An antitumor polypeptide composition, its preparation method and application

Abstract

The application belongs to the technical field of biological medicine, and discloses an antitumor polypeptide composition, a preparation method and application thereof; the composition is composed of the following components in parts by weight: 10-30 parts of mucopolysaccharide combined mixed peptide (polypeptide A) extracted from sea cucumber body wall, 5-15 parts of angiogenesis inhibiting peptide RGD-4C, 5-10 parts of tumor penetrating peptide iRGD, 3-8 parts of thymosin alpha 1, 2-6 parts of ganoderma lucidum polysaccharide and 1-4 parts of vitamin C palmitate. The polypeptide A is prepared by a process comprising specific ultrasonic crushing, ultrafiltration, ion exchange chromatography and reverse phase liquid chromatography purification. The application further provides a preparation method of the composition, comprising the steps of weighing, sterile mixing, grinding and sub-packaging, and can be further prepared into a lyophilized powder injection and the like dosage form. The composition can be used for preparing a drug for treating solid tumors such as lung cancer, breast cancer, colorectal cancer, liver cancer and the like, and can be used in combination with chemotherapeutic drugs such as paclitaxel.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a polypeptide composition with antitumor activity, its preparation method, and its application in the preparation of antitumor drugs. Background Technology

[0002] Sea cucumbers, as a precious marine biological resource, are rich in proteins, polysaccharides, and various bioactive substances in their body walls and viscera. Studies have shown that peptides derived from sea cucumbers possess a variety of pharmacological activities, including antitumor, anticoagulant, and immunomodulatory effects, demonstrating great potential in the development of functional foods and drugs.

[0003] Various active ingredients from sea cucumbers have been developed and applied in the prior art. For example, an existing invention (CN120535572A) discloses a single-sequence antitumor active peptide (FNPDTFD) screened from sea cucumber flowers, which exerts its inhibitory effect on non-small cell lung cancer by targeting the EGFR signaling pathway. This technology focuses on single, well-defined small molecule peptides, but its screening process is complex and does not involve the synergistic utilization of naturally occurring mixed polypeptide components in the sea cucumber body wall.

[0004] On the other hand, an existing invention (CN115948492A) discloses a method for preparing sea cucumber peptides with high sea cucumber polysaccharide content. This method utilizes enzymatic hydrolysis, ultrafiltration, and integration processes to obtain a complex product of sea cucumber polysaccharides and peptides, aiming to exert comprehensive health benefits. This technology focuses on obtaining polysaccharide-peptide complexes, rather than the precise design and formulation of multi-component active ingredients for specific anti-tumor effects.

[0005] In addition, as shown in the prior art (CN106267141A), the combination of sea cucumber intestinal polypeptides with other plant extracts for anti-tumor purposes has also been reported. However, its composition is complex, its mechanism of action is not clear, and it does not involve the targeted extraction and application of sea cucumber body wall polypeptide components with specific functions (such as mucopolysaccharide binding activity).

[0006] Therefore, existing technologies lack systematic research on mixed polypeptide components in the sea cucumber body wall that possess specific biological functions (such as mucopolysaccharide-binding activity). Related findings are mostly focused on screening single active peptides or preparing polysaccharide-peptide complexes. Furthermore, there are few studies on scientifically combining such mixed polypeptides from sea cucumbers with other functional polypeptides and non-peptide active substances with well-defined mechanisms to construct multi-target, synergistic antitumor compositions. Summary of the Invention

[0007] To address the shortcomings of the existing technology, this invention aims to provide a novel antitumor polypeptide composition. The core innovation of this composition lies in: This invention marks the first time that two novel peptides with characteristic sequences (VTPALPWMK and GDREPSGFLRKYC) have been directionally isolated and identified from the body wall of sea cucumbers. Using this "dual-characteristic peptide complex" as the core active unit, it is scientifically formulated with other functional components with well-defined mechanisms. The aim is to address the problems of insufficient activity of single components or unclear mechanisms in complex mixtures in existing technologies, providing a highly efficient antitumor composition with relatively well-defined components and significant synergistic effects.

[0008] The present invention also provides a method for preparing the composition and its application in the preparation of antitumor drugs.

[0009] To achieve the above objectives, the present invention includes the following technical solutions.

[0010] An antitumor polypeptide composition comprising the following components in parts by weight: Polypeptide A: 10-30 parts, wherein polypeptide A is a mucopolysaccharide-binding peptide extracted from the marine echinoderm sea cucumber, containing the characteristic peptide VTPALPWMK as shown in SEQ ID NO: 1 and the characteristic peptide GDREPSGFLRKYC as shown in SEQ ID NO: 2, and based on the total mass of polypeptide A, the content of the characteristic peptide shown in SEQ ID NO: 1 is 20-40 wt%, and the content of the characteristic peptide shown in SEQ ID NO: 2 is 25-45 wt%. The polypeptide A was prepared by the following steps: fresh sea cucumber body wall tissue was taken, homogenized, and then added to a phosphate buffer solution with a pH of 6.5-7.5. The mixture was ultrasonically disrupted at 0-4℃ for 30-60 minutes. The supernatant was centrifuged and then separated sequentially through an ultrafiltration membrane with a molecular weight cutoff of 30 kDa. The permeate was collected. The permeate was loaded onto an anion exchange chromatography column and eluted with a linear gradient containing 0.1-0.5 M NaCl. The eluted fraction with a conductivity corresponding to 15-25 mS / cm was collected. The fraction was purified by reversed-phase high-performance liquid chromatography with a gradient elution of acetonitrile-water solution at a volume ratio of 20:80 to 50:50. The chromatographic peak fraction with a retention time of 15-18 minutes was collected and lyophilized to obtain the final product. Polypeptide B: 5-15 parts, which is the angiogenesis inhibitory peptide RGD-4C; Polypeptide C: 5-10 parts, is the tumor-penetrating peptide iRGD; Polypeptide D: 3-8 parts, which is thymosin α1; Non-peptide active substance E: 2-6 parts, wherein the non-peptide active substance E is Ganoderma lucidum polysaccharide with a molecular weight of 50-200 kDa; Non-peptide active substance F: 1-4 parts, wherein the non-peptide active substance F is vitamin C palmitate.

[0011] Furthermore, in the above-mentioned antitumor polypeptide composition, during the extraction of polypeptide A, anion exchange chromatography is performed using a DEAE-Sepharose Fast Flow column with an elution flow rate of 1-2 mL / min.

[0012] Furthermore, in the above-mentioned antitumor polypeptide composition, the C-terminus of polypeptide B is a carboxyl or amide group.

[0013] Furthermore, in the above-mentioned antitumor polypeptide composition, the non-peptide active substance E of Ganoderma lucidum polysaccharide is derived from the fruiting body of Ganoderma lucidum, and its polysaccharide content is not less than 90%.

[0014] Furthermore, the above-mentioned antitumor polypeptide composition further includes a pharmaceutically acceptable carrier selected from at least one of mannitol, sucrose, or trehalose, in parts by weight of 20-50.

[0015] This invention also discloses a method for preparing the above-mentioned antitumor polypeptide composition, comprising the following steps: (1) Weigh out polypeptide A, polypeptide B, polypeptide C, polypeptide D, non-peptide active substance E and non-peptide active substance F according to their weight parts; (2) Mix the components under aseptic conditions and add a pharmaceutically acceptable carrier; (3) Grind and mix thoroughly using the equal-increment method until the material is uniform; (4) Dispense the mixture into vials or capsules to obtain the final product.

[0016] Furthermore, the above preparation method, which prepares the polypeptide composition into a lyophilized powder injection, further includes the following steps after step (2): dissolving the mixture in water for injection, sterilizing it through a 0.22 μm filter membrane, dispensing it, pre-freezing it at -40℃ for 4-6 hours, and then freeze-drying it under a vacuum of less than 10 Pa for 24-36 hours.

[0017] The present invention also discloses the use of the above-mentioned antitumor polypeptide composition in the preparation of a medicament for treating solid tumors, wherein the solid tumors include lung cancer, breast cancer, colorectal cancer, or liver cancer.

[0018] Furthermore, in the above application, the drug is used in combination with the chemotherapy drug paclitaxel.

[0019] Compared with the prior art, the present invention has the following outstanding advantages: 1. The core components are clearly defined, quantified, and novel: For the first time, the novel sea cucumber characteristic peptides VTPALPWMK (20-40%) and GDREPSGFLRKYC (25-45%), which coexist in specific amounts, are defined as the core active substances. The material basis is clear, novel, and quantifiable, which is different from the ambiguous mixtures in existing technologies.

[0020] 2. The synergistic effect is extremely significant and attributable: Based on the aforementioned quantitatively defined dual-characteristic peptides, the constructed multi-target combination produced a good synergistic effect, demonstrating significant in vitro antitumor activity (IC50). 50 The concentration of < 1 μg / mL reached a very high level, and the effect clearly depended on complete compatibility.

[0021] 3. Controllable quality and good reproducibility: The core active ingredient (dual characteristic peptide) has a clear sequence and content standard, and the remaining components are all commercially available and clearly defined substances, ensuring a high degree of controllability of the composition formulation and quality, as well as production reproducibility, and a clear prospect for industrialization. Attached Figure Description

[0022] Figure 1 The inhibitory effect of each composition on scratch healing of A549 cells after 24 hours (migration rate %). Figure 2 The apoptosis-inducing effect (%) of each composition on HepG2 cells after 24 hours of treatment. Detailed Implementation

[0023] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0024] Table 1: Raw Material List

[0025] Example 1: An antitumor polypeptide composition, made from the following raw materials in parts by weight: 10 parts of polypeptide A, 5 parts of polypeptide B (RGD-4C), 5 parts of polypeptide C (iRGD), 3 parts of polypeptide D (thymosin α1), 2 parts of Ganoderma lucidum polysaccharide (molecular weight 50-200 kDa), and 1 part of vitamin C palmitate. The polypeptide A was prepared by the following method: 200g of fresh sea cucumber body wall tissue was homogenized and then 800mL of phosphate buffer (pH 6.8) was added. The mixture was ultrasonically disrupted at 0℃ for 60 minutes. The supernatant was centrifuged and then separated sequentially through an ultrafiltration membrane with a molecular weight cutoff of 30 kDa. The permeate was collected. The permeate was loaded onto a DEAE-Sepharose Fast Flow anion exchange chromatography column and eluted with a linear gradient containing 0.1 M to 0.5 M NaCl at a flow rate of 1.0 mL / min. The eluent fraction with a conductivity corresponding to 18 mS / cm was collected. The fraction was purified by reversed-phase high-performance liquid chromatography (RP-HPLC) with a gradient elution of acetonitrile-water solution at a volume ratio of 20:80 to 50:50. The chromatographic peak fraction with a retention time of 17 minutes was collected and lyophilized to obtain polypeptide A lyophilized powder.

[0026] Amino acid composition analysis and peptide sequencing were performed on the prepared lyophilized peptide A powder, and it was determined that the content of the characteristic peptide VTPALPWMK (SEQ ID NO: 1) was about 30 wt%, the content of the characteristic peptide GDREPSGFLRKYC (SEQ ID NO: 2) was about 33 wt%, and the total content of the two characteristic peptides was about 63 wt%.

[0027] The preparation method of the Ganoderma lucidum polysaccharide includes: taking the fruiting body of Ganoderma lucidum, crushing it, adding 10 times the amount of water and extracting it at 90°C for 2 hours, filtering, concentrating, precipitating with alcohol, removing the protein from the precipitate by the Sevag method, and then freeze-drying it to obtain Ganoderma lucidum polysaccharide with a polysaccharide content of 92%.

[0028] The preparation method of the above-mentioned antitumor polypeptide composition includes the following steps: (1) Weigh out the lyophilized powder of polypeptide A, polypeptide B, polypeptide C, polypeptide D, Ganoderma lucidum polysaccharide and vitamin C palmitate according to the above weight parts; (2) Mix the components under sterile conditions and add 20 parts of mannitol as a pharmaceutically acceptable carrier; (3) Grind and mix thoroughly for 30 minutes using the equal-increment method until the material is uniform; (4) Dispense the mixture into vials to obtain the powder of the composition.

[0029] Example 2: An antitumor polypeptide composition, made from the following raw materials in parts by weight: 20 parts of polypeptide A, 10 parts of polypeptide B (RGD-4C), 7 parts of polypeptide C (iRGD), 5 parts of polypeptide D (thymosin α1), 4 parts of Ganoderma lucidum polysaccharide (molecular weight 50-200 kDa), and 3 parts of vitamin C palmitate. The polypeptide A was prepared by the following method: 200g of fresh sea cucumber body wall tissue was homogenized and then 800mL of phosphate buffer (pH 6.8) was added. The mixture was ultrasonically disrupted at 0℃ for 60 minutes. The supernatant was centrifuged and then separated sequentially through an ultrafiltration membrane with a molecular weight cutoff of 30 kDa. The permeate was collected. The permeate was loaded onto a DEAE-Sepharose Fast Flow anion exchange chromatography column and eluted with a linear gradient containing 0.1 M to 0.5 M NaCl at a flow rate of 1.0 mL / min. The eluent fraction with a conductivity corresponding to 18 mS / cm was collected. The fraction was purified by reversed-phase high-performance liquid chromatography (RP-HPLC) with a gradient elution of acetonitrile-water solution at a volume ratio of 20:80 to 50:50. The chromatographic peak fraction with a retention time of 17 minutes was collected and lyophilized to obtain polypeptide A lyophilized powder.

[0030] Amino acid composition analysis and peptide sequencing were performed on the obtained lyophilized peptide A powder, and it was determined that the content of the characteristic peptide VTPALPWMK (SEQ ID NO: 1) was about 30 wt%, the content of the characteristic peptide GDREPSGFLRKYC (SEQ ID NO: 2) was about 35 wt%, and the total content of the two characteristic peptides was about 65 wt%.

[0031] The preparation method of the Ganoderma lucidum polysaccharide includes: taking the fruiting body of Ganoderma lucidum, crushing it, adding 15 times the amount of water and extracting it at 95°C for 1.5 hours, filtering, concentrating, precipitating with alcohol, removing the protein from the precipitate by the Sevag method, and then freeze-drying it to obtain Ganoderma lucidum polysaccharide with a polysaccharide content of 94%.

[0032] The preparation method of the above-mentioned antitumor polypeptide composition includes the following steps: (1) Weigh out the lyophilized powder of polypeptide A, polypeptide B, polypeptide C, polypeptide D, Ganoderma lucidum polysaccharide and vitamin C palmitate according to the above weight parts; (2) Mix the components under sterile conditions and add 35 parts of trehalose as a pharmaceutically acceptable carrier; (3) Grind and mix thoroughly for 40 minutes using the equal-increment method until the material is uniform; (4) The mixture is packaged into enteric capsule shells to obtain oral capsule preparations.

[0033] Example 3: An antitumor polypeptide composition, made from the following raw materials in parts by weight: 30 parts of polypeptide A, 15 parts of polypeptide B (RGD-4C), 10 parts of polypeptide C (iRGD), 8 parts of polypeptide D (thymosin α1), 6 parts of Ganoderma lucidum polysaccharide (molecular weight 50-200 kDa), and 4 parts of vitamin C palmitate. The polypeptide A was prepared by the following method: 200g of fresh sea cucumber body wall tissue was homogenized and then 800mL of phosphate buffer (pH 6.8) was added. The mixture was ultrasonically disrupted at 0℃ for 60 minutes. The supernatant was centrifuged and then separated sequentially through an ultrafiltration membrane with a molecular weight cutoff of 30 kDa. The permeate was collected. The permeate was loaded onto a DEAE-Sepharose Fast Flow anion exchange chromatography column and eluted with a linear gradient containing 0.1 M to 0.5 M NaCl at a flow rate of 1.0 mL / min. The eluent fraction with a conductivity corresponding to 18 mS / cm was collected. The fraction was purified by reversed-phase high-performance liquid chromatography (RP-HPLC) with a gradient elution of acetonitrile-water solution at a volume ratio of 20:80 to 50:50. The chromatographic peak fraction with a retention time of 17 minutes was collected and lyophilized to obtain polypeptide A lyophilized powder.

[0034] Amino acid composition analysis and peptide sequencing were performed on the prepared lyophilized peptide A powder, and the content of the characteristic peptide VTPALPWMK (SEQ ID NO: 1) was approximately 31 wt%, the content of the characteristic peptide GDREPSGFLRKYC (SEQ ID NO: 2) was approximately 36 wt%, and the total content of the two characteristic peptides was approximately 67 wt%.

[0035] The preparation method of the Ganoderma lucidum polysaccharide includes: taking the fruiting body of Ganoderma lucidum, crushing it, adding 20 times the amount of water and extracting it at 100℃ for 1 hour, filtering, concentrating, precipitating with alcohol, removing the protein from the precipitate by the Sevag method, and then freeze-drying it to obtain Ganoderma lucidum polysaccharide with a polysaccharide content of 95%.

[0036] The preparation method of the above-mentioned antitumor polypeptide composition includes the following steps: (1) Weigh out the lyophilized powder of polypeptide A, polypeptide B, polypeptide C, polypeptide D, Ganoderma lucidum polysaccharide and vitamin C palmitate according to the above weight parts; (2) Mix the components under sterile conditions and add 50 parts of sucrose as a pharmaceutically acceptable carrier; (3) Grind and mix thoroughly for 50 minutes using the equal-increment method until the material is uniform; (4) Dispense the mixture into vials to obtain the powder of the composition.

[0037] Comparative Example 1: A composition, by weight, made from the following raw materials: 20 parts of polypeptide A, 10 parts of polypeptide B (RGD-4C), 7 parts of polypeptide C (iRGD), 5 parts of polypeptide D (thymosin α1), and 3 parts of vitamin C palmitate; (excluding Ganoderma lucidum polysaccharides).

[0038] The preparation methods of the polypeptide A and the composition are the same as in Example 2.

[0039] Comparative Example 2: A composition, by weight, made from the following raw materials: 20 parts of polypeptide A, 10 parts of polypeptide B (RGD-4C), 7 parts of polypeptide C (iRGD), 5 parts of polypeptide D (thymosin α1), and 4 parts of Ganoderma lucidum polysaccharide (excluding vitamin C palmitate).

[0040] The preparation methods of the polypeptide A and the composition are the same as in Example 2.

[0041] Comparative Example 3: A composition, by weight, made from the following raw materials: 20 parts of polypeptide A, 10 parts of polypeptide B (RGD-4C), 7 parts of polypeptide C (iRGD), 4 parts of Ganoderma lucidum polysaccharide, and 3 parts of vitamin C palmitate; (excluding polypeptide D).

[0042] The preparation methods of the polypeptide A and the composition are the same as in Example 2.

[0043] Comparative Example 4: A composition, by weight, made from the following raw materials: 20 parts of polypeptide A, 10 parts of polypeptide B (RGD-4C), 5 parts of polypeptide D (thymosin α1), 4 parts of Ganoderma lucidum polysaccharide, and 3 parts of vitamin C palmitate; (excluding polypeptide C).

[0044] The preparation methods of the polypeptide A and the composition are the same as in Example 2.

[0045] Comparative Example 5: A composition, by weight, made from the following raw materials: 20 parts of polypeptide A, 7 parts of polypeptide C (iRGD), 5 parts of polypeptide D (thymosin α1), 4 parts of Ganoderma lucidum polysaccharide, and 3 parts of vitamin C palmitate; (excluding polypeptide B).

[0046] The preparation methods of the polypeptide A and the composition are the same as in Example 2.

[0047] Test Example 1

[0048] In vitro anti-tumor cell proliferation inhibition experiment.

[0049] Objective: To evaluate the inhibitory activity of the polypeptide composition of the present invention on the proliferation of different tumor cell lines and to verify its multi-component synergistic effect.

[0050] method: 1. Cell Culture: Human non-small cell lung cancer cells A549, human liver cancer cells HepG2, and human breast cancer cells MCF-7 were selected. Cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum and incubated at 37°C in a 5% CO2 incubator.

[0051] 2. Sample preparation: The powdered compositions prepared in Examples 1-3 and Comparative Examples 1-5 were prepared into a 100 mg / mL stock solution using DMSO, and then diluted with complete culture medium to working solutions with final concentrations of 0.1, 0.5, 1, 2.5, 5, and 10 μg / mL. An equal volume of DMSO (final concentration <0.1%) was used as a solvent control group.

[0052] 3. CCK-8 assay: Cells in logarithmic growth phase were cultured at 5 × 10⁶ cells per well. 3 Inoculate samples at a density of 10 μL each into 96-well plates. After 24 hours of incubation and adhesion, discard the original culture medium and add fresh culture medium containing different concentrations of the sample, with 6 replicates for each concentration. Continue incubation for 48 hours, then add 10 μL of CCK-8 solution to each well and incubate for 2 hours.

[0053] 4. Data Reading and Analysis: The absorbance (OD value) at 450 nm was measured using a microplate reader. Cell viability (%) = (OD value of experimental group - OD value of blank well) / (OD value of solvent control group - OD value of blank well) × 100%. The half-maximal inhibitory concentration (IC50) was calculated. 50 The experiment was repeated three times independently.

[0054] result: As shown in Table 2 below, the composition of Example 2 exhibited the strongest inhibitory activity against all three tumor cell lines, with an IC50 value of [missing value]. 50 The IC50 value of Example 2 was the lowest. Compared to Comparative Examples 1-5, which lacked any active component, the IC50 value of Example 2 was the lowest. 50 The values ​​were all significantly reduced (P<0.01). Examples 1 and 3 also showed strong activity, indicating that the compositions of the present invention are effective over a wide range of formulations.

[0055] Table 2: IC50 inhibitory effects of each composition on the proliferation of different tumor cell lines 50 Value (μg / mL, mean ± SD).

[0056]

[0057] Conclusion: The core of the complete composition of this invention lies in the use of two sea cucumber characteristic peptides (SEQ ID NO: 1 & 2) with clearly defined contents and novel sequences as basic active units. The composition exhibits high antitumor activity at low concentrations, indicating a synergistic effect between these two characteristic peptides and other functional components.

[0058] Test Example 2

[0059] Scratch healing inhibition experiment based on A549 lung cancer cells.

[0060] Objective: To evaluate the effect of the composition of the present invention on the migration ability of tumor cells and to simulate its potential to inhibit tumor invasion and metastasis.

[0061] method: 1. Cell preparation and scratching: A549 cells were prepared at a density of 2 × 10⁶ cells per well. 5 Cells were seeded at a density of 1,000 cells / well in 24-well plates and cultured until a monolayer was formed. Straight scratches were made on the monolayer using a 200 μL sterile pipette tip, and the cells were gently washed twice with PBS to remove any detached cells.

[0062] 2. Drug administration: Replace with low-serum culture medium containing 1% FBS containing the compositions of Examples 2 and Comparative Examples 1-5 (concentration of 1 μg / mL). Use low-serum culture medium containing 1% FBS as the control group.

[0063] 3. Image Acquisition and Analysis: Scratch images were captured at a fixed position under an inverted microscope at 0 and 24 hours. The scratch width was measured using ImageJ software. Cell migration rate (%) = [(0-hour scratch width - 24-hour scratch width) / 0-hour scratch width] × 100%. The experiment was independently repeated three times.

[0064] result: As shown in Table 3 and Figure 1 As shown, compared with the control group, all treatment groups were able to inhibit the migration of A549 cells to some extent. Among them, the composition of Example 2 showed the most significant inhibitory effect, with the lowest migration rate after 24 hours. Compared with Comparative Examples 1-5, the inhibitory effect of Example 2 was superior, especially compared with Comparative Example 4 which lacked peptide C (tumor penetrating peptide) and Comparative Example 5 which lacked peptide B (angiogenesis inhibitory peptide), its advantage in inhibiting cell migration was more obvious.

[0065] Table 3: Inhibitory effect of each composition on scratch healing of A549 cells after 24 hours (migration rate, mean ± SD).

[0066] Note: P<0.01 vs control group and each comparative group.

[0067] Conclusion: The complete composition of this invention significantly inhibits the migration ability of tumor cells. Peptide B may play a key role by inhibiting pro-migration angiogenesis signals, and peptide C may play a key role by interfering with integrin-mediated adhesion and movement. The absence of either key peptide leads to a decrease in the ability to inhibit migration, verifying the functional synergy between the components.

[0068] Test Example 3: Induction of apoptosis in HepG2 liver cancer cells.

[0069] Objective: To detect the ability of the composition of the present invention to induce tumor cell apoptosis and to explore one of the mechanisms by which it exerts its anti-tumor effect.

[0070] method: Cell treatment: HepG2 cells were seeded into 6-well plates and cultured for 24 hours. Then, culture medium containing the compositions of Example 2, Comparative Example 1 (lacking Ganoderma lucidum polysaccharides), and Comparative Example 3 (lacking thymosin α1) was added to a final concentration of 2 μg / mL. A solvent control group was established. The cells were cultured for another 24 hours.

[0071] Annexin V-FITC / PI double staining assay: Cells were collected, washed twice with pre-chilled PBS, and resuspended in 1× Binding Buffer. 100 μL of cell suspension was added, along with 5 μL of Annexin V-FITC and 10 μL of PI staining solution, and incubated at room temperature in the dark for 15 minutes. Then, 400 μL of Binding Buffer was added, and the cells were analyzed by flow cytometry within 1 hour.

[0072] Data Analysis: FlowJo software was used to analyze early apoptosis (Annexin V). + / PI - ) and late apoptosis / necrosis (Annexin V) + / PI + The percentage of cells and the total apoptosis rate are the sum of the two. The experiment was independently repeated three times.

[0073] result: As shown in Table 4 and Figure 2 As shown, the treatment in Example 2 significantly induced apoptosis in HepG2 cells, with a total apoptosis rate as high as 45.6%. In contrast, the apoptosis rates induced by Comparative Example 1 (lacking Ganoderma lucidum polysaccharides with immunomodulatory and potential pro-apoptotic effects) and Comparative Example 3 (lacking the immunomodulatory peptide thymosin α1) were significantly reduced (P<0.01).

[0074] Table 4: Apoptosis induction effect of each composition on HepG2 cells after 24 hours of treatment (%, mean ± SD).

[0075] Note: P<0.01 vs solvent control group, comparative example 1 and comparative example 3.

[0076] Conclusion: The complete composition can effectively induce tumor cell apoptosis. The absence of Ganoderma lucidum polysaccharide and thymosin α1 both significantly reduced the apoptosis-inducing ability, suggesting that they may play an important role in regulating the immune status of the tumor microenvironment and synergistically enhancing the pro-apoptotic signaling of other components (such as peptides A and B), further demonstrating the synergistic effect between non-peptide active substances and functional peptides.

[0077] Test Example 4: Effects on the cell cycle distribution of MCF-7 breast cancer cells.

[0078] Objective: To investigate whether the polypeptide composition of the present invention can inhibit the unlimited proliferation of tumor cells by interfering with the normal cell cycle process and blocking them at a specific time phase, and to verify the synergistic effect of the multi-component composition from this perspective.

[0079] method: 1. Cell Synchronization and Treatment: MCF-7 cells were seeded in 6-well plates and cultured in serum-free medium for 24 hours for synchronization, causing most cells to arrest in the G0 / G1 phase. The medium was then replaced with complete medium containing 10% FBS to restart the cell cycle, and the following samples were immediately added: the composition of Example 2, Comparative Example 2 (vitamin C palmitate deficiency), and Comparative Example 4 (tumor-penetrating peptide iRGD deficiency), to a final concentration of 2 μg / mL. A control group containing only the medium was set up. Each group had 3 replicates.

[0080] 2. Cell cycle detection: After 24 hours of treatment, cells were collected by trypsin digestion and washed twice with pre-chilled PBS. Pre-chilled 70% ethanol (-20°C) was slowly added, and the cells were fixed overnight at 4°C. After fixation, the cells were washed with PBS to remove the ethanol, and staining buffer containing 0.1% Triton X-100, 50 μg / mL propidium iodide (PI), and 100 μg / mL RNase A was added. The cells were incubated at 37°C in the dark for 30 minutes.

[0081] 3. Flow cytometry analysis: PI fluorescence intensity was detected using flow cytometry, with at least 10,000 cell events collected for each sample. The DNA content histogram was fitted and analyzed using ModFit LT software to calculate the percentage of cells in G0 / G1, S, and G2 / M phases. The experiment was independently repeated three times.

[0082] result: As shown in Table 5 below, compared with the control group, the treatment with the composition of Example 2 significantly altered the cell cycle distribution of MCF-7 cells, resulting in a significant increase in the proportion of cells in the G0 / G1 phase (from 54.6% to 72.3%), while the proportion of cells in the S phase significantly decreased (from 31.2% to 18.1%), indicating that it mainly arrested cells in the early stage of DNA synthesis (G0 / G1 phase). The cell cycle arrest effects of Comparative Example 2 (lacking the antioxidant vitamin C palmitate) and Comparative Example 4 (lacking the internalization-promoting peptide C) were weaker than those of Example 2, especially in terms of insufficient inhibition of S phase cells.

[0083] Table 5: Effects of each composition on the cell cycle distribution of MCF-7 cells after 24 hours of treatment (%, mean ± SD) Note: P<0.01 vs control group, comparative example 2 and comparative example 4; the sum of proportions within each group is 100%.

[0084] Conclusion: The complete composition of this invention can effectively arrest breast cancer cells in the G0 / G1 phase, inhibiting their entry into the DNA synthesis phase (S phase), thereby fundamentally inhibiting cell proliferation. Vitamin C palmitate may affect the activity of cell cycle regulatory proteins by maintaining intracellular redox homeostasis; while the tumor-penetrating peptide iRGD may enhance the efficiency of the entire composition in entering the cell, allowing the core active ingredients (such as peptides A, B, and D) to act more effectively on cell cycle regulatory targets. The absence of both leads to a weakened cell cycle arrest effect, further demonstrating the indispensable synergistic effect between non-peptide active substances and functional peptides in this invention.

[0085] The above are merely a few preferred embodiments of the present invention, described in a relatively specific and detailed manner, but they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention.

Claims

1. An antitumor polypeptide composition, characterized in that, It consists of the following components in parts by weight: Polypeptide A: 10-30 parts, wherein polypeptide A is a mucopolysaccharide-binding peptide extracted from the marine echinoderm sea cucumber, containing the characteristic peptide VTPALPWMK as shown in SEQ ID NO: 1 and the characteristic peptide GDREPSGFLRKYC as shown in SEQ ID NO: 2, and based on the total mass of polypeptide A, the content of the characteristic peptide shown in SEQ ID NO: 1 is 20-40 wt%, and the content of the characteristic peptide shown in SEQ ID NO: 2 is 25-45 wt%. The polypeptide A was prepared by the following steps: fresh sea cucumber body wall tissue was taken, homogenized, and then added to a phosphate buffer solution with a pH of 6.5-7.

5. The mixture was ultrasonically disrupted at 0-4℃ for 30-60 minutes. The supernatant was centrifuged and then separated sequentially through an ultrafiltration membrane with a molecular weight cutoff of 30 kDa. The permeate was collected. The permeate was loaded onto an anion exchange chromatography column and eluted with a linear gradient containing 0.1-0.5 M NaCl. The eluted fraction with a conductivity corresponding to 15-25 mS / cm was collected. The fraction was purified by reversed-phase high-performance liquid chromatography with a gradient elution of acetonitrile-water solution at a volume ratio of 20:80 to 50:

50. The chromatographic peak fraction with a retention time of 15-18 minutes was collected and lyophilized to obtain the final product. Polypeptide B: 5-15 parts, which is the angiogenesis inhibitory peptide RGD-4C; Polypeptide C: 5-10 parts, is the tumor-penetrating peptide iRGD; Polypeptide D: 3-8 parts, which is thymosin α1; Non-peptide active substance E: 2-6 parts, wherein the non-peptide active substance E is Ganoderma lucidum polysaccharide with a molecular weight of 50-200 kDa; Non-peptide active substance F: 1-4 parts, wherein the non-peptide active substance F is vitamin C palmitate.

2. The antitumor polypeptide composition according to claim 1, characterized in that, During the extraction of polypeptide A, anion exchange chromatography was performed using a DEAE-Sepharose Fast Flow column with an elution flow rate of 1-2 mL / min.

3. The antitumor polypeptide composition according to claim 1, characterized in that, The C-terminus of the polypeptide B is a carboxyl or amide group.

4. The antitumor polypeptide composition according to claim 1, characterized in that, The non-peptide active substance E is derived from the fruiting body of Ganoderma lucidum, and its polysaccharide content is not less than 90%.

5. The antitumor polypeptide composition according to claim 1, characterized in that, The composition further includes a pharmaceutically acceptable carrier selected from at least one of mannitol, sucrose, or trehalose, in parts by weight of 20-50.

6. The method for preparing the antitumor polypeptide composition according to any one of claims 1-5, characterized in that, Includes the following steps: (1) Weigh out the polypeptide A, polypeptide B, polypeptide C, polypeptide D, non-peptide active substance E and non-peptide active substance F as described in claim 1 by weight. (2) Mix the components under aseptic conditions and add a pharmaceutically acceptable carrier; (3) Grind and mix thoroughly using the equal-increment method until the material is uniform; (4) Dispense the mixture into vials or capsules to obtain the final product.

7. The method according to claim 6, characterized in that, The polypeptide composition is prepared into a lyophilized powder injection. After step (2), the mixture is further prepared by dissolving it in water for injection, sterilizing it through a 0.22 μm filter membrane, dispensing it, pre-freezing it at -40℃ for 4-6 hours, and then freeze-drying it under a vacuum of less than 10 Pa for 24-36 hours.

8. The use of the antitumor polypeptide composition according to any one of claims 1-5 in the preparation of a medicament for treating solid tumors, characterized in that, The solid tumors include lung cancer, breast cancer, and liver cancer.

9. The application according to claim 8, characterized in that, The drug was used in combination with the chemotherapy drug paclitaxel.

Images