A deer antler polypeptide with anti-tumor effects and its preparation method

Abstract

This invention discloses a deer antler polypeptide with anti-tumor effects and its preparation method, belonging to the field of biomedical technology. The amino acid sequence of this deer antler polypeptide is PADLAGNENCN. It is derived from deer antler and is prepared by a two-step enzymatic hydrolysis process using trypsin and neutral protease. After purification by ultrafiltration, gel filtration chromatography, and HPLC, a high-purity active polypeptide is obtained. The deer antler polypeptide prepared by this invention exhibits significant inhibitory activity against the proliferation of human lung cancer cells, with low toxicity and good biocompatibility. Compared with existing anti-tumor drugs, it has the advantages of natural source and fewer side effects, and can be widely used in the research and development and production of anti-tumor drugs, providing a new candidate substance and technical pathway for tumor treatment.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a deer antler polypeptide with anti-tumor effects and its preparation method. Background Technology

[0002] Cancer is a major disease that seriously threatens human health and life, with lung cancer being one of the malignant tumors with the highest incidence and mortality rates worldwide. Currently, clinical treatments for lung cancer mainly include surgical resection, chemotherapy, radiotherapy, targeted therapy, and immunotherapy. However, these treatments all have certain limitations: surgical resection is only suitable for early-stage lung cancer patients, and its efficacy is limited for patients in the middle and late stages; chemotherapy drugs are mostly cytotoxic drugs, which, while killing tumor cells, severely damage normal cells, leading to serious side effects such as hair loss, nausea, and bone marrow suppression; targeted therapy and immunotherapy, although having certain specificity, have problems such as a narrow applicable population, easy development of drug resistance, and high treatment costs, making them difficult to widely apply.

[0003] Therefore, developing a naturally derived, low-side-effect, and highly effective anti-tumor substance has become a research hotspot in the current biomedical field. Deer antler, the unossified, densely haired young antler of male sika deer or red deer, is a traditional and precious Chinese medicinal material with effects such as warming the kidneys and strengthening yang, nourishing essence and blood. Modern pharmacological studies have shown that deer antler contains various active ingredients such as polypeptides, amino acids, and polysaccharides. Among them, deer antler polypeptides possess various biological activities such as antioxidant, immunomodulatory, and anti-inflammatory effects. However, there are currently few reports on the clear anti-tumor effects of deer antler polypeptides (especially against lung cancer), and existing extraction methods for deer antler polypeptides mostly employ single enzymatic hydrolysis or traditional extraction processes, resulting in low extraction efficiency, low purity of active ingredients, and weak anti-tumor activity, which cannot meet the needs of clinical applications.

[0004] Based on this, the present invention optimizes the extraction process and adopts stepwise enzymatic hydrolysis combined with separation and purification technology to extract a high-purity deer antler polypeptide from deer antler and verify its significant inhibitory activity against lung cancer cells. This solves the problems of unclear anti-tumor activity of deer antler polypeptide and imperfect extraction process in the prior art, and provides new technical support for the research and development of anti-tumor drugs. Summary of the Invention

[0005] To solve the above-mentioned technical problems, the technical solution of the present invention is implemented as follows: This invention provides a deer antler polypeptide with anti-tumor effects, the amino acid sequence of which is shown in SEQ ID NO:1, namely PADLAGNENCN.

[0006] Furthermore, the deer antler polypeptide is prepared by chemical solid-phase synthesis.

[0007] Furthermore, the deer antler polypeptide is obtained by extraction and separation from deer antler tissue.

[0008] The present invention also provides a method for preparing deer antler polypeptide with anti-tumor effects. The preparation method includes raw material pretreatment, two-step enzymatic hydrolysis, and separation and purification steps. The two-step enzymatic hydrolysis uses trypsin and neutral protease, respectively.

[0009] Further, the first enzymatic hydrolysis step is as follows: mix deer antler powder with deionized water at a mass-to-volume ratio of 1:20 (g / mL), adjust the pH to 7.5-8.0, add 1.5% of trypsin by mass of deer antler powder, and enzymatically hydrolyze for 3 hours at 50℃ and 180r / min. After enzymatic hydrolysis, inactivate the enzyme, centrifuge, and collect the supernatant.

[0010] Further, the second enzymatic hydrolysis step is as follows: adjust the pH of the supernatant collected in the first enzymatic hydrolysis step to 6.5-7.0, add 1.0% of neutral protease by weight of deer antler powder, and enzymatically hydrolyze for 2 hours at 45℃ and 180r / min. After enzymatic hydrolysis, inactivate the protease, centrifuge, and collect the supernatant.

[0011] The present invention also provides a pharmaceutical preparation containing the aforementioned deer antler polypeptide.

[0012] Furthermore, the pharmaceutical preparation also includes pharmaceutically acceptable excipients.

[0013] Furthermore, the deer antler polypeptide includes its pharmaceutically acceptable salt.

[0014] Further, the pharmaceutically acceptable salt includes at least one of the following: hydrochloride, sulfate, acetate, methanesulfonate, succinate, fumarate, citrate, malate, organic amine salt, phosphate, nitrate, carbonate, bicarbonate, tartrate, maleate, benzenesulfonate, p-toluenesulfonate, lactate, and gluconate.

[0015] Furthermore, the pharmaceutical formulation can be formulated into any pharmacologically acceptable dosage form.

[0016] Furthermore, the dosage form of the pharmaceutical preparation is selected from one or more of liquid preparations, solid preparations, and semi-solid preparations.

[0017] The present invention also provides the use of the aforementioned deer antler polypeptide or the aforementioned pharmaceutical preparation in the preparation of antitumor drugs.

[0018] Furthermore, the tumor is lung cancer.

[0019] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention employs a two-step synergistic enzymatic hydrolysis process using trypsin and neutral protease, followed by ultrafiltration, gel filtration chromatography, and HPLC purification to obtain a deer antler polypeptide with the amino acid sequence PADLAGNENCN (SEQ ID NO.1). In vitro experiments confirmed its dose-dependent inhibitory effect on A549 lung cancer cells, and nude mouse experiments demonstrated its significant in vivo antitumor effect, with an inhibition rate of 78.24% in the high-dose group. This polypeptide exhibits excellent stability, retaining ≥90% purity at room temperature, refrigeration, and high temperature, facilitating storage, transportation, and industrial application. Furthermore, it has fewer side effects and higher safety compared to chemical drugs, providing a new natural candidate drug and treatment strategy for the clinical treatment of lung cancer, possessing significant clinical application value and broad market prospects. Attached Figure Description

[0020] Figure 1 This is a predicted three-dimensional structure diagram of the deer antler polypeptide of the present invention. Detailed Implementation

[0021] Unless otherwise specified, the raw materials, reagents, or apparatus used in the following embodiments are all available from conventional commercial sources or can be obtained by existing known methods. Those skilled in the art should understand that the present invention can be practiced even without certain specific details. In other embodiments, methods, means, equipment, and steps well known to those skilled in the art have not been described in detail in order to highlight the spirit of the invention.

[0022] The experimental data of this invention were statistically analyzed using SPSS 26.0 software. The data are expressed as mean ± standard deviation (x ± s). The t-test was used for comparison between groups, and P < 0.05 indicated that the difference was statistically significant.

[0023] Example 1: Preparation of deer antler polypeptide A method for preparing deer antler polypeptide with anti-tumor effects, comprising the following steps: (1) Raw material pretreatment: Take fresh deer antler (legally purchased from Dongfeng County Sika Deer Antler Trading Market), rinse it with clean water, remove the skin and surface impurities, cut it into small pieces of 0.5-1cm, place it in a freeze dryer, freeze dry it for 12 hours at -50℃ and 0.01MPa. After drying, crush it with a pulverizer, pass it through an 80-mesh sieve, collect the deer antler powder, and place it in a desiccator for later use.

[0024] (2) First step of enzymatic hydrolysis: Weigh 100g of deer antler powder, place it in a 5000mL beaker, add 2000mL of deionized water, adjust the pH to 7.8 with 0.1mol / L NaOH solution, add 1.5g of trypsin (product number: T8003, Shanghai Yuanye Biotechnology Co., Ltd.) at 1.5% of the mass of deer antler powder, place it in a constant temperature water bath shaker, and enzymatically hydrolyze for 3h at 50℃ and 180r / min. After the enzymatic hydrolysis is completed, place the beaker in a 95℃ water bath and heat for 15min to inactivate trypsin. After naturally cooling to room temperature, transfer the mixture to a centrifuge tube, centrifuge at 8000r / min and 4℃ for 20min, collect the supernatant, discard the precipitate, and keep the supernatant for later use.

[0025] (3) Second step of enzymatic hydrolysis: Adjust the pH of the supernatant collected in the first step of enzymatic hydrolysis to 6.8 with 0.1 mol / L HCl solution, add 1.0 g of neutral protease (product number: N8002, Shanghai Yuanye Biotechnology Co., Ltd.), place it in a constant temperature water bath shaker, and enzymatically hydrolyze for 2 h at 45℃ and 180 r / min. After the enzymatic hydrolysis is completed, heat in a 95℃ water bath for 15 min to inactivate the neutral protease. After naturally cooling to room temperature, centrifuge at 8000 r / min and 4℃ for 20 min, collect the supernatant and discard the precipitate.

[0026] (4) Separation and purification: The supernatant collected from the second enzymatic hydrolysis was ultrafiltered through an ultrafiltration membrane with a molecular weight cutoff of 1500 Da (product number: UF3000, Millipore) at an ultrafiltration pressure of 0.1 MPa, and the permeate was collected. The permeate was purified by filtration chromatography through a Sephadex G-15 gel (product number: 17-0030-01, GE Healthcare) with a pH 7.4 0.01 mol / L PBS buffer, an elution flow rate of 1.0 mL / min, and a detection wavelength of 220 nm. The target elution peak solution was collected using a chromatography workstation.

[0027] (5) HPLC purification: The target elution peak solution was further purified by HPLC. The HPLC purification conditions were as follows: mobile phase A was 0.1% TFA-ultrapure water, mobile phase B was 0.1% TFA-acetonitrile, elution gradient: 0-15 min, mobile phase B volume fraction from 8% to 30%; 15-20 min, mobile phase B volume fraction from 30% to 70%; 20-35 min, mobile phase B volume fraction from 70% to 8%, column equilibration; flow rate was 1.0 mL / min, detection wavelength was 220 nm, column temperature was 30 ℃, injection volume was 20 μL, the eluent corresponding to the target absorption peak was collected and freeze-dried in a freeze dryer to obtain white powdered deer antler polypeptide. The amino acid sequence was PADLAGNENCN (SEQ ID NO.1, molecular weight 1.12 KD), and the purity was ≥98%.

[0028] (6) Structure prediction: The tertiary structure prediction tool PEP-FOLD (https: / / bioserv.rpbs.univ-paris-diderot.fr / services / PEP-FOLD3 / ) was used to predict the antler polypeptide of SEQ ID NO. 1. The prediction results are as follows: Figure 1 As shown, it exhibits a distinct spiral shape.

[0029] Example 2: Cytotoxicity detection of deer antler polypeptide Cell culture: After reviving normal human lung epithelial cells BEAS-2B (catalog number: THu177, Cell Bank of Chinese Academy of Sciences), they were seeded in DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin mixture (catalog number: 15140122, Gibco). The cells were cultured in a 37°C, 5% CO2 cell culture incubator. When the cell confluence reached 80%-90%, the cells were digested and passaged with trypsin-EDTA digestion solution (catalog number: 25200056, Gibco). Cells in the logarithmic growth phase were selected for subsequent experiments.

[0030] Grouping setup: After digesting BEAS-2B cells in logarithmic growth phase, the cell concentration was adjusted to 1×10⁻⁶ cells using DMEM medium. 5Cells / mL were seeded into 96-well plates, 100 μL per well. After culturing for 24 h, the cells were divided into 4 groups with 6 replicates per group. The specific groupings are as follows: (1) Blank control group: 100 μL of DMEM medium (without cells and drugs) was added; (2) Normal cell group: 100 μL of DMEM medium containing cells (without drugs) was added; (3) Low concentration of deer antler polypeptide group: 100 μL of medium containing deer antler polypeptide was added, with a final concentration of deer antler polypeptide of 50 μg / mL; (4) High concentration of deer antler polypeptide group: 100 μL of medium containing deer antler polypeptide was added, with a final concentration of deer antler polypeptide of 100 μg / mL.

[0031] Cell viability assay: After culturing cells for 48 hours, 10 μL of CCK-8 reagent was added to each well, and the cells were incubated for another 2 hours at 37℃ in a 5% CO2 incubator. The OD value of each well was measured at 450 nm using a microplate reader, and the cell viability was calculated using the following formula: Cell viability (%) = (OD value of drug-treated group - OD value of blank control group) / (OD value of normal cell group - OD value of blank control group) × 100% Experimental results: Table 1. Cytotoxicity test results of the deer antler polypeptide of the present invention

[0032] The toxicity of deer antler polypeptide to normal human lung epithelial cells BEAS-2B is shown in Table 1. Compared with the normal cell group, the cell survival rates of the low concentration group (50 μg / mL) and the high concentration group (100 μg / mL) of deer antler polypeptide were 98.96% and 97.01%, respectively, with no significant difference (P>0.05). This indicates that the deer antler polypeptide prepared in this invention has no obvious toxicity to normal lung epithelial cells and has good biocompatibility, providing a safety guarantee for its subsequent application in anti-tumor drugs.

[0033] Example 3: Stability detection of deer antler polypeptide Sample preparation: Take the deer antler polypeptide prepared in Example 1 and prepare a polypeptide solution with a concentration of 1 mg / mL using 0.01 mol / L PBS buffer at pH 7.4. Divide the solution into 3 groups, each with 3 samples, and perform the following stability treatments: (1) Room temperature stability: Place the sample in a 25℃ constant temperature incubator and take samples at 0, 7, 14, 21, and 28 days respectively; (2) Refrigeration stability: Place the sample in a 4℃ refrigerator and take samples at 0, 30, 60, and 90 days respectively; (3) High temperature stability: Place the sample in a 60℃ constant temperature incubator and take samples at 0, 2, 4, 6, and 8 days respectively.

[0034] Purity detection: The elution method of HPLC purification in Example 1 was used to detect the purity of the peptide solution sampled at each time point, and the purity retention rate was calculated using the following formula: Purity retention rate (%) = (Peptide purity at each time point / Initial purity) × 100%. If the purity retention rate is ≥ 90%, the peptide is considered to have good stability under these conditions.

[0035] Experimental results: Table 2. Stability test results of the deer antler polypeptide of the present invention

[0036] The stability test results of deer antler polypeptide under different conditions are shown in Table 2. Under normal temperature (25℃), the purity retention rate of deer antler polypeptide was 94.3% within 28 days, still ≥90%; under refrigeration (4℃), the purity retention rate was 96.7% within 90 days, showing good stability; under high temperature (60℃), the purity retention rate was 91.1% within 8 days, still maintaining high purity. This indicates that the deer antler polypeptide prepared by this invention has good stability, making it easy to store, transport, and use.

[0037] Example 4: Validation of the antitumor activity of deer antler polypeptide in human lung cancer cells A549 Cell culture: Human lung cancer cells A549 (catalog number: THu150, Chinese Academy of Sciences Cell Bank) were revived and cultured in a 37℃, 5% CO2 cell culture incubator. When the cell confluence reached 80%-90%, they were digested and passaged with trypsin-EDTA digestion solution. Cells in the logarithmic growth phase were selected for subsequent experiments.

[0038] Grouping setup: After digesting A549 cells in the logarithmic growth phase, the cell concentration was adjusted to 1×10⁻⁶ cells using DMEM medium. 5The cells / mL were seeded into 96-well plates, 100 μL per well. After culturing for 24 h, the plates were divided into 6 groups, with 6 replicates per group. The specific groupings are as follows: (1) Blank control group: 100 μL of the blank control group was added. (1) DMEM medium (without cells and drugs); (2) Model group: 100 μL of DMEM medium containing cells (without drugs) was added; (3) Positive control group 1 (cisplatin): 100 μL of medium containing cisplatin was added, with a final concentration of 5 μg / mL (product number: P4394, Sigma-Aldrich); (4) Positive control group 2 (paclitaxel): 100 μL of medium containing paclitaxel was added, with a final concentration of 10 μg / mL (product number: T7191, Sigma-Aldrich); (5) Low-dose deer antler polypeptide group: 100 μL of medium containing deer antler polypeptide prepared in Example 1 was added, with a final concentration of 50 μg / mL; (6) High-dose deer antler polypeptide group: 100 μL of medium containing deer antler polypeptide prepared in Example 1 was added, with a final concentration of 100 μg / mL.

[0039] Cell proliferation inhibition rate assay: After culturing cells for 48 h, 10 μL of CCK-8 reagent was added to each well, and the cells were incubated for another 2 h at 37℃ in a 5% CO2 incubator. The OD value of each well was measured at 450 nm using a microplate reader, and the cell proliferation inhibition rate was calculated using the following formula: Cell proliferation inhibition rate (%) = (OD value of model group - OD value of drug treatment group) / (OD value of model group - OD value of blank control group) × 100%.

[0040] Experimental results: Table 3. Results of the antitumor activity of the deer antler polypeptide of the present invention against human lung cancer cells A549.

[0041] The results of the inhibitory effect of deer antler polypeptide on the proliferation of human lung cancer cells A549 are shown in Table 3. Compared with the model group, the two positive control groups, the low-dose group and the high-dose group of deer antler polypeptide can significantly inhibit the proliferation of A549 cells. Among them, the cell proliferation inhibition rate of the high-dose group of deer antler polypeptide reached 79.56%, which was better than that of positive control group 2 (paclitaxel) and close to that of positive control group 1 (cisplatin). It also showed obvious dose dependence, indicating that the deer antler polypeptide prepared in this invention has significant anti-lung cancer activity, and the higher the dose, the better the inhibitory effect.

[0042] Example 5: Animal model antitumor test of deer antler polypeptide Animal source: Specific pathogen-free (SPF) female BALB / c nude mice, 6 weeks old, with a body mass of 18-20 g, were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., with the use license number SCXK (Jing) 2021-0011. The experiment was started after 1 week of adaptive feeding: the temperature was 22-25°C, the humidity was 50%-60%, the circadian rhythm was 12h / 12h, with sterile feed and drinking water.

[0043] Construction of nude mouse xenograft lung cancer model: Human lung cancer A549 cells in logarithmic growth phase were digested with trypsin-EDTA digest, the cells were collected by centrifugation, washed twice with PBS buffer, and the cell concentration was adjusted to 1×10 7 cells / mL, and set aside for later use. After 1 week of adaptive feeding of BALB / c nude mice, the right axillary region of the nude mice was disinfected, and 0.1 mL of cell suspension (containing 1×10 6 A549 cells) was drawn with a 1 mL sterile syringe and inoculated via subcutaneous injection. After inoculation, the nude mice continued to be fed, and their mental state, diet and the growth of the xenograft tumor were observed daily. When the volume of the xenograft tumor reached 80-100 mm 3 , the model was considered successfully constructed, and the nude mice with successfully constructed model were selected for subsequent experiments.

[0044] Grouping settings: The successfully modeled nude mice were randomly divided into 5 groups with 6 mice in each group. The specific grouping is as follows: (1) Blank control group: no tumor inoculation, intraperitoneal injection of normal saline, 0.2 mL per mouse each time, once a day, continuous administration for 14 days; (2) Model control group: inoculated with lung cancer xenograft, intraperitoneal injection of normal saline, 0.2 mL per mouse each time, once a day, continuous administration for 14 days; (3) Positive control group (cisplatin): inoculated with lung cancer xenograft, intraperitoneal injection of cisplatin solution, the final concentration of cisplatin was 5 mg / kg, 0.2 mL per mouse each time, once a day, continuous administration for 14 days; (4) Low-dose velvet antler polypeptide group: inoculated with lung cancer xenograft, intraperitoneal injection of velvet antler polypeptide solution, the final concentration of velvet antler polypeptide was 50 mg / kg, 0.2 mL per mouse each time, once a day, continuous administration for 14 days; (5) High-dose velvet antler polypeptide group: inoculated with lung cancer xenograft, intraperitoneal injection of velvet antler polypeptide solution, the final concentration of velvet antler polypeptide was 100 mg / kg, 0.2 mL per mouse each time, once a day, continuous administration for 14 days.

[0045] Index detection: During the administration period, the body weight change of the nude mice was recorded daily; the long diameter (a) and short diameter (b) of the xenograft tumor were measured with a vernier caliper every 3 days, and the tumor volume was calculated with the following formula: tumor volume (mm 3 ) = (a × b 214 days after administration, nude mice were euthanized by cervical dislocation, and the transplanted tumors were removed (the blank control group had no transplanted tumors and did not require removal). The tumor weight was measured using an electronic balance, and the tumor inhibition rate was calculated using the following formula: Tumor inhibition rate (%) = (average tumor weight of the model control group - average tumor weight of the administered group) / average tumor weight of the model control group × 100%. Simultaneously, the morphology of the nude mice's organs (heart, liver, spleen, lungs, and kidneys) was observed to preliminarily assess drug toxicity.

[0046] Experimental results: Table 4. Effect of the deer antler polypeptide of the present invention on the tumor inhibition rate in mice.

[0047] Note: Compared with the blank control group, #p<0.05, ##p<0.01; compared with the model control group, *p<0.05, **p<0.01.

[0048] During the administration period, the weight of nude mice in the blank control group steadily increased, their mental state was good, and their diet was normal; the weight of nude mice in the model control group gradually decreased, their mental state was lethargic, and their food intake decreased. After 14 days of administration, the results of tumor volume and weight and tumor inhibition rate of each group of nude mice are shown in Table 4. No tumor growth was observed in the blank control group, which was significantly different from the model control group (P<0.01), confirming the successful establishment of the nude mouse lung cancer xenograft model. Compared with the model control group, the positive control group, the low-dose group and the high-dose group of deer antler polypeptide all significantly reduced the volume and weight of the xenograft (P<0.01). Among them, the tumor inhibition rate of the high-dose group of deer antler polypeptide reached 78.24%, which is close to the 80.15% of the positive control group (cisplatin). This indicates that the deer antler polypeptide prepared in this invention has significant anti-tumor effects in animals, can effectively inhibit the growth of lung cancer xenografts in nude mice, and shows obvious dose dependence.

[0049] The basic principles, main features, and advantages of this invention have been described above. Those skilled in the art should understand that this invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely principles of the invention. Various changes and modifications can be made to this invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the invention as claimed. The scope of protection claimed by this invention is defined by the appended claims and their equivalents.

Claims

1. A deer antler polypeptide with anti-tumor effects, characterized in that, The amino acid sequence of the deer antler polypeptide is shown in SEQ ID NO:

1.

2. A pharmaceutical preparation, characterized in that, The pharmaceutical preparation contains the deer antler polypeptide as described in claim 1.

3. The pharmaceutical preparation according to claim 2, characterized in that, The pharmaceutical preparation also includes pharmaceutically acceptable excipients.

4. The pharmaceutical preparation according to claim 2, characterized in that, The deer antler polypeptide includes its pharmaceutically acceptable salt.

5. The pharmaceutical preparation according to claim 4, characterized in that, The pharmaceutically acceptable salts include at least one of the following: hydrochloride, sulfate, acetate, methanesulfonate, succinate, fumarate, citrate, malate, organic amine salt, phosphate, nitrate, carbonate, bicarbonate, tartrate, maleate, benzenesulfonate, p-toluenesulfonate, lactate, and gluconate.

6. The pharmaceutical preparation according to claim 2, characterized in that, The pharmaceutical preparation can be formulated into any pharmacologically acceptable dosage form.

7. The pharmaceutical preparation according to claim 6, characterized in that, The dosage form of the pharmaceutical preparation is selected from one or more of liquid preparations, solid preparations, and semi-solid preparations.

8. Use of the deer antler polypeptide of claim 1 or the pharmaceutical preparation of any one of claims 2-7 in the preparation of an antitumor drug, wherein the tumor is lung cancer.

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