A liposome preparation of five-grain worm and a preparation method and application thereof

Abstract

The application discloses a liposome preparation of pentatomidae, a preparation method and application thereof, and belongs to the technical field of medicine.The liposome preparation of pentatomidae comprises pentatomidae active ingredients and liposomes, the pentatomidae active ingredients include pentatomidae water-soluble extracts and pentatomidae fat-soluble extracts, and the liposomes are formed by phospholipids and cholesterols.The application simultaneously loads the two types of pentatomidae active ingredients in the liposomes, realizes the co-delivery of the two types of active ingredients, and has good physical stability and tumor targeting property.The particle size of the preparation is 150-200nm, and the absolute value of Zeta potential is greater than 20mV.The application further provides a preparation method of the preparation and application of the preparation in the preparation of medicines for resisting lung cancer, improving peripheral blood leukopenia and protecting the immune organ function of the spleen.In addition, the application further provides a plant exosome complex loaded with pentatomidae extracts, a preparation method and application thereof.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a liposome preparation of *Polygonum multiflorum*, its preparation method and application, and particularly to a liposome preparation simultaneously loaded with water-soluble and fat-soluble extracts of *Polygonum multiflorum*, and the application of this preparation in the preparation of drugs for treating lung cancer, improving peripheral blood leukopenia and protecting the function of the spleen immune organ. Background Technology

[0002] Lung cancer is one of the leading causes of cancer-related morbidity and mortality worldwide, with non-small cell lung cancer (NSCLC) accounting for approximately 85% of all lung cancer cases. Despite significant advancements in targeted therapy and immunotherapy in recent years, the overall five-year survival rate for lung cancer remains low, and drug resistance and treatment-related toxicities continue to pose major clinical challenges. Therefore, developing novel, highly effective, and low-toxicity anti-lung cancer drugs is of significant clinical importance.

[0003] Maggot, also known as grain worm, is the larva of the big-headed golden fly (Chrysomya megacephala (Fabricius)) or other closely related insects, and is one of the traditional Chinese medicinal materials. Modern pharmacological studies have shown that Maggot extract possesses various biological activities, including anti-tumor, anti-inflammatory, antibacterial, and antioxidant effects. In the treatment of lung cancer, Maggot extract can inhibit the proliferation, migration, and invasion of lung cancer cells. Its mechanism involves inhibiting the interaction between HSP90AB1 and IGF1R, activating the p38 mitogen-activated protein kinase signaling pathway, regulating the expression of inflammatory factors, and inducing ferroptosis. In addition, Maggot extract also has anti-inflammatory effects, inhibiting the expression of inflammatory factors such as IL-6 and CXCL8, and has certain anti-infective activity.

[0004] However, the clinical application of *Polygonum multiflorum* extract still faces several challenges. First, the active components of *Polygonum multiflorum* are complex, comprising two main categories: water-soluble peptides and fat-soluble fatty acids. A single extraction method cannot simultaneously preserve both types of active components, leading to a loss of effective ingredients. Second, current methods of administration for *Polygonum multiflorum* are relatively limited, primarily relying on traditional decoctions, powders, or simple extracts, resulting in low bioavailability and poor targeting. Third, the stability issue of *Polygonum multiflorum* extract has not been effectively resolved; the active components are easily and rapidly eliminated from the body, making it difficult to maintain a sustained therapeutic effect.

[0005] Currently, some studies have attempted to develop extracts from *Pseudolarix amabilis* into pharmaceutical formulations. For example, Chinese patent CN109464468A discloses a method for preparing *Pseudolarix amabilis* powder, which involves alkali treatment and ultrafine pulverization to obtain the powder; Chinese patent CN108392496B discloses an extraction process for fatty acids from *Pseudolarix amabilis*; and Chinese patent CN106344615A discloses the application of *Pseudolarix amabilis* products in the preparation of drugs for treating non-small cell lung cancer. However, these technical solutions have failed to solve the challenge of simultaneously and targetedly delivering both water-soluble and lipid-soluble active ingredients from *Pseudolarix amabilis*.

[0006] Liposomes, as a mature nanomedicine delivery system, have advantages such as good biocompatibility, the ability to simultaneously encapsulate hydrophilic and lipophilic drugs, the ability to prolong drug circulation time in vivo, and improved drug targeting. The particle size and zeta potential of liposomes are key parameters affecting their stability and in vivo behavior. When the absolute value of the zeta potential is greater than 20 mV, the electrostatic repulsion between liposomes is sufficient to overcome van der Waals forces, which is beneficial to system stability.

[0007] Therefore, how to efficiently co-deliver both water-soluble and fat-soluble active ingredients of the five grain insect and improve the targeted retention ability and anti-lung cancer activity of the formulation is a technical problem that needs to be solved by those skilled in the art. Summary of the Invention

[0008] The purpose of this invention is to overcome the technical defects of existing *Pteris vittata* preparations, such as single active ingredient content, difficulty in simultaneously delivering water-soluble and fat-soluble active ingredients, low bioavailability, and lack of targeted delivery systems. The invention provides a liposomal preparation that can simultaneously load both water-soluble and fat-soluble active ingredients of *Pteris vittata*, has good stability and anti-lung cancer activity, as well as its preparation method and application.

[0009] Another object of the present invention is to provide a plant exosome complex loaded with the extract of *Ixodes spp.*, its preparation method and application.

[0010] Inventive concept

[0011] The antitumor active ingredients in *Pteris vittata* are mainly distributed in two polarity components: water-soluble portions (such as polypeptides and proteins) and lipid-soluble portions (such as fatty acids and oils). Existing drug delivery routes make it difficult to deliver both types of active ingredients simultaneously. This invention uses both water-soluble and lipid-soluble extracts of *Pteris vittata* as active ingredients, utilizing the structural characteristic of liposomes' phospholipid bilayers to simultaneously encapsulate both hydrophilic and lipophilic drugs, thus achieving the co-delivery of both types of active ingredients. Specifically, the lipid-soluble extract is co-formed with phospholipids and cholesterol to embed the lipid-soluble components within the lipid bilayer; then, the water-soluble extract is dissolved in an aqueous phase for hydration, encapsulating it within the hydrophilic lumen of the liposome, thereby constructing a liposome formulation simultaneously loading both types of active ingredients. This liposome formulation has a particle size of 150–200 nm, an absolute Zeta potential greater than 20 mV, and can passively target tumor tissue through the EPR effect, while also exhibiting good physical stability.

[0012] Furthermore, this invention also provides a delivery scheme for the active ingredients of *Ceratophyllum demersum* based on plant exosomes. Plant exosomes are natural nanovesicles derived from plant cells, possessing advantages such as small particle size, high biocompatibility, and low immunogenicity. This invention co-incubates exosomes extracted from cucumber with the active ingredients of *Ceratophyllum demersum*, loading the active ingredients onto the surface or interior of the exosomes through the affinity between the exosome membrane and the active ingredients, forming a plant exosome complex loaded with *Ceratophyllum demersum* extract (particle size 60–100 nm, absolute Zeta potential greater than 20 mV), thus achieving another nano-delivery method for the active ingredients of *Ceratophyllum demersum*.

[0013] Five-grain insect liposome preparation

[0014] This invention provides a liposome formulation of *Polygonum multiflorum*, comprising the active ingredient of *Polygonum multiflorum* and liposomes. The active ingredient of *Polygonum multiflorum* consists of a water-soluble extract and a fat-soluble extract of *Polygonum multiflorum*. The water-soluble extract of *Polygonum multiflorum* is prepared by water extraction and mainly contains *Polygonum multiflorum* polypeptides; the fat-soluble extract of *Polygonum multiflorum* is prepared by organic solvent extraction and mainly contains *Polygonum multiflorum* oil.

[0015] Liposomes are composed of phospholipids and cholesterol as membrane materials. Phospholipids serve as the backbone material of liposomes, while cholesterol acts as a membrane stabilizer embedded in the phospholipid bilayer, regulating the fluidity and rigidity of the membrane.

[0016] As a preferred option, the liposome formulation of *Polygonum multiflorum* has a particle size of 150–200 nm and an absolute Zeta potential value >20 mV. The nanoscale particle size endows the formulation with the ability to accumulate in tumor tissues through the EPR effect; when the absolute Zeta potential value is greater than 20 mV, the electrostatic repulsion between particles can effectively prevent aggregation, ensuring the storage stability and in vivo circulation stability of the formulation.

[0017] As a preferred option, the water-soluble extract of *Corydalis esculenta* achieves an encapsulation rate of 70%–85% in liposomes, while the fat-soluble extract achieves an encapsulation rate of 80%–95%. This high encapsulation rate ensures effective loading of the active ingredient and reduces the toxic side effects of free drugs.

[0018] Preparation method of liposome preparation of five grain insects

[0019] This invention provides a method for preparing the above-mentioned five-grain insect liposome preparation, comprising the following steps:

[0020] (1) Preparation of water-soluble extract of five grain insects: Five grain insect powder was taken, water was added as a solvent for ultrasonic extraction, the supernatant was collected by centrifugation, and the supernatant was freeze-dried under vacuum to obtain water-soluble extract of five grain insects.

[0021] (2) Preparation of fat-soluble extract of five grain insects: Five grain insect powder was extracted with organic solvent, the solvent was removed and dried to obtain fat-soluble extract of five grain insects.

[0022] (3) Liposome encapsulation: Phospholipids, cholesterol and the lipophilic extract of *Corydalis esculenta* obtained in step (2) are dissolved together in an organic solvent. After removing the solvent, a drug-containing film is formed on the container wall. The water-soluble extract of *Corydalis esculenta* obtained in step (1) is dissolved in an aqueous phase and hydrated with the film, so that the film falls off and spontaneously forms liposomes. The particle size is controlled by ultrasonic treatment to obtain the *Corydalis esculenta* liposome preparation.

[0023] In the preparation method, the process parameters of each step can be adjusted according to actual needs. In the water extraction step, the ratio of *Pseudolarix amabilis* powder to water can be 1:10 to 1:80, the pH can be 5.5 to 6.5, and the ultrasonic treatment time can be 20 to 60 min. In the lipid extraction step, the organic solvent can be ethyl acetate, and Soxhlet extraction can be used. The ratio of *Pseudolarix amabilis* powder to ethyl acetate can be 1:3 to 1:10, and the extraction time can be 1 to 4 h. In the encapsulation step, the mass ratio of phospholipids to cholesterol can be 5:1 to 35:1, the mass ratio of phospholipids to *Pseudolarix amabilis* lipid-soluble extract can be 3:1 to 10:1, the mass ratio of *Pseudolarix amabilis* water-soluble extract to *Pseudolarix amabilis* lipid-soluble extract can be 0.5:1 to 3:1, the ultrasonic frequency can be 100 to 150 kHz, and the ultrasonic time can be 5 to 20 min. The solvent removal can be done by rotary evaporation at a temperature of 40 to 50 °C, and the hydration temperature can be 40 to 50 °C.

[0024] Plant exosome complex

[0025] This invention provides a plant exosome complex loaded with extract of *Ceratophyllum demersum*, comprising the active ingredient of *Ceratophyllum demersum* and plant exosomes.

[0026] The active ingredients of *Polygonum multiflorum* include water-soluble and fat-soluble extracts. Plant exosomes are derived from the fruits of Cucurbitaceae plants. Cucurbitaceae plants have the advantages of abundant sources and mature extraction processes. As a preferred option, cucumber exosomes are selected.

[0027] As a preferred option, the plant exosome complex has a particle size of 60–100 nm and an absolute value of Zeta potential >20 mV.

[0028] Preparation method of plant exosome complex

[0029] This invention provides a method for preparing a plant exosome complex loaded with *Pteris vittata* extract, comprising the following steps:

[0030] (1) Extraction of plant exosomes: Take plant fruits, homogenize them, remove cell debris and large vesicles by differential centrifugation, and then obtain plant exosomes by ultracentrifugation.

[0031] (2) Preparation of active ingredients of the five grain insect: Take the five grain insect powder and extract the water-soluble extract and / or the fat-soluble extract of the five grain insect respectively.

[0032] (3) Co-incubation loading: The plant exosomes obtained in step (1) are mixed with the active ingredient of the five grain insect obtained in step (2) and then co-incubated to load the active ingredient onto the exosomes. The unloaded components are then removed by ultracentrifugation and washing to obtain the plant exosome complex loaded with the five grain insect extract.

[0033] In the preparation method, differential centrifugation includes sequential centrifugation at 200–500 × g for 5–20 min, 1000–3000 × g for 10–30 min, and 8000–12000 × g for 20–40 min; ultracentrifugation is performed at 100000–150000 × g for 50–90 min. In the co-incubation loading step, the mass ratio of plant exosomes to the active ingredient of *Gnaphalium affine* is 1:2–1:5, the co-incubation temperature is 25–40℃, and the time is 2–6 h.

[0034] Medical Use

[0035] This invention also relates to the application of liposome preparations of *Pteris vittata* in the preparation of drugs.

[0036] Specifically, the liposome preparation of *Pteris vittata* can be used to prepare drugs for treating lung cancer, preferably for preparing drugs for treating non-small cell lung cancer.

[0037] Furthermore, based on the observed effects of improving peripheral blood leukocyte levels in tumor-bearing nude mice and protecting the structure of the spleen's immune organ, the liposome preparation of *Eriocaulon buergerianum* can also be used to prepare drugs for improving peripheral blood leukopenia and drugs for protecting the function of the spleen's immune organ.

[0038] The present invention also relates to the use of plant exosome complexes loaded with *Pteris vittata* extract in the preparation of medicaments for treating lung cancer, preferably in the preparation of medicaments for treating non-small cell lung cancer.

[0039] The present invention, by adopting the above-described technical solution, has the following beneficial effects:

[0040] 1. This invention integrates the water-soluble and lipid-soluble extracts of *Polygonum multiflorum* as a single active component, simultaneously encapsulated in liposomes to achieve co-delivery of both active ingredients. Experimental data show that the encapsulation rate of the water-soluble extract in the *Polygonum multiflorum* liposome formulation reaches 75.79±2.18%, and the encapsulation rate of the lipid-soluble extract reaches 87.38±3.19%. Both active ingredients are efficiently encapsulated in the same delivery system, enabling them to fully exert their synergistic anti-lung cancer effect.

[0041] 2. The liposome formulation of *Polygonum multiflorum* prepared by this invention has a particle size of 179.02±1.20 nm (preferably within the range of 150–200 nm) and a Zeta potential of -53.1±0.12 mV (absolute value > 20 mV), exhibiting good physical stability. When the absolute value of the Zeta potential is greater than 20 mV, the electrostatic repulsion between liposomes is sufficient to overcome van der Waals forces, preventing particle aggregation and precipitation, which is beneficial for the long-term storage of the formulation and its structural integrity during in vivo circulation. In contrast, the active ingredients in traditional *Polygonum multiflorum* decoctions and powders are easily degraded and inactivated; the liposome formulation of this invention effectively solves this technical problem.

[0042] 3. Liposomes, as nanodelivery carriers, with a particle size of 150–200 nm, can passively target tumor tissue through the EPR effect (enhanced permeability and retention effect). The *Polygonum multiflorum* liposome formulation prepared in this invention has a particle size of 179.02 nm, which falls precisely within the optimal particle size window for the EPR effect. This effectively prolongs the drug's circulation time in vivo, increases the accumulation of *Polygonum multiflorum* active ingredients at the tumor site, thereby enhancing the anti-tumor effect while reducing toxic side effects on normal tissues. Cell retention experiments showed that after entering lung cancer PC9 cells, the *Polygonum multiflorum* liposome formulation remained largely retained within the cancer cells for 12 hours, indicating that the formulation has good affinity for lung cancer cells and can achieve long-term release of active ingredients within target cells.

[0043] 4. In the present invention, cucumber exosomes are used as a delivery vehicle for *Sarcophaga peregrina* larvae extract to prepare a plant exosome complex loaded with *Sarcophaga peregrina* larvae extract. Experiments show that the embedding rate of the complex for water-soluble extracts is 71.39±1.81%, the embedding rate for fat-soluble extracts is 66.28±3.10%, the particle size is 78.13±3.77 nm, and the Zeta potential is -47.59±1.33mV (absolute value > 20mV), indicating the system is stable. Plant exosomes have the advantages of wide sources, low cost, high biocompatibility, low immunogenicity, and large-scale preparation, which provide a brand-new technical solution for the delivery of active ingredients of *Sarcophaga peregrina* larvae and enrich the product forms of *Sarcophaga peregrina* larvae nano-preparations.

[0044] 5. The results of in vitro CCK8 assay show that the inhibitory effect of *Sarcophaga peregrina* larvae liposome preparation and cucumber exosome complex on lung cancer PC9 cells is significantly superior to that of the traditional *Sarcophaga peregrina* larvae compound decoction, suggesting that the nano-delivery system can effectively enhance the in vitro anti-tumor activity of the active ingredients of *Sarcophaga peregrina* larvae. In vivo experiments on tumor-bearing nude mice further confirmed that the tumor volumes of the *Sarcophaga peregrina* larvae liposome preparation group and the cucumber exosome complex group were both significantly smaller than that of the blank control group (p < 0.01), and significantly smaller than that of the traditional compound decoction group (p < 0.05), indicating that the two nano-preparations have better in vivo anti-lung cancer effects than the traditional decoction. Meanwhile, the peripheral blood white blood cell counts of all *Sarcophaga peregrina* larvae treatment groups were significantly higher than that of the blank control group (p < 0.05), and the splenic reticular fiber structure was clearer and more complete than that of the control group, suggesting that while exerting the anti-tumor effect, the *Sarcophaga peregrina* larvae preparation can also improve the peripheral blood white blood cell level of tumor-bearing organisms and protect the function of spleen immune organs, which has the dual benefit of immune protection. This feature is particularly important for lung cancer patients receiving chemotherapy.

[0045] 6. It is confirmed by ELISA and Western blot experiments that the *Sarcophaga peregrina* larvae preparation can significantly inhibit the release of inflammatory factor TNF-α (p < 0.01), down-regulate the expression level of Phospho-NF-κB p65 protein, and up-regulate the expression level of p53 protein. The above results show that the *Sarcophaga peregrina* larvae preparation exerts the anti-lung cancer effect from two aspects: inhibiting the inflammatory microenvironment and promoting tumor cell apoptosis, by inhibiting the TNF-α-mediated inflammatory response, blocking the abnormal activation of the NF-κB signaling pathway, and restoring the p53-mediated apoptosis pathway. This discovery of the mechanism provides a theoretical basis at the molecular level for the clinical application of *Sarcophaga peregrina* larvae, and also points out the direction for the subsequent expansion of indications. Description of Drawings

[0046] The present invention is further described below with reference to the accompanying drawings:

[0047] Figure 1This image shows the retention effect of the *Pteris vittata* liposome formulation in lung cancer cells. DAPI (blue) labels the cell nuclei, C-6 (green) labels the liposomes, and MERGE is an overlay of the two. Scale bar: 50 μm.

[0048] Figure 2 Bar chart showing the effect of different *Pteris vittatae* preparations on PC9 cell viability (CCK8 experiment results). The ordinate represents normalized cell viability (mean ± SD, n = 5).

[0049] Figure 3 The effects of the *Five Grains Worm* preparation on inflammatory factors and the expression of NF-κB and p53 proteins are shown in Figure A (TNF-α ELISA results), Figure B (IL-6 ELISA results), and Figure C (NF-κB and p53 protein expression results from Western blot analysis).

[0050] Figure 4 The results show the detection of tumors and related indicators in nude mice under different treatment groups. A shows the gross specimens of tumors dissected from each group; B shows the statistical results of tumor volume in each group; C shows the statistical results of peripheral blood white blood cell count (WBC) in each group; D shows the statistical results of spleen index in each group; E shows the H&E staining results of tumor tissue in each group; and F shows the Gomori silver staining results of spleen tissue in each group. Detailed Implementation

[0051] To make the objectives, technical solutions, and advantages of this invention clearer, the embodiments of this invention will be described in further detail below with reference to the accompanying drawings. It should be noted that the following embodiments are only for a better understanding of this invention and do not constitute a limitation on the scope of protection of this invention.

[0052] Example 1: Preparation and Characterization of Five-Grain Worm Liposome Formulation

[0053] 1.1 Quality Control of Grain Worm Raw Materials

[0054] The grain worms used in this embodiment are dried larvae of the large-headed golden fly (Chrysomya megacephala (Fabricius)) of the family Blisteridae, which are processed into stir-fried grain worms and produced by Jiaxing Dongfang Guoyao Pharmaceutical Co., Ltd. (product batch number: 25100903, production date: 20251009). The standard implemented is the "Quality Standard of Grain Worm Chinese Medicinal Material" in the "Announcement of Zhejiang Provincial Drug Administration" (No. 3 of 2022).

[0055] The quality control indicators for this batch of raw materials are as follows: moisture 9.0% (standard stipulates not to exceed 10.0%); acid-insoluble ash 0.1% (standard stipulates not to exceed 3.0%); alcohol-soluble extract 44.4% (standard stipulates not to be less than 20.0%); sulfur dioxide residue 11 mg / kg (standard stipulates not to exceed 150 mg / kg); total aerobic bacteria count <10 cfu / g (standard stipulates not to exceed 100,000 cfu / g); mold and yeast count <10 cfu / g (standard stipulates not to exceed 1,000 cfu / g); bile salt-tolerant Gram-negative bacteria, Salmonella, and Escherichia coli were not detected.

[0056] Take the above-mentioned stir-fried grain insects that meet the quality standards, freeze-dry them under vacuum until constant weight, pulverize them and pass them through an 80-mesh sieve to obtain grain insect dry powder, seal and store at 4°C for later use. The grain insects used in subsequent examples all come from this batch of raw materials.

[0057] 1.2 Preparation of water-soluble extract of *Polygonum multiflorum*

[0058] Accurately weigh the dried powder of *Polygonum multiflorum*, add purified water at a ratio of 1:50, adjust the pH to 6.0 with dilute hydrochloric acid, sonicate at 25 Hz for 40 min, centrifuge at 4000 rpm for 10 min, collect the supernatant, freeze dry it into powder using a freeze dryer to obtain a water-soluble extract of *Polygonum multiflorum* (mainly containing *Polygonum multiflorum* polypeptides), for later use.

[0059] 1.3 Preparation of fat-soluble extract of *Pteris vittata*

[0060] Accurately weigh the dried powder of *Polygonum multiflorum* and add it to a Soxhlet extractor. Add ethyl acetate (the ratio of *Polygonum multiflorum* powder to ethyl acetate is 1:5). Heat in a water bath and reflux for 2 hours. Recover the ethyl acetate and remove the organic solvent by rotary evaporation to obtain the fat-soluble extract of *Polygonum multiflorum* (mainly containing *Polygonum multiflorum* oil), which is then ready for use.

[0061] 1.4 Preparation of Five-Grain Worm Liposome Formulation

[0062] Liposome preparations of *Strombus haematocephala* were prepared using a combination of thin-film dispersion and ultrasonication.

[0063] (1) Formation of film: 100mg soybean lecithin, 10mg cholesterol and 20mg fat-soluble extract of five grain insects were placed in a flask, 5mL ethyl acetate was added and dissolved completely, and the flask was placed in a rotary evaporator. The ethyl acetate was removed by rotary evaporation at 45℃ and under negative pressure, and a uniform film was formed on the inner wall of the flask.

[0064] (2) Hydration: Dissolve 20 mg of water-soluble extract of the five grain insect in 3 mL of purified water. After fully dissolving, preheat to 45 °C and transfer to a flask containing the above-mentioned film. Vortex for 10 min to allow the film to hydrate and detach, forming a crude liposome suspension.

[0065] (3) Ultrasonic treatment: Place the crude liposome suspension in a probe ultrasonic instrument and sonicate for 10 minutes under ice bath conditions (ultrasonic frequency 130kHz, 5s on, 3s off) to obtain the Wugu Chong liposome preparation.

[0066] 1.5 Characterization of the Five Grain Worm Liposome Preparation

[0067] Encapsulation rate detection: The free active ingredients of *Eriocheir sinensis* and the active ingredients of *Eriocheir sinensis* encapsulated in liposomes were separated by ultrafiltration centrifugation.

[0068] Preparation of the standard curve: For the water-soluble fraction, bovine serum albumin (BSA) was used as a reference standard, and detection was performed at 750 nm using the Folin-Ciocalteu colorimetric method. A standard curve was plotted with BSA solutions at concentrations ranging from 0.01 to 1.51 mg / mL, plotted on the x-axis (concentration) and y-axis (absorbance). The regression equation was: y = 2.0576x + 0.2544 (R²). 2 =0.9993). For the lipid-soluble fraction, using *Pteris vittata* oil as a reference standard, reference solutions with concentrations of 0.20–2.00 mg / mL were prepared with anhydrous ethanol. The absorbance was measured at 233 nm, and a standard curve was plotted, yielding the regression equation: y = 0.2138x + 0.004 (R² = 0.9993). 2 =0.9998).

[0069] Determination of total drug content: Accurately pipette 100 μL of the Wugu Chong liposome preparation into a 10 mL volumetric flask, add 70% methanol to break the emulsion and dilute to the mark, and use a UV-Vis spectrophotometer to determine the total content Q_total of the Wugu Chong active ingredients in the liposome preparation, and calculate the concentration according to the above standard curve.

[0070] Determination of free drug content: Accurately pipette 100 μL of the *Corydalis esculenta* liposome preparation into a 100 kDa ultrafiltration centrifuge tube, centrifuge at 4℃ and 8000 rpm for 40 min, collect the lower layer filtrate of the ultrafiltration centrifuge tube, transfer it to a 10 mL volumetric flask, dilute with 70% methanol and bring to the mark, and determine the content Q_free of the free *Corydalis esculenta* active ingredient using a UV-Vis spectrophotometer, and calculate the concentration based on the above standard curve.

[0071] The encapsulation rate is calculated using the following formula: Encapsulation rate (%) = (1 - Q_spontaneous / Q_total) × 100%.

[0072] Particle size and zeta potential detection: The particle size and zeta potential of the Wugu Chong liposome preparation were detected by dynamic light scattering (DLS) at 25℃, and each sample was measured in triplicate.

[0073] The characterization results are shown in Table 1. Encapsulation efficiency and particle size distribution of the Wugu Chong liposome formulation (mean±SD, n=3).

[0074] Table 1:

[0075] Blank liposomes — — 168.45±3.79 -41.9±0.48 Five-grain insect liposome preparation 75.79±2.18 87.38±3.19 179.02±1.20 -53.1±0.12

[0076] Table 1 shows that the encapsulation rate of the water-soluble extract of *Corydalis esculenta* by liposomes was 75.79±2.18%, and the encapsulation rate of the fat-soluble extract was 87.38±3.19%, indicating that liposomes have a good encapsulation effect on both types of active ingredients. The particle size of the *Corydalis esculenta* liposome formulation was 179.02±1.20 nm, and the Zeta potential was -53.1±0.12 mV. The absolute value of the Zeta potential was greater than 20 mV, indicating that the electrostatic repulsion between liposomes was sufficient to maintain the stability of the system, which is beneficial to the long-term storage of the formulation.

[0077] Example 2: Preparation and Characterization of Cucumber Exosome Complex Loaded with Five Grains Insect Extract

[0078] 2.1 Extraction of cucumber exosomes

[0079] Cucumber exosomes were extracted using differential ultracentrifugation at 4°C throughout the process. Fresh cucumbers were washed, cut into pieces, and homogenized with pre-cooled PBS at a 1:1 mass ratio. The homogenate was then filtered through gauze to remove coarse residue. The filtrate was centrifuged sequentially at 300×g for 10 min, 2000×g for 20 min to remove cell debris, and then centrifuged at 10000×g for 30 min to remove large vesicles. The supernatant was then ultracentrifuged at 120000×g for 70 min to collect the precipitate. The precipitate was resuspended in PBS and washed and purified again by ultracentrifugation at 100000×g for 70 min. The final product was aliquoted and stored at -80°C.

[0080] 2.2 Preparation of cucumber exosome complex loaded with *Ceratophyllum demersum* extract

[0081] Purified cucumber exosomes were mixed with water-soluble and lipid-soluble extracts of *Ceratophyllum demersum* at a mass ratio of 1:3:3 in sterile PBS to achieve a final exosome concentration of 0.25 mg / mL. The mixture was incubated at 37°C and 120 rpm in the dark for 4 hours, followed by sonication in an ice bath for 5 minutes (125 kHz, 5 s on, 3 s off). After incubation, the free extract was removed by ultracentrifugation at 100,000 × g and 4°C for 70 minutes. The precipitate was resuspended and centrifuged and washed again to obtain the cucumber exosome complex loaded with *Ceratophyllum demersum* extract, which was then aliquoted and stored at -80°C.

[0082] 2.3 Characterization of cucumber exosome complex

[0083] The encapsulation efficiency, particle size, and zeta potential of the cucumber exosome complex loaded with *Ceratophyllum demersum* extract were detected using the same method as in Example 1. The results are shown in Table 2: Encapsulation efficiency and particle size distribution of the cucumber exosome complex loaded with *Ceratophyllum demersum* extract (mean ± SD, n = 3).

[0084] Table 2:

[0085] Cucumber exosomes — — 66.79±1.54 -40.21±1.30 Cucumber exosome complex containing five grain insects 71.39±1.81 66.28±3.10 78.13±3.77 -47.59±1.33

[0086] Table 2 shows that the encapsulation rate of cucumber exosomes for the water-soluble extract of *Corydalis esculenta* was 71.39±1.81%, and the encapsulation rate for the fat-soluble extract of *Corydalis esculenta* was 66.28±3.10%, indicating that cucumber exosomes have good loading capacity for both types of active ingredients. The particle size of the drug-loaded complex was 78.13±3.77 nm, and the Zeta potential was -47.59±1.33 mV. The absolute value of the Zeta potential was greater than 20 mV, indicating that the complex system was stable.

[0087] 2.4 Validation of Fluorescence Colocalization

[0088] The binding mechanism of the active ingredient of *Gnaphalium affine* to cucumber exosomes was verified by fluorescence colocalization assay. Cucumber exosomes were labeled with C-6 green fluorescent dye, co-incubated with the active ingredient of *Gnaphalium affine*, and then thoroughly washed by ultracentrifugation to remove free components. The results were observed under a fluorescence microscope.

[0089] The results showed that the fluorescence signal of the C-6 labeled cucumber exosomes (green fluorescence) and the active ingredient of the ginkgo worm were spatially overlapped, indicating that the active ingredient of the ginkgo worm and cucumber exosomes formed a stable complex.

[0090] Example 3: Study on the in vitro anti-lung cancer activity, cell retention and mechanism of action of *Pteris vittata* liposome preparation.

[0091] 3.1 In vitro anti-lung cancer activity assay (CCK8 assay)

[0092] Logarithmic growth phase PC9 cells (human non-small cell lung cancer cells) were collected, digested, and counted. Cells were seeded at a density of 4000 cells / well in 96-well plates, with 90 μL of fresh complete culture medium added to each well. The plates were incubated overnight at 37°C in a 5% CO2 cell culture incubator. After cell attachment, the following treatments were added (final concentration based on the amount of *Cynanchum paniculatum* extract: blank control group (equal volume of PBS), *Cynanchum paniculatum* compound decoction group (MS, prepared according to traditional decoction method), *Cynanchum paniculatum* liposome preparation group (ME, prepared in Example 1), and cucumber exosome complex group loaded with *Cynanchum paniculatum* extract (MC, prepared in Example 2). Each group was configured with 5 replicates. After culturing for 24 h, 10 μL of CCK-8 reagent was added to each well, and the plates were incubated at 37°C for 2 h. The absorbance at 450 nm was measured using a multi-mode microplate reader, and cell viability was calculated (data statistical methods are described in Example 6). Cell viability (%) = (absorbance of drug-treated group / absorbance of blank control group) × 100%.

[0093] The results are as follows Figure 2As shown, compared with the blank control group, the cell viability of each treatment group decreased. Among them, the inhibitory effect of the *Eriocaulon buergerianum* liposome preparation group (ME) and the cucumber exosome complex group (MC) on PC9 cells was significantly better than that of the *Eriocaulon buergerianum* compound decoction group (MS). The results indicate that both the *Eriocaulon buergerianum* liposome preparation and the cucumber exosome complex loaded with *Eriocaulon buergerianum* extract have good in vitro anti-lung cancer activity.

[0094] 3.2 Observation of cell retention effect

[0095] The retention effect of *Pteris vittata* liposome preparation in lung cancer PC9 cells was observed using fluorescent labeling technology. Liposomes were labeled with C-6 green fluorescent dye, and cell nuclei were labeled with DAPI (blue). PC9 cells were seeded in confocal culture dishes, and co-incubated with C-6-labeled *Pteris vittata* liposome preparation. At different time points (0h, 12h), cells were washed three times with PBS, fixed with 4% paraformaldehyde for 15 min, stained with DAPI for 10 min, and observed and photographed using a laser confocal microscope.

[0096] The results are as follows Figure 1 As shown, after entering PC9 cells (0h), the liposome preparation of *Eriocaulon buergerianum* remained largely within the cancer cells for 12 hours, indicating that the preparation has a good affinity for lung cancer cells and can prolong the retention time of the active ingredients in the cells, which is beneficial for sustained anti-tumor effects.

[0097] 3.3 Research on the mechanism of action against lung cancer

[0098] 3.3.1 ELISA method for detecting inflammatory factors

[0099] Cell culture supernatants from each group under section 3.1 were collected, and the levels of TNF-α and IL-6 were detected according to the ELISA kit instructions. The kits used were: Mouse TNF-α ELISA kit (catalog number: PT512, Beyotime) and Mouse IL-6 ELISA kit (catalog number: PT326, Beyotime). Each sample group was tested in triplicate, and the experiment was independently repeated three times. Data analysis methods are described in Example 6.

[0100] 3.3.2 Western blot detection of protein expression

[0101] Cells from each group were collected, and total protein was extracted using RIPA lysis buffer. Protein concentration was determined using the BCA method. Equal amounts of protein (30 μg / well) were subjected to SDS-PAGE electrophoresis. The separating gel concentration was determined based on the molecular weight of the target protein (8% separating gel for NF-κB p65, 10% for p53, and 12% for β-actin). After electrophoresis, the protein was transferred to a PVDF membrane (0.45 μm pore size) using a wet transfer method and blocked with 5% skim milk powder (prepared with 1×TTBS) at room temperature for 1 h. Primary antibodies were added to each membrane: Phospho-NF-κB p65 antibody (catalog number: AF5875, Beyotime, dilution 1:1000), p53 antibody (catalog number: 2524, Cell Signaling Technology, dilution 1:1000), and β-actin antibody (catalog number: 4970, Cell Signaling Technology, dilution 1:2000), and incubated overnight at 4°C on a shaker. Wash the membrane three times with 1×TTBS, 10 min each time. Add HRP-labeled secondary antibody (dilution ratio 1:5000) and incubate on a shaker at room temperature for 1 h. Wash the membrane three times with 1×TTBS, 10 min each time. Develop with ECL chemiluminescence staining solution, and perform grayscale analysis using ImageJ software. The relative expression level is expressed as the ratio of the grayscale value of the target protein to that of the internal control β-actin.

[0102] The regulatory effects of *Strombus haematocephala* on the secretion of inflammatory factors and the expression of related proteins in model mice were analyzed using ELISA and Western blot techniques. Figure 3 ).

[0103] ELISA test results ( Figure 3 As shown in A and 3B, compared with the control group (Ctl), the serum levels of the inflammatory factor TNF-α in mice in the *Eriocaulon buergerianum* liposome preparation group (ME) and the cucumber exosome complex group (MC) were significantly decreased (p < 0.01), indicating that the *Eriocaulon buergerianum* preparation has anti-inflammatory activity; while the IL-6 level did not differ significantly among the groups (p > 0.05), indicating that the *Eriocaulon buergerianum* preparation has selective regulation of inflammatory factors.

[0104] Western blot results ( Figure 3 C) showed that the expression levels of Phospho-NF-κB p65 protein in the *Phospho* liposome preparation group and the cucumber exosome complex group were significantly downregulated and the expression level of p53 protein was significantly upregulated compared with the control group. These results indicate that the *Phospho* preparation exerts anti-inflammatory and anti-tumor effects by inhibiting TNF-α release, downregulating NF-κB signaling pathway activation, and upregulating p53 protein expression.

[0105] Example 4: Evaluation of the in vivo anti-lung cancer activity of the *Pteris vittata* liposome preparation.

[0106] 4.1 Establishment of a tumor-bearing nude mouse model

[0107] Five-week-old female SPF-grade BALB / c nu nude mice were used for experiments after one week of acclimatization. PC9 cells in the logarithmic growth phase were collected, digested, resuspended in sterile PBS, and the cell concentration was adjusted to 5 × 10⁻⁶. 7 After anesthetizing the mice with a respiratory anesthesia machine, 100 μL of PC9 cell suspension (containing 5 × 10⁶ cells / mL) was subcutaneously injected into the right axilla. 6 (cells). Tumor formation was observed every 3 days after inoculation, and the longest diameter (a) and shortest diameter (b) of the tumor were measured with calipers. The tumor volume was increased to approximately 100 mm². 3 Start administering medication at that time.

[0108] 4.2 Animal grouping and administration

[0109] Tumor-bearing nude mice were randomly divided into four groups of five mice each, and the experiment was repeated three times independently: a blank control group (administered with an equal volume of physiological saline), a *Wuguchong* compound decoction group (MS), a *Wuguchong* liposome preparation group (ME, prepared in Example 1), and a cucumber exosome complex group loaded with *Wuguchong* extract (MC, prepared in Example 2). All three *Wuguchong* treatment groups started with 3g of *Wuguchong* and were administered the corresponding preparations according to their respective methods. The dosage for each group was 150mg / kg / day based on the raw *Wuguchong* amount, administered via tail vein injection once daily for 14 consecutive days.

[0110] 4.3 Tumor volume measurement

[0111] Starting from day 1 of drug administration, the longest diameter (a) and shortest diameter (b) of the tumor were measured every 3 days using precision vernier calipers. The tumor volume was calculated using the following formula: V = a × b² × π / 6. The day after the last drug administration, the mice were sacrificed, the tumor tissue was dissected, weighed, and the tumor inhibition rate was calculated: Tumor inhibition rate (%) = (1 - average tumor weight of the drug administration group / average tumor weight of the control group) × 100%.

[0112] The results are as follows Figure 4 As shown in A and 4B, the tumor volume in each of the five-grain insect treatment groups was significantly smaller than that in the blank control group (p < 0.05 or p < 0.01). Among them, the tumor volume in the five-grain insect liposome preparation group (ME) and the cucumber exosome complex group (MC) was significantly smaller than that in the five-grain insect compound decoction group (MS) (p < 0.05), indicating that the five-grain insect liposome preparation and the cucumber exosome complex have better in vivo anti-lung cancer effects than traditional decoctions. The data statistics method is shown in Example 6.

[0113] 4.4 Peripheral blood leukocyte count

[0114] The day after the last administration, blood was collected from the orbital cavity of mice, blood smears were prepared, and white blood cells were counted under an optical microscope after Wright staining.

[0115] The results are as follows Figure 4 As shown in Figure C, the peripheral blood leukocyte counts in each of the five-grain insect treatment groups were significantly higher than those in the blank control group (p < 0.05), indicating that the five-grain insect preparation can improve the leukocyte level in tumor-bearing nude mice and has a positive regulatory effect on hematopoietic function under tumor-bearing conditions.

[0116] 4.5 Spleen Index Measurement

[0117] The day after the last administration, the mice were sacrificed, and the spleens were weighed and the spleen index was calculated: spleen index = spleen weight (mg) / body weight (g).

[0118] The results are as follows Figure 4 As shown in Figure D, the spleen index of the cucumber exosome complex (MC) group was significantly higher than that of the blank control group and the other two grain worm treatment groups (p < 0.05), indicating that this component can improve the relative spleen quality of tumor-bearing nude mice and has a protective effect on immune organs.

[0119] 4.6 Histopathological examination

[0120] Tumor and spleen tissues from each group were collected, fixed in 4% paraformaldehyde for at least 72 hours, dehydrated with a gradient of ethanol (75%, 85%, 95%, 100%), cleared with xylene, and embedded in liquid paraffin at 60°C. Thin sections of 4 μm thickness were cut using a microtome and baked at 60°C for 2 hours. Tumor tissue sections were stained with H&E, and spleen tissue sections were stained with Gomori silver. Observation and photography were performed under a biological microscope.

[0121] The results are as follows Figure 4 As shown in Figure E, H&E staining revealed that, compared with the blank control group, the tumor tissues of each *Eriocheir sinensis* treatment group showed inhibitory changes in tumor cell proliferation-related structural features, with varying degrees of tumor cell necrosis and apoptosis observed. Gomori silver staining results ( Figure 4 F) shows that the reticular fiber structure of the spleen in each of the five-grain worm treatment groups was clearer and more complete than that in the blank control group, suggesting that the five-grain worm preparation can maintain the structural integrity of the spleen under tumor-bearing conditions and has a protective effect on the function of immune organs.

[0122] Example 5: Screening of preparation process parameters

[0123] 5.1 Screening of water extraction feed-liquid ratio

[0124] With other extraction conditions kept constant (pH 6.0, ultrasonic treatment for 40 min, 25 Hz), the effect of solid-liquid ratios of *Polygonum multiflorum* powder to water of 1:20, 1:30, 1:50, 1:80, and 1:100 on the yield of water-soluble extract was investigated. The results showed that the extraction efficiency was higher within the solid-liquid ratio range of 1:30–1:80, with the optimal extraction effect (highest yield) at a solid-liquid ratio of 1:50. When the solid-liquid ratio was below 1:30, insufficient extraction solvent led to incomplete extraction; when the solid-liquid ratio was above 1:80, the extraction rate did not improve significantly and increased the burden on subsequent concentration.

[0125] 5.2 Screening of pH values ​​for water extraction

[0126] With other extraction conditions fixed (solid-to-liquid ratio 1:50, ultrasonic treatment for 40 min, 25 Hz), the effects of extraction pH at 4.0, 5.0, 5.5, 6.0, 6.5, 7.0, and 8.0 on the yield and activity of the water-soluble extract were investigated. The results showed that the extract yield and activity were higher within the pH range of 5.5–6.5, with pH 6.0 being the optimal value. Below pH 5.5, peptides may undergo acid degradation; above pH 6.5, peptide solubility decreases, and the extraction rate decreases.

[0127] 5.3 Screening based on the phospholipid to cholesterol ratio

[0128] With other liposome preparation conditions fixed (phospholipid to lipophilic extract mass ratio 5:1, hydration temperature 45℃, ultrasonic frequency 130kHz, ultrasonic time 10min), the effects of phospholipid to cholesterol mass ratios of 3:1, 5:1, 8:1, 10:1, 12:1, 15:1, and 20:1 on the particle size, encapsulation efficiency, and stability of the liposome formulation were investigated. The results showed that within the phospholipid to cholesterol mass ratio range of 5:1 to 15:1, the liposome formulation exhibited superior particle size (150–200 nm) and encapsulation efficiency, with the best overall performance observed at a mass ratio of 10:1. When the cholesterol ratio was too low (>15:1), excessive liposome membrane fluidity led to drug leakage and a decrease in encapsulation efficiency; when the cholesterol ratio was too high (<5:1), the liposome membrane rigidity increased, resulting in larger particle size and a lower encapsulation efficiency.

[0129] Example 6 Statistical Analysis

[0130] All experimental data are expressed as mean ± SD. Statistical analysis was performed using GraphPad Prism 9.0 software. One-way ANOVA was used for comparisons among multiple groups, and Tukey's post-hoc test was used for pairwise comparisons between groups. The statistical significance level was set as follows: p < 0.05 was considered statistically significant, p < 0.01 was considered highly significant, and p < 0.001 was considered highly significant.

[0131] The raw material used in this invention, *Pteris vittata*, is widely available and inexpensive, allowing for large-scale farming or procurement. The liposome preparation process employs a combination of thin-film dispersion and ultrasonication, which is simple to operate, highly reproducible, and suitable for scale-up production. All excipients used are pharmaceutical-grade and meet the requirements of Good Manufacturing Practices (GMP). The *Pteris vittata* liposome preparation provided by this invention can be prepared into injection solutions or lyophilized powder injections using conventional pharmaceutical processes. It shows clear application prospects in the preparation of anti-lung cancer drugs, drugs to improve peripheral blood leukopenia, and drugs to protect the function of the spleen and other immune organs, and is suitable for industrial-scale mass production.

[0132] The above are merely specific embodiments of the present invention, but the technical features of the present invention are not limited thereto. Any simple changes, equivalent substitutions, or modifications made based on the present invention to solve essentially the same technical problems and achieve essentially the same technical effects are all covered within the protection scope of the present invention.

Claims

1. A liposome preparation of *Pteris vittata*, characterized in that, The five-grain insect liposome preparation contains the active ingredient of five-grain insect and liposomes; The active ingredients of the five-grain insect include water-soluble extract and fat-soluble extract of the five-grain insect; The liposomes are formed from phospholipids and cholesterol.

2. The five-grain insect liposome preparation according to claim 1, characterized in that: The water-soluble extract of the five-grain insect contains five-grain insect polypeptide, and the fat-soluble extract of the five-grain insect contains five-grain insect oil.

3. The five-grain insect liposome preparation according to claim 1, characterized in that: The particle size of the liposome preparation of the five grain insect is 150-200 nm, and the absolute value of the zeta potential is >20 mV.

4. The five-grain insect liposome preparation according to claim 1, characterized in that: The water-soluble extract of *Polygonum multiflorum* was encapsulated in liposomes at a rate of 70%–85%, and the fat-soluble extract of *Polygonum multiflorum* was encapsulated in liposomes at a rate of 80%–95%.

5. A method for preparing a liposome preparation of *Pteris vittata* as described in any one of claims 1 to 4, characterized in that, Includes the following steps: (1) Preparation of water-soluble extract of five grain insects: Take five grain insect powder, add solvent to extract, centrifuge to collect the supernatant, dry to obtain water-soluble extract of five grain insects; (2) Preparation of fat-soluble extract of five grain insects: Take five grain insect powder, extract with organic solvent, remove solvent and dry to obtain fat-soluble extract of five grain insects; (3) Liposome encapsulation: Phospholipids, cholesterol and the fat-soluble extract of the five grain insects obtained in step (2) are dissolved in an organic solvent, and the solvent is removed to form a film; The water-soluble extract of *Ceratophyllum demersum* obtained in step (1) is dissolved in an aqueous phase, added to the film for hydration, and then subjected to ultrasonic treatment to obtain the *Ceratophyllum demersum* liposome preparation.

6. The method for preparing a liposome preparation of *Pteris vittata* according to claim 5, characterized in that: The solvent mentioned in step (1) is water, and the extraction conditions are: the ratio of grain insect powder to water is 1:10 to 1:80, pH is 5.5 to 6.5, and ultrasonic treatment is performed for 20 to 60 minutes.

7. The method for preparing a liposome preparation of *Pteris vittata* according to claim 5, characterized in that: The organic solvent mentioned in step (2) is ethyl acetate. The extraction is carried out by Soxhlet extraction. The ratio of the grain insect powder to ethyl acetate is 1:3 to 1:10, and the extraction time is 1 to 4 hours.

8. The method for preparing a liposome preparation of *Pteris vittata* according to claim 5, characterized in that: In step (3), the mass ratio of phospholipid to cholesterol is 5:1 to 35:1, the mass ratio of phospholipid to fat-soluble extract of *Polygonum multiflorum* is 3:1 to 10:1, and the mass ratio of water-soluble extract of *Polygonum multiflorum* to fat-soluble extract of *Polygonum multiflorum* is 0.5:1 to 3:

1.

9. The method for preparing a liposome preparation of *Pteris vittata* according to claim 5, characterized in that: The frequency of the ultrasonic treatment in step (3) is 100-150 kHz and the time is 5-20 min.

10. The method for preparing a liposome preparation of *Pteris vittata* according to claim 5, characterized in that: In step (3), solvent removal to form a thin film is carried out by rotary evaporation at a temperature of 40-50°C under negative pressure conditions; the hydration temperature is 40-50°C.

11. The use of a liposome preparation of *Pteris vittata* as described in any one of claims 1 to 4 in the preparation of a medicament for treating lung cancer.

12. The application according to claim 11, characterized in that: The lung cancer in question is non-small cell lung cancer.

13. The use of a liposome formulation of *Gnaphalium affine* as described in any one of claims 1 to 4 in the preparation of a medicament for improving peripheral blood leukopenia and / or protecting the function of the spleen as an immune organ.

14. A plant exosome complex loaded with extract of *Pteris vittata*, characterized in that, The plant exosome complex contains the active ingredient of *Pteris vittata* and plant exosomes; The active ingredients of the five-grain insect include water-soluble extract and fat-soluble extract of the five-grain insect; The plant exosomes are derived from the fruits of Cucurbitaceae plants.

15. A plant exosome complex loaded with *Pteris vittata* extract according to claim 14, characterized in that: The plant exosomes mentioned are cucumber exosomes.

16. The plant exosome complex loaded with *Pteris vittata* extract according to claim 14, characterized in that: The particle size of the plant exosome complex is 60–100 nm, and the absolute value of the Zeta potential is >20 mV.

17. A method for preparing a plant exosome complex loaded with *Pteris vittata* extract as described in any one of claims 14 to 16, characterized in that, Includes the following steps: (1) Extraction of plant exosomes: Take plant fruits, homogenize them, and then centrifuge them by differential centrifugation and ultracentrifugation to collect the precipitate and obtain plant exosomes; (2) Preparation of active ingredients of five grain insects: Take five grain insect powder and extract five grain insect water-soluble extract and five grain insect fat-soluble extract; (3) Co-incubation loading: The plant exosomes obtained in step (1) are co-incubated with the active ingredient of the five grain insect obtained in step (2), and the free components are removed by ultracentrifugation and washing to obtain the plant exosome complex loaded with the five grain insect extract.

18. The method for preparing the plant exosome complex loaded with *Pteris vittata* extract according to claim 17, characterized in that: The differential centrifugation in step (1) includes centrifugation at 200-500×g for 5-20 min, 1000-3000×g for 10-30 min, and 8000-12000×g for 20-40 min in sequence; the ultracentrifugation is centrifugation at 100000-150000×g for 50-90 min.

19. The method for preparing the plant exosome complex loaded with *Pteris vittata* extract according to claim 17, characterized in that: In step (3), the mass ratio of the plant exosomes to the active ingredient of the grain insect is 1:2 to 1:5, and the co-incubation temperature is 25 to 40°C for 2 to 6 hours.

20. Use of a plant exosome complex loaded with *Pteris vittata* extract as described in any one of claims 14 to 16 in the preparation of a medicament for treating lung cancer.

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