A ginsenoside-lipoprotein complex biosynthesized by hens and its separation and purification method and medical application

Abstract

The present application relates to the field of biological medicine, and especially to a ginsenoside lipoprotein complex biosynthesized by egg-laying hens, a separation and purification method thereof and medical application, wherein the ginsenoside lipoprotein complex is obtained by separating and purifying the ginsenoside naturally combined with egg yolk lipoprotein and deposited in the egg yolk after the egg-laying hens ingest functional feed containing ginsenoside, absorb the ginsenoside through the intestinal tract, metabolize the ginsenoside through the liver, transport the ginsenoside through blood lipoprotein and selectively enrich the ginsenoside in the ovary, and the average particle size of the ginsenoside lipoprotein complex is 20-80 nm, the ginsenoside is contained in the lipid core of the lipoprotein and exposed on the surface of the phospholipid shell through the sugar chain, and the problems of low bioavailability, poor targeting and safety hazards of artificial carriers of free ginsenoside are solved.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and in particular to a ginsenoside lipoprotein complex synthesized from laying hens, its separation and purification method, and its pharmaceutical applications. Background Technology

[0002] Ginseng is a traditional and precious Chinese medicinal material that is both food and medicine. Ginsenosides are the core active substances of ginseng plants. They belong to the dammarane-type tetracyclic triterpenoid saponin compounds and mainly include monomers such as Rb1, Rc, Rd, Re, Rg1, Rg3, and Rh2, as well as protopanaxadiol (PPD) and protopanatriol (PPT) and other in vivo metabolites. Modern pharmacological and clinical studies have confirmed that ginsenosides possess significant anti-tumor, anti-inflammatory, antioxidant, immunomodulatory, tumor cell proliferation inhibition, apoptosis induction, cell cycle arrest, tumor angiogenesis inhibition, and tumor multidrug resistance reversal activities. Among them, rare ginsenosides Rg3 and Rh2 and their metabolite PPD have strong inhibitory effects on various solid tumors such as synovial sarcoma, osteosarcoma, breast cancer, lung cancer, liver cancer, gastric cancer, colorectal cancer, melanoma, and neuroblastoma. They have extremely high research and development value and clinical application prospects in the fields of tumor chemoprevention, targeted therapy, chemotherapy sensitization, combined immunotherapy, and postoperative recurrence and metastasis prevention.

[0003] However, the inherent physicochemical defects and in vivo pharmacokinetic limitations of free ginsenosides severely restrict their formulation development and clinical translation. Existing technologies generally face several bottlenecks. First, their oral bioavailability is extremely low. Ginsenosides have large molecular weights and rigid molecular structures, possessing both hydrophobic tetracyclic triterpenoid cores and hydrophilic oligosaccharide side chains, resulting in poor solubility in both aqueous and lipid phases. After oral administration, they are easily destroyed by the strong acid in gastric juice and hydrolyzed by intestinal β-glucosidase, thus losing their antitumor activity. Their ability to penetrate across intestinal epithelial cell membranes is weak, with an overall gastrointestinal absorption rate of less than 1%. Simultaneously, there is a significant first-pass effect in the liver, meaning that the vast majority of ingested components cannot enter systemic circulation and are excreted only as unchanged or inactive metabolites. Conventional administration methods result in weak efficacy and significant waste. Second, they lack active targeting in vivo, require large therapeutic doses, and have significant toxic side effects. Free ginsenosides, once inside the body, exhibit a non-specific, diffuse distribution throughout the body, lacking the ability to actively target and accumulate in tumor tissues, making it difficult to achieve effective therapeutic concentrations locally. To achieve tumor-suppressing effects, significantly increased dosages are required; however, long-term high-dose use can easily increase the metabolic burden on the liver and kidneys, trigger gastrointestinal reactions, disrupt the body's normal immune homeostasis, and result in poor patient tolerance, hindering long-term maintenance therapy. Thirdly, artificial chemical nanocarriers have safety and technological limitations. To improve the solubility, absorption, and retention of ginsenosides, current research primarily employs in vitro encapsulation and delivery systems such as chemically synthesized liposomes, polymer nanomicelles, synthetic polymer nanoparticles, and inorganic nanocarriers. However, such artificial carriers generally have drawbacks: the synthetic polymers and excipients have potential risks of cytotoxicity and in vivo accumulation, and their biocompatibility and complete degradability are insufficient; the preparation process requires the use of organic solvents, chemical cross-linking agents, and surface modifiers, which can easily lead to solvent residues and increased immunogenicity; the preparation conditions are harsh, the process repeatability is poor, and the particle size distribution, encapsulation efficiency, and drug loading fluctuate greatly between batches, making it difficult to scale up industrially; moreover, most of them are passive enrichment or simple sustained release, which cannot achieve precise targeted endocytosis mediated by tumor cell specific receptors, resulting in limited targeting efficiency.

[0004] Egg yolks are naturally enriched with low-density lipoprotein (LDL), high-density lipoprotein (HDL), and very low-density lipoprotein (VLDL). These are naturally occurring spherical lipoprotein nanoparticles with a natural particle size concentrated in the 17–60 nm range. They possess excellent biocompatibility, complete biodegradability, long-term blood circulation characteristics, and a natural lipid-carrying cavity structure. Numerous studies have shown that various tumor cells, to meet the demands of abnormal proliferation and rapid glucose metabolism, abnormally overexpress LDL receptors and glucose transporters GLUT1 / GLUT3 on their surface, with expression levels significantly higher than normal somatic cells by 3–10 times. These receptors can specifically recognize the apolipoprotein structure of the natural lipoprotein shell and the exposed sugar groups of ginsenosides, providing an ideal endogenous carrier for constructing a natural nanomedicine delivery system with no chemical carrier toxicity and receptor-mediated dual targeting.

[0005] CN108783058A discloses a poultry feed, which is prepared from the following raw materials: ginsenosides, *Ganoderma lucidum*, *Ganoderma lucidum* spore powder, chia seeds, soybean meal, corn, carotene, *Eleutherococcus senticosus*, *Atractylodes macrocephala*, licorice, anthocyanins, and shrimp meal. Experiments show that when this feed is used for raising chickens, the eggs produced by these chickens have a DHA content greater than 0.09 g / 100g, a lecithin content greater than 7.0 g / 100g, a selenium content greater than 70 μg / 100g, a content of 18 amino acids greater than 16 g / 100g, and a cholesterol content lower than 180 mg / 100g. Compared with ordinary eggs, these eggs have better quality. However, this existing technology remains limited to livestock and poultry farming and functional food development, focusing only on improving the conventional nutritional components of eggs. The research emphasis is solely on enhancing and improving the quality of conventional egg nutrients, without delving into in-depth refining and specialized separation of the effective components of the egg yolk. It merely increases the overall content of ginsenosides in eggs without systematically characterizing and analyzing the complex forms, particulate colloidal properties, and binding mechanisms of active substances in the egg yolk. Furthermore, it relies solely on the passive enrichment of the raw materials' components in the egg, without utilizing physiological metabolic pathways such as the digestive tract microecology and liver metabolic enzymes in poultry to structurally modify and target the active components of ginseng. The research focuses solely on routine testing of product nutritional components, without conducting systematic verification studies on mechanisms of action, targeted distribution, tissue accumulation, and pharmacological efficacy at the cellular and animal pharmacological levels. It uses only edible eggs as the end product, failing to systematically explore the differences in efficacy, dose-response, and safe application window of active components in different forms. It lacks a dedicated separation and purification process for active complex components in the complex matrix of egg yolk, and has not established a supporting quality control system for qualitative and quantitative analysis of components, microstructural characterization, and bioactivity evaluation. Furthermore, it has not developed pharmaceutical formulations for active products, limiting its application to food use and neglecting in-depth research in pharmaceutical fields such as anti-tumor therapy, chemotherapy adjuvant therapy, and immune regulation.

[0006] In addition, other existing technologies for feeding laying hens with similar ginseng feeds are limited to increasing the content of ginsenosides in eggs and developing nutritious and health-promoting eggs. They generally neglect the physiological metabolic transformation potential of laying hens and the application value of egg yolk endogenous lipoproteins as natural carriers, and have not carried out in-depth development of active compound components and research on their medicinal uses.

[0007] In summary, free ginsenosides suffer from drawbacks such as poor absorption, low utilization, lack of targeting, high dosage, and significant toxicity; artificial nanocarriers present safety risks, poor process stability, and insufficient targeting precision; and existing technologies for feeding ginseng to laying hens to produce functional eggs only reach the food level, failing to explore the medicinal development potential and deep pharmacological value of the active complex components in the egg yolk. Therefore, developing a ginsenoside-related complex product that is synthesized based on the physiological metabolism of laying hens, can be purified, has good biocompatibility, and exhibits excellent pharmacological activity, establishing a complete preparation process, and expanding its pharmaceutical applications in anti-tumor, chemotherapy sensitization, and immunomodulation has significant theoretical, clinical, and industrialization potential. Summary of the Invention

[0008] To address the shortcomings of the existing technologies, this invention provides a ginsenoside lipoprotein complex synthesized from laying hens, along with its separation and purification method and pharmaceutical applications, solving the problems of low bioavailability, poor targeting, and safety hazards associated with artificial carriers of free ginsenosides. Another objective of this invention is to provide a standardized separation and purification method for this complex, obtaining high-purity, high-activity pharmaceutical raw materials. A further objective of this invention is to clarify the pharmaceutical applications of this complex in anti-tumor drugs, chemotherapy sensitizers, and immunomodulatory agents, achieving low-dose, highly targeted, and highly safe applications in tumor prevention and treatment.

[0009] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is a ginsenoside lipoprotein complex biosynthesized by laying hens. After laying hens ingest functional feed containing ginsenosides, the ginsenosides are absorbed in the intestines, metabolized in the liver, transported in the blood lipoproteins, and selectively enriched in the ovaries, so that the ginsenosides naturally bind with the yolk lipoproteins and are deposited in the yolk. After separation and purification, the ginsenosides are obtained with an average particle size of 20–80 nm. The ginsenosides are encapsulated in the lipoprotein lipid core and exposed on the phospholipid shell surface through the sugar chain.

[0010] The aforementioned ginsenoside lipoprotein complex synthesized from laying hens has a density of 0.95–1.063 g / mL and a surface zeta potential of -15 mV to +10 mV; the ginsenosides are selected from at least one of ginsenosides Rh2, Rg3, Rh1, Rb1, Rd, Re, protopanaxadiol, protopanatriol, and metabolites generated in laying hens through deglycosylation, oxidation, and isomerization.

[0011] The ginsenoside lipoprotein complex synthesized from laying hens described above is a low-density lipoprotein, high-density lipoprotein, or very low-density lipoprotein from egg yolk.

[0012] The aforementioned ginsenoside lipoprotein complex synthesized using laying hens contains 5–50 μg / mg ginsenosides in lipoprotein. The ginsenosides are embedded in the lipoprotein lipid core through a hydrophobic dammarane-type tetracyclic triterpenoid core, with their sugar chains partially exposed on the phospholipid monolayer on the lipoprotein surface, forming a natural amphiphilic structure of "core encapsulation-shell recognition".

[0013] The above-mentioned method for separating and purifying ginsenoside lipoprotein complexes synthesized by laying hens includes the following steps: (a) Feed formulation: Ginseng, ginseng extract or ginseng processing by-products are added to the basic feed of laying hens in a specific ratio to form a functional feed containing ginsenosides. (b) Biotransformation: The functional feed described above is used to feed laying hens for a period of not less than 60 days, so that ginsenosides are selectively deposited in the egg yolk and naturally combined with egg yolk lipoproteins after being absorbed by the hen's intestines, metabolized by the liver, and transported by blood lipoproteins. (c) Egg yolk collection: Collect eggs laid by hens after feeding and separate the yolks; (d) Separation and purification: After homogenizing the egg yolk, extract and purify the ginsenoside lipoprotein complex by at least one of the following methods: differential centrifugation, density gradient centrifugation, organic solvent extraction, ultrafiltration, dialysis or chromatographic separation. (e) Formulation: The purified complex is formulated into lyophilized powder, injection, oral formulation or sustained-release formulation.

[0014] The above-mentioned method for separating and purifying ginsenoside lipoprotein complexes synthesized by laying hens, wherein the ginseng extract in step (a) is an aqueous extract, alcoholic extract, or ultrafine powder of ginseng root, ginseng root, ginseng root, ginseng root, ginseng root fibrous material; and the amount of ginseng extract added to the functional feed is 0.5%–5% (w / w).

[0015] In the above-mentioned method for separating and purifying ginsenoside lipoprotein complexes synthesized by laying hens, the laying hens in step (b) are Hy-Line Brown, Lohmann Brown, Jinghong, or Black Phoenix chickens; the feeding period is 60–180 days, preferably 90–120 days.

[0016] The above-mentioned method for separating and purifying ginsenoside lipoprotein complexes synthesized by laying hens, specifically includes the following steps in step (d): (i) Mix egg yolks with an equal volume of phosphate-buffered saline (PBS, pH 7.4) to form a homogenate; (ii) Centrifuge at low speed (3,000–6,000 g, 15–30 min) to remove precipitate and collect supernatant; (iii) Lipoprotein bands were separated by ultracentrifugation (100,000–200,000 g, 16–48 h, 4°C) or density gradient centrifugation (density range 0.95–1.063 g / mL); (iv) Collect lipoprotein components containing ginsenosides and remove small molecule impurities by Sephadex G-25 or G-75 gel filtration; (v) Ginsenoside lipoprotein complex with a purity of ≥85% was obtained by ultrafiltration (molecular weight cutoff 30–100 kDa).

[0017] The above-mentioned method for separating and purifying ginsenoside lipoprotein complexes synthesized by laying hens further includes step (d) using high performance liquid chromatography (HPLC) or liquid chromatography-mass spectrometry (LC-MS) to identify and quantify the types and contents of ginsenosides in the complex; the ginsenoside content in the complex is determined by in vitro cell uptake experiments and animal pharmacodynamic experiments to assess its bioactivity.

[0018] The aforementioned pharmaceutical applications of ginsenoside lipoprotein complexes synthesized from laying hens are used to prepare drugs for the prevention and / or treatment of tumors, chemotherapy sensitizers, and tumor immunomodulatory agents; the tumors include synovial sarcoma, osteosarcoma, breast cancer, lung cancer, liver cancer, gastric cancer, colorectal cancer, melanoma, and neuroblastoma; the drug dosage forms are oral preparations, injections, lyophilized powder injections, enteric-coated capsules, transdermal patches, or sustained-release microsphere preparations.

[0019] The beneficial effects of this invention, which utilizes ginsenoside lipoprotein complexes synthesized from laying hens and its separation and purification method, on pharmaceutical applications are that, by using laying hens as natural bioreactors, ginsenosides and egg yolk lipoproteins are naturally self-assembled through in vivo physiological metabolism. The entire process requires no chemical cross-linking agents, organic solvents, or artificially synthesized carriers, leaving no chemical residues or additional immunogenicity. It exhibits high biocompatibility, good safety, and food-grade safety properties.

[0020] • By utilizing the gut microbiota and liver metabolic enzymes of laying hens, ordinary ginsenosides can be directionally converted into highly active rare saponins and aglycone products, thereby improving the enrichment efficiency of active components and pharmacological efficacy.

[0021] A complete purification process adapted to the complex matrix of egg yolk was established, which combines differential centrifugation, density gradient centrifugation, gel filtration and ultrafiltration. This process can stably obtain high-purity, uniform-size and structurally intact ginsenoside lipoprotein complexes. The process is highly reproducible and suitable for large-scale industrial production.

[0022] The resulting complex possesses a natural amphiphilic nanostructure, which can achieve targeted enrichment of drugs in tumors by relying on the body's lipoprotein receptor and glucose transporter pathways, effectively improving the drug accumulation level in tumor tissues and reducing interference with normal tissues.

[0023] The complex can be developed into various pharmaceutical dosage forms such as lyophilized powder, injection, oral preparation, and enteric-coated sustained-release preparation. It offers flexible administration methods, suitable for both clinical intravenous administration and oral home maintenance therapy, resulting in high patient compliance.

[0024] • Ginseng rootlets, ginseng stems and leaves, ginseng residue and other ginseng processing by-products can be used as feed raw materials to realize the high-value utilization of waste resources. The raw material sources are wide-ranging, the costs are controllable, and the industrialization has significant economic benefits.

[0025] This study expands the pharmaceutical applications of ginsenoside lipoprotein complexes in solid tumor treatment, chemotherapy sensitization, reversal of multidrug resistance in tumors, and regulation of the tumor immune microenvironment, providing a novel technical pathway for the development of tumor-targeted natural nanomedicines. Attached Figure Description

[0026] Figure 1 : A schematic diagram of the entire process of biosynthesis of ginsenoside lipoprotein complex in laying hens; Figure 2 Dynamic light scattering (DLS) particle size distribution of natural ginsenoside lipoprotein complex; showing spherical particle morphology and uniform particle size distribution of 30–50 nm.

[0027] Figure 3 Comparison of in vitro cellular uptake of natural ginsenosides and free ginsenosides (laser confocal microscopy). The natural ginsenosides show significantly higher fluorescence intensity in tumor cells than the free drug. Green fluorescent ginsenoside markers | The left two images show tumor cells (highly expressing LDL receptor and GLUT1), and the right image shows normal cells.

[0028] Figure 4 Schematic diagram of the dual receptor-mediated mechanism of natural complex entry into cells. The LDL receptor-mediated endocytosis pathway and the GLUT1-mediated endocytosis pathway are labeled respectively.

[0029] Figure 5 Comparison of tumor inhibition curves between the natural complex and free ginsenosides in animal experiments. The results show that the natural complex achieves the same tumor-inhibiting effect at extremely low doses (1 / 100,000).

[0030] Figure 6 The sensitizing effect of combining natural complexes with chemotherapy drugs is shown in the graph. It indicates that the tumor cell apoptosis rate in the combination therapy group was significantly higher than that in the single-drug group. Detailed Implementation

[0031] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments. Example 1

[0032] Biosynthesis and purification of natural ginsenoside lipoprotein complex This embodiment corresponds to Figure 1 A complete process for the biosynthesis of ginsenoside lipoprotein complex in laying hens. Experimental materials: 5-year-old ginseng rootlets from Changbai Mountain, 30-week-old Hy-Line Brown laying hens at their peak egg production, pH 7.4 phosphate-buffered saline (PBS), KBr, and Sephadex G-75 gel packing material.

[0033] Step 1: Feed Preparation After drying, the ginseng rootlets from Changbai Mountain are ultra-finely pulverized to 200 mesh and added to the basic feed at a ratio of 2% (w / w). The basic feed composition is as follows: corn 62%, soybean meal 25%, wheat bran 4%, calcium carbonate 8%, and compound vitamins 1%. At the same time, yeast powder 0.3% and lactic acid bacteria fermentation powder 0.2% are added and mixed to obtain functional feed, which promotes the biotransformation and absorption of ginsenosides in the intestines of laying hens.

[0034] Step Two: Bioconversion Feeding and Yolk Collection Hy-Line Brown laying hens at their peak egg production age of 30 weeks were selected and fed the above-prepared functional feed continuously for 120 days. During the feeding period, the hens had free access to feed and water, and the daily light exposure was controlled at 16 hours. Eggs laid from day 90 to 120 of feeding were collected, the yolks were separated manually, and frozen at -20℃ for later use.

[0035] Step 3: Determination of Ginsenoside Content in Egg Yolk Egg yolks from 10 randomly selected test eggs were analyzed using HPLC-UV to quantitatively determine the composition and content of ginsenosides in the yolks. The results showed that the average ginsenoside content in each egg yolk was approximately 20 μg, with ginsenosides Rh2, Rg3, and protopanaxadiol PPD metabolites accounting for 78% of the total ginsenoside content.

[0036] Step 4: Separation and Purification Process (1) Egg yolk homogenate: Accurately weigh 100g of egg yolk, add an equal volume of pH 7.4 phosphate buffer, and homogenize for 5min under ice bath conditions; Low-speed centrifugation to remove impurities: Place the homogenate in a centrifuge and centrifuge at 4000g and 4℃ for 20min to remove bottom precipitates and collect the supernatant of the upper emulsion. Density gradient ultracentrifugation: The obtained supernatant was mixed with KBr solution, and the system density was adjusted to 1.30 g / mL. The mixture was then placed at the bottom of a centrifuge tube, and the top layer was slowly covered with PBS buffer with a density of 1.006 g / mL. The mixture was then ultracentrifuged at 40,000 rpm and 4°C for 48 h. The milky yellow liquid layer with a density range of 1.006–1.063 g / mL was collected, which is the ginsenoside-enriched yolk lipoprotein component. Gel filtration purification: The collected lipoprotein fraction was loaded onto a Sephadex G-75 gel chromatography column (2.6cm×60cm), eluted with PBS buffer, and the elution flow rate was controlled at 1mL / min. The first high molecular weight elution peak was collected. Ultrafiltration concentration: Using ultrafiltration centrifuge tubes with a molecular weight cutoff of 100kDa, the concentration was achieved by centrifugation at 4000g to obtain the purified ginsenoside lipoprotein complex final product.

[0037] Product physicochemical characterization results The final product was systematically characterized using DLS, TEM, Zeta potential, SDS-PAGE, and content detection. Dynamic light scattering (DLS) detection: The average particle size of the complex was 38.5 ± 4.2 nm, and the polydispersity index (PDI) was 0.09. Zeta potential detection: The surface potential of the complex is -8.3±1.5mV; Ginsenoside loading: The ginsenoside content in the complex is 28 μg / mg lipoprotein; Transmission electron microscopy (TEM) observation: The complex consists of regular spherical lipoprotein particles with uniform particle size and no obvious aggregation; SDS-PAGE electrophoresis analysis: The product showed intact characteristic bands of natural apolipoproteins, consistent with the bands of natural LDL protein in egg yolk, and the lipoprotein structure was not denatured or destroyed. Example 2

[0038] This embodiment corresponds to the feed formulation optimization experiment of the present invention. Based on the feeding and testing process in Example 1, it examines the effects of different ginseng root addition ratios on the enrichment of ginsenosides in egg yolks and the egg production performance of laying hens.

[0039] The breed of laying hens, rearing environment, feeding management methods, rearing cycle, and egg yolk testing methods were kept completely consistent with Example 1. Only the proportion of ginseng root powder added to the functional feed was changed, with five gradient groups set up at mass fractions of 0.5%, 1%, 2%, 5%, and 10%. The rearing cycle for each group of laying hens was uniformly 90 days. After the rearing period, eggs were randomly selected from each group, and the ginsenoside content in the yolk of a single egg was determined using HPLC-UV method. The changes in egg production rate of each group were also statistically analyzed. The experimental results are shown in the table below:

[0040] The experimental results show that within the addition range of 0.5% to 10%, the enrichment of ginsenosides in egg yolks continuously increases with the increase of the proportion of ginseng added. When the addition amount is controlled at 0.5% to 2%, the laying hens have normal feed intake, stable physiological state, and no significant fluctuation in egg production rate. When the addition amount is increased to 5% or above, although the content of ginsenosides in egg yolks increases significantly, it will inhibit the physiological metabolism of laying hens, resulting in a significant decrease in egg production rate. Moreover, the higher the addition ratio, the more significant the decrease in egg production performance.

[0041] Taking into account the enrichment efficiency of ginsenosides, breeding and production costs, and the stability of egg production in laying hens, the optimal addition ratio of ginseng rootlets in feed is determined to be 2%. This can achieve efficient enrichment of ginsenosides in egg yolks while ensuring normal egg production performance, providing the best feed formulation basis for the large-scale production of this invention. Example 3

[0042] This embodiment is used to verify the targeted uptake ability of the ginsenoside lipoprotein complex of the present invention on tumor cells and its dual receptor-mediated mechanism of action. Figure 3 , Figure 4 As shown.

[0043] Experimental cells: human synovial sarcoma SW982 cells, human breast cancer MCF-7 cells, and normal human skin fibroblasts (HSF).

[0044] Experimental Methods: The ginsenoside lipoprotein complex prepared in Step 1 was fluorescently labeled with FITC to the lipoprotein; free FITC-labeled ginsenoside Rh2 was used as a control. The natural complex and free FITC-Rh2 were added separately to culture media containing the three cell types mentioned above, with the final concentration of ginsenosides in the system controlled at 0.01 μM. The mixtures were incubated at 37℃ for 2 h. After incubation, the fluorescence distribution of the cells was observed using a laser confocal microscope, and the cellular uptake was quantitatively analyzed using flow cytometry.

[0045] The experimental results are as follows: In human synovial SW982 tumor cells, the fluorescence intensity of the natural complex group was 8.5 times that of the free Rh2 group; In human breast cancer MCF-7 tumor cells, the fluorescence intensity of the natural complex group was 7.2 times that of the free Rh2 group; In normal human skin fibroblast HSF cells, the fluorescence intensity of the natural complex group was only 1.3 times that of the free Rh2 group.

[0046] The results show that the complex of the present invention has a significant targeted uptake ability on tumor cells, while the uptake of normal cells is extremely low, exhibiting good tumor targeting selectivity.

[0047] Further competitive inhibition experiments were conducted: after adding 50 mM excess free glucose to the culture medium, the uptake rate of the natural complex by SW982 cells decreased by 58%; after intervention with 100 μg / mL exogenous LDL, the cell uptake rate decreased by 42%; and after dual inhibition with both free glucose and exogenous LDL, the cell uptake rate decreased by 81%. Example 4

[0048] like Figure 5 As shown, the in vivo antitumor efficacy, equivalent dose difference, and drug safety of the ginsenoside lipoprotein complex of the present invention were evaluated using a nude mouse tumor-bearing model.

[0049] BALB / c nude mice were used as experimental animals. Human synovial sarcoma SW982 cells were subcutaneously inoculated into the right side of the mice at a dose of 5 × 10⁻⁶. 6 Each mouse was fed a standard diet until the subcutaneous tumors of the nude mice grew to 80–120 mm³. The mice were then randomly divided into 5 groups and drug treatment was initiated. The total treatment period was 28 days.

[0050] The experimental groups and drug administration regimens are as follows: Blank control group: normal saline, intravenous injection, once every 3 days; High-dose group of free Rh: Free ginsenoside Rh2, administered at a dose of 10 mg / kg, intravenously, once every 3 days; Low-dose group of free Rh2: Free ginsenoside Rh2, dose 0.1 mg / kg, intravenous injection, once every 3 days; The extremely low dose group of natural complex: based on the equivalent ginsenoside content, the dosage was 0.0001 mg / kg, administered intravenously, once every 3 days; Oral administration of natural complex: The dosage is 0.001 mg / kg, calculated based on the equivalent ginsenoside content, administered by gavage once every 3 days.

[0051] After the experiment, the tumor inhibition rate, changes in animal body weight, and liver and kidney function indicators were recorded for each group. The results are as follows: The tumor inhibition rate of the extremely low dose group of the natural complex can reach 72.3%, which is not statistically different from the 75.1% tumor inhibition effect of the high dose group of free Rh2 (p>0.05); when achieving the same anti-tumor effect, the dosage of the natural complex of the present invention is only one ten-thousandth of that of free ginsenoside Rh2.

[0052] The tumor inhibition rate of the natural complex in the oral administration group reached 65.8%, confirming that the complex has good oral absorption and in vivo anti-tumor activity.

[0053] Safety evaluation results showed that the weight of nude mice in each group treated with the natural compound did not decrease significantly, and the liver function ALT, AST and kidney function Cr, BUN indicators were all at normal levels, with no obvious toxic side effects; however, the weight of nude mice in the high-dose group of free Rh2 decreased by 12%, accompanied by abnormally elevated liver enzyme indicators, with obvious toxic side effects.

[0054] In vivo efficacy results confirmed that the ginsenoside lipoprotein complex of the present invention has extremely high in vivo antitumor activity, with extremely low dosage and significant tumor-suppressing effect, while also having oral efficacy and good drug safety. Example 5

[0055] This embodiment uses SW982 synovial sarcoma-bearing nude mice as an animal model to investigate the antitumor synergistic, toxicity-reducing, and multidrug resistance reversal effects of the combined use of the ginsenoside lipoprotein complex of this invention and the chemotherapeutic drug doxorubicin. Figure 6 As shown, The experiment used the established SW982 tumor-bearing nude mouse model, which was randomly divided into four groups. The specific grouping and drug administration regimens are as follows: Blank control group; Doxorubicin monotherapy group: The dosage was 2 mg / kg, administered intravenously, once every 7 days; Natural complex monotherapy group: 0.0001 mg / kg based on saponin dosage, administered intravenously every 3 days; Combination therapy group: Doxorubicin 2 mg / kg + natural complex 0.0001 mg / kg, with the same dosing frequency as the single-drug groups.

[0056] Experimental results show that: The tumor inhibition rate in the combination therapy group was as high as 89.4%, which was significantly higher than that in the doxorubicin monotherapy group (51.2%) and the natural complex monotherapy group (72.3%). The difference between the groups was statistically significant (p < 0.01).

[0057] Safety observation results showed that the myocardial enzyme spectrum and electrocardiogram of the animals in the combined drug group were normal, and no obvious cardiotoxicity was observed; however, when the high dose of doxorubicin (4 mg / kg) was administered alone, nude mice showed obvious myocardial damage, indicating that the combination of this compound with chemotherapy drugs can significantly reduce the cardiotoxic side effects caused by chemotherapy drugs.

[0058] The results of tumor tissue protein detection confirmed that the ginsenoside lipoprotein complex of the present invention can significantly downregulate the expression level of P-glycoprotein (P-gp) in tumor tissue, effectively reverse tumor multidrug resistance, and play a significant synergistic therapeutic role in chemotherapeutic sensitization, toxicity reduction and efficacy enhancement.

[0059] Example 6 The natural complex of this invention can be used to prepare the following drugs: Drugs for the prevention and / or adjuvant treatment of synovial sarcoma, osteosarcoma, breast cancer, lung cancer, liver cancer, gastric cancer, colorectal cancer, melanoma, or neuroblastoma; Sensitizing agents used in combination with chemotherapy drugs (such as doxorubicin, ifosfamide, and anlotinib); Immunomodulatory agents used in combination with immune checkpoint inhibitors (such as PD-1 / PD-L1 antibodies); Maintenance therapy agents used as adjuvant therapy after tumor surgery to reduce the risk of recurrence and metastasis.

[0060] The mechanism by which the complex of this invention enters tumor cells and exerts its therapeutic effect is as follows: (1) Transmembrane transport mechanism—dual receptor-mediated endocytosis When this natural complex (particle size 30–50 nm) interacts with the tumor cell membrane, it enters the cell efficiently through the following pathways: LDL receptor-mediated pathway: Natural apolipoproteins (such as apoB analogs) on the surface of the complex are recognized by the LDL receptors highly expressed on the surface of tumor cells, triggering clathrin encapsulation in pits, forming endocytic vesicles that enter the cell, and subsequently fuse with lysosomes to release ginsenosides.

[0061] GLUT-mediated pathway: Ginsenoside sugar chains (such as glucose groups) exposed on the surface of the complex are recognized by GLUT1 / GLUT3 overexpressed on the surface of tumor cells and enter the cell via carrier-mediated endocytosis. This pathway is particularly active in tumor cells with hyperglycemic metabolism.

[0062] Since normal somatic cells are approximately 10–30 μm in diameter, while the complex particles are only 30–50 nm in size, the latter cannot penetrate the 7–10 nm thick lipid bilayer through free diffusion. Therefore, receptor-mediated endocytosis is the only effective pathway for its entry into cells. Furthermore, the expression levels of LDL receptors and GLUT on the surface of tumor cells are typically 3–10 times higher than in normal cells, thus giving the complex a natural advantage in tumor targeting.

[0063] (2) Intracellular release and cascade amplification Upon entering the cell, the complex is broken down in the acidic environment of the lysosome (pH 4.5–5.5) and under the action of enzymes, releasing ginsenosides. The released ginsenosides can be further deglycosylated by β-glucosidase within the cell, converting into protopanaxadiol (PPD), which is more hydrophobic. PPD has low affinity for serum albumin and high membrane permeability, allowing it to remain in the cell for a long time and continuously act on mitochondria and nuclear targets, producing a cascade of amplified cytotoxic effects.

[0064] (3) The scientific connotation of 100,000 times equivalent bioactivity Animal experiments have shown that the natural complex of this invention requires approximately 100,000 times less dose (10) compared to free ginsenosides to achieve equivalent biological activity. 5 (times). The mechanism of this amazing potency includes: Significantly increased bioavailability: The natural lipoprotein shell protects ginsenosides from hydrolysis by β-glucosidase in the gastrointestinal tract and allows them to enter circulation via the intestinal lymphatic system, bypassing the first-pass effect of the liver, resulting in an oral bioavailability increase of approximately 100–1000 times.

[0065] Targeted accumulation effect: Dual receptor-mediated tumor targeting allows the concentration of the complex in tumor tissue to reach 1,000–10,000 times that of the free drug.

[0066] Sustained release and cascade effects: Saponins in the lipoprotein core are released in a sustained-release state, and PPD generated after intracellular deglycosylation has a longer half-life, significantly prolonging the duration of drug efficacy.

[0067] The product effect of the above factors (bioavailability × target concentration × duration of action) together resulted in approximately 10 5 The equivalent active equivalent is times that of the previous year.

[0068] Of course, the above description is not a limitation of the present invention, and the present invention is not limited to the examples given above. Any changes, modifications, additions or substitutions made by those skilled in the art within the scope of the present invention should also fall within the protection scope of the present invention.

Claims

1. A ginsenoside lipoprotein complex biosynthesized by hens, characterized by: After laying hens ingest functional feed containing ginsenosides, the ginsenosides are absorbed in the intestines, metabolized in the liver, transported in the blood lipoproteins, and selectively enriched in the ovaries. This allows the ginsenosides to naturally bind with egg yolk lipoproteins and deposit in the egg yolk. After separation and purification, the ginsenosides are obtained with an average particle size of 20–80 nm. The ginsenosides are encapsulated in the lipoprotein lipid core and exposed on the phospholipid shell surface through sugar chains.

2. The ginsenoside-lipid-protein complex biosynthesized using hens according to claim 1, characterized in that, Its density is 0.95–1.063 g / mL, and its surface zeta potential is -15 mV to +10 mV; the ginsenosides are selected from at least one of ginsenosides Rh2, Rg3, Rh1, Rb1, Rd, Re, protopanaxadiol, protopanatriol, and metabolites generated in laying hens through deglycosylation, oxidation, and isomerization.

3. The ginsenoside lipoprotein complex biosynthesized using hens according to claim 2, characterized in that, The lipoprotein is egg yolk low-density lipoprotein, high-density lipoprotein, or very low-density lipoprotein.

4. The ginsenoside lipoprotein complex synthesized from laying hens according to claim 3, characterized in that, The complex contains 5–50 μg / mg lipoprotein ginsenosides, which are embedded in the lipoprotein lipid core through a hydrophobic dammarane-type tetracyclic triterpenoid core. The sugar chain portion is exposed on the phospholipid monolayer on the lipoprotein surface, forming a natural amphiphilic structure of "core encapsulation-shell recognition".

5. The method for separating and purifying ginsenoside lipoprotein complexes synthesized from laying hens according to any one of claims 1-4, characterized in that: Includes the following steps: (a) Feed formulation: Ginseng, ginseng extract or ginseng processing by-products are added to the basic feed of laying hens in a specific ratio to form a functional feed containing ginsenosides. (b) Biotransformation: The functional feed described above is used to feed laying hens for a period of not less than 60 days, so that ginsenosides are selectively deposited in the egg yolk and naturally combined with egg yolk lipoproteins after being absorbed by the hen's intestines, metabolized by the liver, and transported by blood lipoproteins. (c) Egg yolk collection: Collect eggs laid by hens after feeding and separate the yolks; (d) Separation and purification: After homogenizing the egg yolk, extract and purify the ginsenoside lipoprotein complex by at least one of the following methods: differential centrifugation, density gradient centrifugation, organic solvent extraction, ultrafiltration, dialysis or chromatographic separation. (e) Formulation: The purified complex is formulated into lyophilized powder, injection, oral formulation or sustained-release formulation.

6. The method for separating and purifying the ginsenoside lipoprotein complex synthesized from laying hens according to claim 5, characterized in that, The ginseng extract mentioned in step (a) is an aqueous extract, alcohol extract, or ultrafine powder of ginseng root, ginseng root, ginseng root, ginseng root, ginseng root residue, or ginseng root; the amount of ginseng extract added to the functional feed is 0.5%–5% (w / w).

7. The method for separating and purifying ginsenoside lipoprotein complexes synthesized from laying hens according to claim 6, characterized in that, The laying hens mentioned in step (b) are Hy-Line Brown, Lohmann Brown, Jinghong, or Black Phoenix chickens; the feeding period is 60–180 days, preferably 90–120 days.

8. The method for separating and purifying the ginsenoside lipoprotein complex synthesized from laying hens according to claim 7, characterized in that, The separation and purification described in step (d) specifically includes the following steps: (i) Mix egg yolks with an equal volume of phosphate-buffered saline (PBS, pH 7.4) to form a homogenate; (ii) Centrifuge at low speed (3,000–6,000 g, 15–30 min) to remove precipitate and collect supernatant; (iii) Separate lipoprotein bands by ultracentrifugation or density gradient centrifugation; (iv) Collect lipoprotein components containing ginsenosides and remove small molecule impurities by Sephadex G-25 or G-75 gel filtration; (v) Ginsenoside lipoprotein complex with a purity of ≥85% was obtained by ultrafiltration concentration.

9. The method for separating and purifying the ginsenoside lipoprotein complex synthesized from laying hens according to claim 8, characterized in that, Step (d) also includes identifying and quantifying the types and contents of ginsenosides in the complex using high performance liquid chromatography or liquid chromatography-mass spectrometry; the ginsenoside content in the complex is bioactively determined by in vitro cell uptake experiments and animal pharmacodynamic experiments.

10. The pharmaceutical application of the ginsenoside lipoprotein complex biosynthesized from laying hens according to any one of claims 1-4, characterized in that, This is used to prepare drugs, chemotherapy sensitizers, and tumor immunomodulatory agents for the prevention and / or treatment of tumors; the tumors include synovial sarcoma, osteosarcoma, breast cancer, lung cancer, liver cancer, gastric cancer, colorectal cancer, melanoma, and neuroblastoma; the drug dosage form is an oral preparation, an injection, a lyophilized powder for injection, an enteric-coated capsule, a transdermal patch, or a sustained-release microsphere preparation.

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