effective drug components, low toxicity, and solubility.

Claims

1. The use of a Nothapodytes pithecellobium exosome in the preparation of an anti-colorectal cancer drug, characterized in that, The Anoectochilus roxburghii exosome can directly target colorectal tissue to play a role. The preparation method of the Anoectochilus roxburghii exosome comprises the following steps: (1) crushing fresh Anoectochilus roxburghii to obtain a crushing liquid; (2) filtering the crushing liquid, and separating and collecting the precipitate by using an ultrahigh-speed centrifugation method; (3) resuspending the precipitate with pre-cooled PBS to obtain the Anoectochilus roxburghii exosome; In step (2), the ultrahigh-speed centrifugation method comprises the following steps: a) centrifuging the filtrate at 1000-2000 g for 10-15 min at 4 ℃, and collecting supernatant I; b) centrifuging supernatant I at 3000-4000 g for 15-20 min at 4 ℃, and collecting supernatant II; c) centrifuging supernatant II at 10000-15000 g for 40-60 min at 4 ℃, and collecting supernatant III; d) centrifuging supernatant III at 100000-150000 g for 70-90 min at 4 ℃, and collecting the precipitate; e) resuspending the precipitate in each tube with pre-cooled PBS, repeating 3-5 times, and centrifuging the resuspension at 100000-150000 g for 70-90 min at 4 ℃ to collect the precipitate again; After the precipitate in step (3) is resuspended with pre-cooled PBS, a filtering step of using a 0.22 µm filter membrane is further included, and the filtrate is collected to obtain the Anoectochilus roxburghii exosome.

2. Use according to claim 1, characterized in that, The colorectal cancer is azoxymethane / dextran sulfate sodium-induced colorectal cancer.

3. Use according to claim 1, characterized in that, The Anoectochilus roxburghii exosome comprises proteins, and the proteins comprise resveratrol O-methyltransferase, a protein containing a FAS1 domain, a pyrophosphate-primed vacuole membrane proton pump, a large subunit of ribulose-1,5-bisphosphate carboxylase / oxygenase, a protein containing a C-terminal domain of large ribosomal subunit protein uL4, 60S ribosomal protein L3, a propylene oxide synthase, a vacuole ATP synthase catalytic subunit, a protein containing a N-terminal domain of large ribosomal subunit protein uL6, small ribosomal subunit protein uS5, and 40S ribosomal protein S2.

4. Use according to claim 1, characterized in that, The Anoectochilus roxburghii exosome has a particle size of 150-200 nm.

5. The use according to claim 1, characterized in that, The Anoectochilus roxburghii exosome has a Zeta potential of -21.63± 1.34 mV, is spherical under an electron microscope, and has a double-membrane structure.

6. The use according to claim 1, characterized in that, The Anoectochilus roxburghii exosome does not contain roxburghin.

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