Use of lycoris radiata exosome in preparation of a medicament for treating heterotopic ossification

Abstract

This invention discloses the application of Lycoris radiata exosomes in the preparation of drugs for treating heterotopic ossification, belonging to the field of biomedical technology. The Lycoris radiata dried exosomes (La-dEXOs) prepared by this invention, verified through osteoclast and osteoblast differentiation experiments and animal experiments, show that the La-dEXOs prepared by this invention can improve heterotopic ossification in rats by promoting osteoclast differentiation and inhibiting osteoblast differentiation. Experimental results indicate that La-dEXOs have the potential to be developed into related pharmaceutical products for the prevention and treatment of heterotopic ossification.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of Lycoris radiata exosomes in the preparation of drugs for the treatment of heterotopic ossification. Background Technology

[0002] In recent years, extracellular vesicles (EVs), especially exosomes, have attracted widespread attention as novel drug delivery systems. Exosomes are naturally derived nanoscale (30-150 nm) lipid bilayer vesicles that can be secreted by almost all types of cells. As natural intercellular messengers, they can carry abundant functional substances such as proteins, nucleic acids, and lipids for long-distance transport within the body. Recent studies have shown that plant-derived exosome-like nanoparticles (PELNs) not only play important biological functions in their parent plants but also exhibit significant cross-boundary regulatory activity, capable of being absorbed by mammalian cells and regulating their physiological state. For example, nanoparticles isolated from grapefruit can effectively alleviate symptoms of ulcerative colitis in mice; ginger-derived nanovesicles show good hepatoprotective effects in an alcoholic liver disease model; and nanoparticles extracted from honeysuckle decoction carrying miRNA2911 can target influenza A virus and inhibit its replication. Plant-derived exosomes not only possess excellent biocompatibility, low immunogenicity, and low toxicity, but also exhibit higher stability and intestinal permeability. Furthermore, they are easy to mass-produce and inexpensive, making them ideal novel drug delivery carriers. Therefore, systematically exploring medicinally valuable plant exosomes and investigating their cross-species regulatory mechanisms has become an innovative strategy for developing next-generation natural nanomedicines.

[0003] Lycoris radiata ( LycorisLycoris radiata, with its dried bulbs, is a traditional Chinese medicine with a long history. Ancient records and modern pharmacological studies both indicate that it is rich in various alkaloids (such as galantamine, lycorine, and lecithin), polysaccharides, and other bioactive components, possessing multiple pharmacological activities including anti-inflammatory, anti-tumor, and neuroprotective effects. Notably, traditional Chinese medicine often uses dried Lycoris radiata rather than fresh. Modern research confirms that proper drying is not only a necessary preservation method but also alters the state, proportion, and bioavailability of active ingredients within Lycoris radiata through a series of biochemical reactions such as Maillard reaction and enzymatic transformation, even generating new active compounds, thereby significantly enhancing or altering its pharmacological effects. Therefore, using dried Lycoris radiata as raw material, rather than fresh Lycoris radiata, for in-depth development is more in line with traditional Chinese medicine theory and can better tap its potential medicinal value, distinct from that of fresh Lycoris radiata. However, how to efficiently deliver the active ingredients in dried Lycoris radiata to the lesion site remains a key technological bottleneck in its development into a modern drug.

[0004] Heterotopic ossification (HO) refers to the formation of mature bone tissue outside of bone tissue, which can severely restrict joint movement, cause chronic pain, and even endanger life. Current treatment strategies for HO still have limitations, including insufficient efficacy and significant side effects. Inflammation plays a crucial role in its development, and nonsteroidal anti-inflammatory drugs (NSAIDs) are currently effective in preventing HO in patients with neurological paralysis or trauma. However, NSAIDs cannot cure the disease; they can only relieve symptoms and cannot stop its progression. Furthermore, NSAIDs may cause adverse reactions. Gastrointestinal reactions are also common side effects, such as abdominal pain, nausea, and vomiting. For patients whose HO causes severe functional impairment, surgical resection is the only treatment. Therefore, developing novel therapeutic agents from traditional medicinal plants and further researching drugs that can effectively improve HO has become an important clinical problem that urgently needs to be solved. Summary of the Invention

[0005] In view of the problems existing in the treatment of heterotopic ossification, the purpose of this invention is to provide a dried Lycoris radiata exosome (La-dEXOs) and its preparation method, as well as the application of La-dEXOs in the preparation of functional foods, special dietary foods, health foods and / or pharmaceuticals for improving heterotopic ossification.

[0006] To achieve the above objectives, this invention discloses the use of Lycoris exosomes in the preparation of drugs for treating heterotopic ossification.

[0007] Specifically, the Lycoris radiata exosomes are dried Lycoris radiata exosomes, and the preparation method includes the following steps:

[0008] S1. Mix the air-dried Lycoris tissue with a buffer solution, crush and filter to obtain a crude Lycoris extract.

[0009] S2. Centrifuge and enzymatically hydrolyze the crude extract of Lycoris radiata obtained in step S1 to obtain clear Lycoris radiata extract.

[0010] S3. The Lycoris radiata solution obtained in step S2 is subjected to ultracentrifugation, the precipitate is retained and resuspended in a buffer solution to obtain the resuspension of Lycoris radiata exosomes.

[0011] Furthermore, the process described in step S2 includes the following steps:

[0012] S201. Centrifuge the crude extract of Lycoris radiata obtained in step S1 at 3-5℃ and 900-1100×g for 8-12 min to obtain supernatant 1.

[0013] S202. Centrifuge the supernatant 1 obtained in step S201 at 3-5℃ and 2900-3100×g for 28-32 min to obtain supernatant 2.

[0014] S203. The supernatant 2 obtained in step S202 is mixed with hemicellulase, pectinase and cellulase to obtain an enzyme mixture. After standing at room temperature for 2-3 hours for digestion, it is filtered through a filter membrane to obtain the Lycoris radiata clear liquid.

[0015] In step S203, the mass concentration of the hemicellulase in the enzyme mixture is 0.1%-0.3%, the mass concentration of the pectinase in the enzyme mixture is 0.05%-0.15%, and the mass concentration of the cellulase in the enzyme mixture is 0.05%-0.15%.

[0016] Furthermore, the process described in step S2 includes the following steps:

[0017] S201. Centrifuge the crude extract of Lycoris radiata obtained in step S1 at 4℃ and 1000×g for 10 min to obtain supernatant 1.

[0018] S202. Centrifuge the supernatant 1 obtained in step S201 at 4°C and 3000×g for 30 min to obtain supernatant 2.

[0019] S203. The supernatant 2 obtained in step S202 is mixed with hemicellulase, pectinase and cellulase to obtain an enzyme mixture. After standing at room temperature for 2-3 hours for digestion, it is filtered through a filter membrane to obtain the Lycoris radiata clear liquid.

[0020] In step S203, the mass concentration of the hemicellulase in the enzyme mixture is 0.2%, the mass concentration of the pectinase in the enzyme mixture is 0.1%, and the mass concentration of the cellulase in the enzyme mixture is 0.1%.

[0021] The filter membrane used in the above steps is a 0.45 μm filter membrane.

[0022] Further, in step S3, the Lycoris radiata solution obtained in step S2 is first centrifuged at 3-5℃ and 9000-11000×g for 25-35 min, and then centrifuged at 110000-130000×g for 80-100 min at least twice.

[0023] Preferably, in step S3, the Lycoris radiata solution obtained in step S2 is first centrifuged at 4°C and 10,000×g for 30 min, and then centrifuged twice at 120,000×g for 90 min.

[0024] Furthermore, in step S3, the resuspension is filtered using a 0.22 μm microporous membrane.

[0025] Preferably, in step S1, the air-dried Lycoris radiata tissue is prepared by drying at 55-65°C for 12-24 hours. Further, the air-dried Lycoris radiata tissue is prepared from Lycoris radiata bulbs dried at 60°C for 24 hours.

[0026] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0027] The dried Lycoris radiata exosomes (La-dEXOs) prepared in this invention were verified through osteoclast and osteoblast differentiation experiments, as well as animal experiments. The La-dEXOs prepared in this invention can improve ectopic ossification in rats by promoting osteoclast differentiation and inhibiting osteoblast differentiation. Experimental results indicate that La-dEXOs have the potential to be developed into related pharmaceutical products for the prevention and treatment of ectopic ossification. Attached Figure Description

[0028] Figure 1 Characterization results of dried Lycoris exosome-like nanoparticles, where A: transmission electron microscopy image of La-dEXOs; B: particle size distribution of La-dEXOs.

[0029] Figure 2 Results of toxicity testing of dried Lycoris radiata exosomes, including A: CCK-8 toxicity test results of air-dried Lycoris radiata exosomes at different air-drying times on days 1-5; B: results of primary mononuclear cell live / dead staining.

[0030] Figure 3Effects of dried Lycoris radiata exosomes (La-dEXOs) on osteoclast differentiation: A: TRAP staining results (MΦ: macrophage group, MΦ+RANKL: macrophages induced by RANKL, MΦ+La-dEXOs: macrophages treated with La-dEXOs, MΦ+RANKL+La-dEXOs: macrophages induced by RANKL and treated with La-dEXOs); B: TRAP activity assay results, p<0.0001; C: CTSK immunofluorescence staining.

[0031] Figure 4 Effects of dried Lycoris radiata exosomes (La-dEXOs) on osteoblast differentiation: A: ALP staining results on day 7; B: ALP activity assay results, *** p<0.001.

[0032] Figure 5 The alleviating effect of injecting dried Lycoris exosomes La-dEXOs on ectopic ossification in rats. Detailed Implementation

[0033] This application provides a method for preparing dried Lycoris radiata exosomes La-dEXOs. In this embodiment, the materials used include: air-dried Lycoris radiata bulbs; PBS buffer (pH 7.4, sterile); EP tubes (1.5 / 5 mL); centrifuge tubes (15 / 50 mL); and microporous filter membranes (0.22 / 0.45 μm).

[0034] Example 1: A method for preparing Lycoris exosomes La-dEXOs

[0035] In its specific implementation, this invention includes the following steps:

[0036] Take 60 g of dried Lycoris bulbs, wash them 3 times with pure water, add 100 mL of PBS buffer pre-cooled at 4°C, blend them for 5 min with a high-speed blender, and filter them coarsely with gauze to obtain the preliminary filtrate.

[0037] At 4℃, the preliminary filtrate was centrifuged at 1000×g for 10 min, the supernatant was collected, and the precipitate was discarded.

[0038] At 4℃, centrifuge the filtrate at 3000×g for 30 min, collect the supernatant and discard the precipitate;

[0039] Add 0.2% hemicellulase, 0.1% pectinase and 0.1% cellulase and let it stand at room temperature for 2.5 hours to digest. Filter using 0.45 μm filter, take the supernatant and discard the precipitate.

[0040] At 4℃, centrifuge the filtrate at 10000×g for 30 min, collect the supernatant and discard the precipitate;

[0041] Slowly aspirate the supernatant and transfer it to a new 10 mL ultracentrifuge tube. Add PBS to make up the difference to obtain a dried Lycoris exosome precipitate suspension.

[0042] At 4℃, the suspension was centrifuged at 120000×g for 90 min, and the supernatant was removed to obtain a high-purity exosome precipitate.

[0043] After resuspending the high-purity dried Lycoris radiata exosome precipitate in pre-cooled 1×PBS, the precipitate was transferred to a new 10 mL ultracentrifuge tube, and PBS was added to make up the difference, thus obtaining the dried Lycoris radiata exosome precipitate suspension again.

[0044] At 4℃, the suspension was centrifuged again at 120000×g for 90 min, and the supernatant was removed to obtain a high-purity exosome precipitate.

[0045] The high-purity dried Lycoris exosome precipitate was resuspended in pre-cooled 1×PBS and filtered sequentially through a 0.22 μm microporous membrane. The supernatant was collected. High-purity La-dEXOs were finally obtained.

[0046] Example 2: Identification of Lycoris exosomes La-dEXOs

[0047] 1. Characterization by transmission electron microscopy (TEM)

[0048] Using a pipette, 10 μL of the separated and purified La-dEXOs was added to a copper grid for precipitation for 1 min, and the floating liquid was absorbed with filter paper. 10 μL of uranium acetate was added to the copper grid for precipitation for 1 min, and the floating liquid was absorbed with filter paper. After drying at room temperature for 5 minutes, electron microscopy was performed at 80 kV to obtain transmission electron microscopy (TEM) images. The morphological identification of dried Lycoris radiata exosomes under TEM is as follows: Figure 1 A in the figure. As can be seen from the figure, the obtained La-dEXOs are uniform in shape and size under electron microscopy, and have a cup-shaped vesicle-like structure.

[0049] 2. Particle size distribution and concentration determination

[0050] One μL of the purified La-dEXOs was diluted with 999 μL of 1×PBS. Using a Particulate Matter X PMX120 instrument, the diluted lily exosome sample was aspirated with a new syringe for particle size distribution and concentration analysis. The particle size distribution curve of the dried Lycoris radiata exosomes prepared by this method is shown in the figure. Figure 1B in the figure. As can be seen from the figure, the main peak is around 100 nm, and the obtained La-dEXOs all fall in the 30-200 nm range, which is consistent with the exosome particle size characteristics.

[0051] Example 3: Toxicity evaluation of dried Lycoris radiata exosomes

[0052] 1. Toxicity evaluation of Lycoris radiata exosomes from dried samples with different air-drying times

[0053] Using rat bone marrow-derived mesenchymal stem cells (BMSCs) as a model, the cytotoxicity of exosomes prepared from Lycoris bulbs after different air-drying times (12 hours and 24 hours at 60°C) in Example 1 was evaluated. BMSCs were prepared into 5 × 10⁻⁶ cells / mL. 4 / mL of cell suspension, add 100 μL to each well of a 96-well plate for seeding.

[0054] The following day, the medium was replaced with α-MEM (containing 10% FBS and 1% penicillin-streptomycin) containing different concentrations (0 μg / mL, 0.2 μg / mL, 1 μg / mL, 5 μg / mL) of exosomes, 100 μL per well. Following the CCK-8 kit (Beyotime, C0037) instructions, BMSCs cell viability was assessed on days 1, 3, and 5 after exosome intervention. 10 μL of CCK-8 reagent was added to each well and incubated at room temperature in the dark for 1 hour. The absorbance (OD value) was measured at 450 nm using a microplate reader (see results below). Figure 2 (A) The results showed that 5 μg / mL exosomes dried for 12 hours had a certain inhibitory effect on BMSCs activity, while 0.2 μg / mL and 5 μg / mL exosomes dried for 12 hours and 24 hours had no inhibitory effect on BMSCs activity. Subsequent experiments selected Lycoris bulbs dried for 24 hours to prepare exosomes.

[0055] 2. Biocompatibility evaluation of Lycoris exosomes from air-dried samples after 24 hours

[0056] Primary mononuclear cells (MNCs) derived from mouse bone marrow were prepared into 5×10⁶ cells. 5 / mL of cell suspension was added to each well of a 48-well plate for seeding, and all cells were cultured overnight in a 5% CO2 incubator at 37°C.

[0057] The following day, after removing the culture medium, 500 μL of dried Lycoris radiata exosome solutions with concentration gradients of 0 μg / mL, 0.2 μg / mL, 1 μg / mL, and 5 μg / mL, prepared in α-MEM medium of 10% FBS and 1% penicillin-streptomycin, were added respectively. After 24 h of exosome treatment, staining was performed according to the instructions of the dead / live cell staining kit. The distribution and proportion of live cells (green fluorescence) and dead cells (red fluorescence) were observed under a fluorescence microscope (results are shown in...). Figure 2 (B) Experimental results showed that after intervention with exosomes in the concentration range of 0.2~5 μg / mL, the proportion of live cells in MNCs remained at a high level, and there was no significant increase in dead cells, indicating that the air-dried Lycoris radiata exosomes in this concentration range for 24 hours had no cytotoxicity to MNCs and had good biocompatibility.

[0058] Example 4: Application of dried Lycoris radiata exosomes in the preparation of drugs for treating heterotopic ossification

[0059] 1. Effects of dried Lycoris radiata exosomes on osteoclast differentiation

[0060] (1) TRAP staining and activity detection

[0061] To facilitate osteoclast differentiation, monocytes were divided into groups of 3 × 10⁻⁶ cells. 5 cells / cm 2 Inoculate into culture dishes, add 30 ng / mL M-CSF to the culture medium, change the medium every 48 h to induce macrophages (MΦ). Inoculate macrophages at a rate of 2 × 10⁶ cells / mL. 4 pcs / cm 2 Cells were seeded at a density in α-MEM medium supplemented with 10% FBS and 1% penicillin-streptomycin, and stimulated with 30 ng / mL macrophage colony-stimulating factor (M-CSF) and 50 ng / mL osteoclastogenic factor (RANKL). The medium was changed every 2 days, and mature osteoclasts were obtained from tissue culture plates after 6 days. The medium was then collected, washed once with PBS, and fixed with 4% paraformaldehyde for 15 minutes. Osteoclast viability was assessed using a commercial TRAP staining kit. TRAP activity was determined using the pNPP method according to the manufacturer's instructions. Results are as follows: Figure 3 A and Figure 3 As shown in Figure B, after RANKL stimulation, macrophages significantly differentiated into osteoclasts, exhibiting typical morphological characteristics, including enhanced TRAP activity and multinucleated cell structure, demonstrating successful osteoclast induction. In contrast, the La-dEXOs treatment group (5 μg / mL) showed a significantly increased number of TRAP-positive multinucleated osteoclasts, a significantly increased TRAP activity, and a significantly larger osteoclast volume compared to the RANKL-induced group. This result indicates that the dried Lycoris radiata exosomes prepared by this method can effectively promote osteoclast differentiation.

[0062] (2) CTSK immunofluorescence staining

[0063] The specific procedure for detecting CTSK was as follows: Cells were first washed with PBS, fixed with 4% paraformaldehyde for 15 minutes, permeabilized with 0.25% Triton X-100 for 20 minutes, and then blocked for 30 minutes with blocking buffer containing 2% BSA, 2% normal goat serum, and 0.1M glycine. Next, anti-CTSK primary antibody was added and incubated overnight at 4°C. After washing with PBS, goat anti-mouse secondary antibody was added and incubated at room temperature for 1 hour. Finally, DAPI staining was performed for 10 minutes, and CTSK expression was observed and analyzed using a laser confocal microscope (Nikon). Results are as follows: Figure 3 As shown in Figure C, osteoclasts stimulated by RANKL exhibited strong CTSK positivity, and the CTSK positivity in the La-dEXOs treatment group (5 μg / mL) was significantly stronger than that in the RANKL-induced group. This result indicates that the dried Lycoris radiata exosomes prepared by this method can effectively promote osteoclast differentiation.

[0064] Effects of dried Lycoris exosomes on osteoblast differentiation

[0065] (1) ALP staining and activity detection

[0066] Bone marrow-derived MSCs were used at a rate of 1×10 4 cells / cm 2 Cells were cultured in 48-well plates at high density for osteogenic differentiation assays. After overnight culture, the medium was replaced with osteogenic medium (OM, α-MEM supplemented with 10% FBS, 1% penicillin-streptomycin, 10 nM dexamethasone, 10 mM β-glycerophosphate, and 0.1 mM 1-ascorbic acid-2-phosphate). The medium was then changed twice weekly. After 7 days of induction, the samples were subjected to two freeze-thaw cycles at -80°C to release intracellular alkaline phosphatase (ALP). ALP activity in the samples was then measured using the p-nitrophenyl phosphate method. Figure 4 The results showed that, compared with the control group, the ALP staining positive signal of BMSCs treated with La-dEXOs was significantly reduced, and the ALP staining positive signal of the 10 μg / mL La-dEXOs treatment group was also weaker than that of the 5 μg / mL La-dEXOs treatment group. Figure 4 (A) This intuitively demonstrates that La-dEXOs can effectively inhibit the osteogenic differentiation process of BMSCs. Further quantitative analysis showed that ALP activity was significantly reduced in the La-dEXOs treatment group, and the higher the concentration of La-dEXOs, the lower the ALP activity (A). Figure 4 (B in the text). This result indicates that the dried Lycoris radiata exosomes prepared by this method can effectively inhibit osteoblast differentiation.

[0067] Establishment of a rat model of heterotopic ossification

[0068] (1) Establishment of a model of heterotopic ossification after tendon transection

[0069] Two 6-week-old male Sprague-Dawley rats were used to establish the model. All rats were fasted for 6 hours before anesthesia. The anesthetic dose was 50 mg / kg sodium pentobarbital administered intraperitoneally, followed by immobilization in a prone position. Under aseptic conditions, both hind limbs were positioned posterolaterally. The midpoint of the Achilles tendon was completely severed, and both ends of the ruptured tendon were clamped with hemostatic forceps, repeatedly clamped 10 times to induce some degree of trauma. The skin incision was then sutured. After 8 weeks of routine feeding and observation, Micro-CT scans showed successful model establishment.

[0070] Evaluation of the efficacy of dried Lycoris radiata exosomes in treating ectopic ossification rats

[0071] Male rats with successfully established ectopic bone marrow were treated with local injection of exosomes into the ectopic bone site. The concentration of exosome protein injected was 126 μg / mL, and the injection volume was 100 μL. Eight weeks after exosome injection, experimental animal samples were collected. Micro-CT scans were performed on the obtained rat ectopic bone samples, and the data were analyzed. Figure 5 The results showed that extraosome injection effectively alleviated heterotopic ossification. Compared with the sham surgery group, the control group showed very significant heterotopic bone formation, and the La-dEXOs group showed a significant therapeutic effect.

[0072] The present invention extracts La-dEXO nanoparticles through the above embodiments and conducts pathological experiments on them in a rat model of ectopic ossification. It can improve ectopic ossification in rats by promoting osteoclast differentiation and inhibiting osteoblast differentiation, and it was found that its effect has no toxic side effects.

[0073] Therefore, the La-dEXO nanoparticles extracted using the method of this invention can be considered an ideal choice for preparing drugs to treat heterotopic ossification. Without conflict, the above embodiments and their technical features can be combined with each other. This invention is not limited to the contents disclosed in the specification and embodiments; those skilled in the art can readily realize other advantages and improvements without departing from the general inventive concept defined by the claims and their equivalents. Accordingly, this invention is not limited to the specific details, representative embodiments, and illustrated examples shown herein.

Claims

1. The application of Lycoris exosomes in the preparation of drugs for the treatment of heterotopic ossification; The heterotopic ossification is traumatic heterotopic ossification; The Lycoris exosomes are dried Lycoris exosomes, and the preparation method includes the following steps: S1. Mix the air-dried Lycoris bulbs with a buffer solution, crush them, filter them, and obtain a crude Lycoris extract. S2. Centrifuge and enzymatically hydrolyze the crude extract of Lycoris radiata obtained in step S1 to obtain clear Lycoris radiata extract. S3. The Lycoris radiata solution obtained in step S2 is subjected to ultracentrifugation, the precipitate is retained and resuspended in a buffer solution to obtain the resuspension of the Lycoris radiata exosomes. The air-dried Lycoris bulbs were dried at 55-65℃ for 12-24 hours; The process described in step S2 includes the following steps: S201. Centrifuge the crude extract of Lycoris radiata obtained in step S1 at 3-5℃ and 900-1100×g for 8-12 min to obtain supernatant 1. S202. Centrifuge the supernatant 1 obtained in step S201 at 3-5℃ and 2900-3100×g for 28-32 min to obtain supernatant 2. S203. The supernatant 2 obtained in step S202 is mixed with hemicellulase, pectinase and cellulase to obtain an enzyme mixture. After standing at room temperature for 2-3 hours for digestion, it is filtered through a filter membrane to obtain the Lycoris radiata clear liquid. In step S203, the mass concentration of the hemicellulase in the enzyme mixture is 0.1%-0.3%, the mass concentration of the pectinase in the enzyme mixture is 0.05%-0.15%, and the mass concentration of the cellulase in the enzyme mixture is 0.05%-0.15%. In step S3, the Lycoris radiata solution obtained in step S2 is first centrifuged at 3-5℃ and 9000-11000×g for 25-35 min, and then centrifuged at 110000-130000×g for 80-100 min at least twice.

2. The application according to claim 1, characterized in that, The process described in step S2 includes the following steps: S201. Centrifuge the crude extract of Lycoris radiata obtained in step S1 at 4℃ and 1000×g for 10 min to obtain supernatant 1. S202. Centrifuge the supernatant 1 obtained in step S201 at 4°C and 3000×g for 30 min to obtain supernatant 2. S203. The supernatant 2 obtained in step S202 is mixed with hemicellulase, pectinase and cellulase to obtain an enzyme mixture. After standing at room temperature for 2-3 hours for digestion, it is filtered through a filter membrane to obtain the Lycoris radiata clear liquid. In step S203, the mass concentration of the hemicellulase in the enzyme mixture is 0.2%, the mass concentration of the pectinase in the enzyme mixture is 0.1%, and the mass concentration of the cellulase in the enzyme mixture is 0.1%.

3. The application according to claim 1 or 2, characterized in that, The filter membrane used was a 0.45 μm filter membrane.

4. The application according to claim 1, characterized in that, In step S3, the Lycoris radiata solution obtained in step S2 is first centrifuged at 4°C and 10,000×g for 30 min, and then centrifuged twice at 120,000×g for 90 min.

5. The application according to claim 1, characterized in that, In step S3, the resuspension is filtered using a 0.22 μm microporous membrane.

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