A ginseng oligopeptide with anti-tumor effects and its preparation method

Abstract

This invention provides a ginseng oligopeptide with anti-tumor effects and its preparation method. The ginseng oligopeptide is obtained by enzymatic hydrolysis of ginseng protein using a dual-enzyme approach. Further screening and identification of the oligopeptide yielded an Rs-A-2 active anticancer peptide, which exhibits good inhibitory effects on cancer cell proliferation. Animal experiments have confirmed that the oligopeptide and / or anticancer peptide can effectively reduce the expression of target epigenes and inhibit tumor growth in mice. The anticancer peptide also helps improve the immune function of tumor-bearing mice. The oligopeptide prepared into a drug according to this invention has good anti-tumor effects and broad application prospects.

Description

Technical Field

[0001] This application relates to the field of biology, specifically to a ginseng oligopeptide with anti-tumor effects and its preparation method. Background Technology

[0002] Lung adenocarcinoma is a type of lung cancer, belonging to the non-small cell lung cancer category. Unlike squamous cell lung cancer, lung adenocarcinoma is more likely to occur in women and non-smokers. It originates from the bronchial mucosal epithelium, and a few originate from the mucous glands of the large bronchi. Its incidence is lower than squamous cell carcinoma and undifferentiated carcinoma, and it occurs at a younger age, with a relatively higher incidence in women. Most adenocarcinomas originate from smaller bronchi, making them peripheral lung cancers. Early stages generally lack obvious clinical symptoms and are often discovered during chest X-ray examinations. It presents as a round or oval mass, generally growing slowly, but sometimes hematogenous metastasis occurs early. Lymphatic metastasis occurs later. Adenocarcinoma is a malignant tumor of glandular epithelium, and can exhibit acinar, papillary, bronchioloalveolar, or solid growth patterns. It is often accompanied by mucus production, and the detection of mucus requires special staining, especially in poorly differentiated tumors. Mucus detection can sometimes differentiate solid adenocarcinoma from other morphologically similar large cell carcinomas. Early treatment of lung adenocarcinoma generally involves surgical resection, which can be divided into radical surgery and palliative surgery. Although surgery is one of the effective treatment methods for lung cancer, with radical resection having a high cure rate and effectively prolonging survival, it not only causes significant trauma to patients but also has very limited indications, often only treating the symptoms and not the root cause.

[0003] Studies have found that the occurrence and development of tumors are closely related to the body's overall health. Normal immune function is a major factor in the body's fight against disease and recovery. With the development of biochemistry, various peptides that inhibit or kill tumor cells have been discovered. These peptides can also enhance their anti-tumor effects by activating tumor antigen peptides that activate the body's own immune function, stimulating macrophage phagocytosis, or promoting lymphocyte proliferation. Oligopeptides, due to their low cost, ease of preparation, and lack of biological hazards, are increasingly being used in the anti-tumor field and show promising application prospects.

[0004] Ginseng is a precious medicinal herb with a history of medicinal use spanning thousands of years. Modern medicine, after experimental research, believes that ginseng possesses anti-cancer properties. Using a culture medium containing 20 mg / ml or 100 mg / ml of ginsenosides, transplanted Morris liver cancer cells from rats were cultured. After 25 passages, morphological changes were observed in the Morris liver cancer cells, clearly distinguishing them from normal liver cancer cells. Microscopic examination revealed a single layer of dense cells with clear intercellular spaces, exhibiting a typical epithelial cell shape and resembling normal liver cells. This indicates that the liver cancer cells had undergone "reversal." After another 300 passages, all Morris liver cancer cells showed "reversal," and no further Morris liver cancer cells were detected under microscopic examination. This effect of ginsenosides may bring hope for the treatment of liver cancer using ginseng. Pharmacological studies have shown that ginseng can promote the biosynthesis and metabolism of bone marrow nucleic acids and proteins, increase the proliferation of bone marrow cells, and raise the white blood cell count in cancer patients. Animal experiments have shown that ginseng stem and leaf saponins can significantly increase the number of nucleated cells in bone marrow, indicating that ginseng stem and leaf saponins can stimulate bone marrow hematopoiesis and have a significant effect on increasing white blood cell count. However, currently, there is not enough research on the use of ginseng polypeptides for anti-tumor purposes, especially on oligopeptides, which have gradually gained attention in recent years. Summary of the Invention

[0005] This invention provides a ginseng oligopeptide, which has significant anti-tumor effects.

[0006] On the one hand, the present invention provides a ginseng oligopeptide, which is isolated from Northeast ginseng and prepared by double enzymatic hydrolysis of total protein.

[0007] On the other hand, further screening and identification of ginseng oligopeptides yielded the active anticancer peptide Rs-A-2, whose amino acid sequence is FHAQDM.

[0008] The anticancer peptides can also be modified to prolong their biological activity or half-life.

[0009] The term "extension of half-life" refers to a drug that is optionally covalently linked ("conjugated" or "fused") to a modified polypeptide described herein via a linker, either directly or via a non-naturally encoded amino acid.

[0010] A chemically acceptable portion, domain, or molecule that, compared to a comparative agent such as an unconjugated modified peptide or a wild-type peptide, prevents or slows down in vivo protein degradation or other chemically degrading effects of the modified peptide.

[0011] Modifications, increasing half-life and

[0012] / or improve or alter other pharmacokinetic or biophysical properties, including but not limited to increasing absorption rate, reducing toxicity, improving solubility, reducing protein aggregation, increasing bioactivity and / or target selectivity of the modified peptide, increasing processability and / or reducing the immunogenicity of the modified peptide. The term "half-life extended portion" includes non-protein half-life extended portions, such as water-soluble polymers, such as polyethylene glycol (PEG) or discrete PEG, hydroxyethyl starch (HES), lipids, branched or unbranched acyl groups, branched or unbranched C8-C30 acyl groups, branched or unbranched alkyl groups and branched or unbranched C8-C30 alkyl groups; and protein half-life extended portions, such as serum albumin, transferrin, Adnectin (e.g., albumin-bound or pharmacokinetically extended (PKE) Adnectin), Fc domains and unstructured peptides, such as XTEN and PAS peptides (e.g., conformationally disordered peptide sequences composed of amino acids Pro, Ala and / or Ser), and fragments of any of the foregoing.

[0013] Some amino acid residues of a polypeptide have side chains that can be fully or partially close to the solvent. The side chains of non-naturally encoded amino acids at these positions can point away from the protein surface and into the solvent, thereby attaching to, for example, water-soluble polymers.

[0014] Furthermore, the present invention also provides the use of the active anticancer peptide Rs-A-2 in the preparation of a drug for antitumor purposes.

[0015] The tumors mentioned. Non-limiting examples of cancer include: acute lymphoblastic leukemia, acute myeloid leukemia, adrenocortical carcinoma, HIV-related cancers, HIV-related lymphomas, anal cancer, appendiceal cancer, astrocytoma, neuroblastoma, basal cell carcinoma, bile duct cancer, bladder cancer, bone cancer, brain tumors (such as cerebellar astrocytoma, cerebral astrocytoma / malignant glioma, ependymoma, medulloblastoma, supratentorial primitive neuroectodermal tumors, visual pathway and hypothalamic gliomas), breast cancer, bronchial adenoma, Burkitt lymphoma, cancer of unknown primary origin, central nervous system lymphoma, cerebellar astrocytoma, cervical cancer. Cancer, childhood cancer, chronic lymphocytic leukemia, chronic myeloid leukemia, chronic myeloproliferative disorders, colon cancer, cutaneous T-cell lymphoma, connective tissue proliferative small round cell tumor, endometrial cancer, ependymoma, esophageal cancer, Ewing sarcoma, germ cell tumor, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumors, gastrointestinal stromal tumors, glioma, hairy cell leukemia, head and neck cancer, heart disease, hepatocellular carcinoma, Hodgkin's lymphoma, hypopharyngeal cancer, intraocular melanoma, islet cell carcinoma, Kaposi's sarcoma, kidney cancer, laryngeal cancer, lip and oral cancer, liposarcoma, liver cancer, lung cancer (such as non-small cell and small cell lung cancer) Lymphoma, leukemia, macroglobulinemia, malignant fibrous histiocytoma / osteosarcoma, medulloblastoma, melanoma, mesothelioma, occult primary metastatic squamous neck cancer, oral cancer, multiple endocrine neoplasia syndrome, myelodysplastic syndrome, myeloid leukemia, nasal cavity and sinus cancer, nasopharyngeal carcinoma, neuroblastoma, non-Hodgkin lymphoma, non-small cell lung cancer, oral cancer, oropharyngeal cancer, osteosarcoma / malignant fibrous histiocytoma, ovarian cancer, ovarian epithelial cancer, ovarian germ cell tumor, pancreatic cancer, pancreatic islet cell carcinoma, sinus and nasal cavity cancer, parathyroid carcinoma, penile cancer, nasopharyngeal carcinoma, pheochromocytoma Tumors, pineal astrocytoma, pineal germ cell tumor, pituitary adenoma, pleural pulmonary blastoma, plasma cell tumor, primary central nervous system lymphoma, prostate cancer, rectal cancer, renal cell carcinoma, transitional cell carcinoma of the renal pelvis and ureter, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma, skin cancer, Merkel cell skin cancer, small intestine cancer, soft tissue sarcoma, squamous cell carcinoma, gastric cancer, T-cell lymphoma, laryngeal cancer, thymoma, thymic carcinoma, thyroid cancer, trophoblastic tumor (pregnancy), cancer of unknown primary site, urethral cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenström macroglobulinemia, and nephroblastoma.

[0016] Furthermore, the drug also contains a pharmaceutically acceptable carrier or excipient.

[0017] This invention also relates to pharmaceutical compositions comprising: an ionic complex comprising: a cationic polypeptide; an anionic excipient selected from: PEG-carboxylic acid; a fatty acid having 10 or more carbon atoms; anionic phospholipids; combinations thereof; and a pharmaceutically acceptable carrier. In one embodiment, the pharmaceutical composition further comprises additional excipients as described below (e.g., carboxymethyl cellulose (CMC)). For example, any anionic phospholipids described herein can be combined with carboxymethyl cellulose (CMC), such as a combination of mPEG-2000-DSPE and CMC.

[0018] The concentration ratio of the cationic peptide to the anionic excipient is determined based on the molar ratio of the cationic charge in the peptide to the charge of the anionic excipient. For example, the amount of anionic excipient relative to one positive charge of the peptide can be 1:1 to 1:10. The proportion of anionic excipient can be adjusted accordingly for any additional positive charge in the peptide. By changing the amount of anionic excipient within this ratio, the in vivo release characteristics of the peptide can be modulated. Higher proportions typically produce compositions that provide a slower release of the peptide from the site of application compared to lower proportions.

[0019] The term "pharmaceutically acceptable carrier" in the pharmaceutical compositions of this invention refers to a biocompatible polar liquid. The polarity of the liquid helps maintain the ionic form of the complex. Biocompatible polar liquids include, but are not limited to, PEG (polyethylene glycol, e.g., polyethylene glycol having an average molecular weight of 100-5000), polyols (e.g., propylene glycol (PG), tripropylene glycol, glycerol), ethanol, benzyl alcohol, DMSO, NMP, DMF, water, pH buffer solutions, and mixtures thereof. It should be understood that additional diluents and excipients, as described below, may be included in the pharmaceutical compositions.

[0020] In some embodiments, the pharmaceutical compositions of the present invention form a drug reservoir upon injection into an individual. In some embodiments, the drug reservoir releases an active compound over time after injection into the individual. In one aspect, at least a portion of the pharmaceutical composition precipitates to form a drug reservoir and releases a pharmacologically active compound over time upon injection into the individual. In some embodiments, the compositions of the present invention are contemplated for high concentrations of peptides suitable for generating a drug reservoir in vivo for sustained-release steady-state therapeutically effective levels. Examples of peptide concentration ranges in the formulation are about 0.0001 mg / mL to about 100 mg / mL.

[0021] The pharmaceutical compositions of the present invention may include additional excipients (also referred to herein as co-excipients). Suitable examples of additional excipients include pH-stabilizing buffers, preservatives, surfactants, stabilizers, antioxidants, tensioning agents, and ionic and nonionic polymers as defined herein.

[0022] These types of excipients can be added to help form homogeneous suspensions and dispersions of ionic complexes, and even hydrophobic anionic excipients, such as lipids or fatty acids. Types of excipients include dispersants and emulsifiers, such as lecithin, soybean oil, castor oil, migliol, polyethylene glycol (MW range 200-5,000), methylcellulose, and carboxymethylcellulose.

[0023] As used herein, the term "surfactant" refers to a surface-active agent or substance that tends to reduce the surface tension of a liquid in which it is dissolved. Suitable surfactants include polysorbates, poloxamers, tritons, sodium lauryl sulfate, sodium lauryl sulfate, and betaine. For example, surfactants include polyoxyethylene (20) lauryl sorbitan (20, e.g. from Sigma-Aldrich), polyoxyethylene (20) palmitoleitan (40), polyoxyethylene (20) dehydrated sorbitol monooleate (80), poloxamer 188, polyoxyethylene-polyoxypropylene block copolymer (F-68, e.g. from Sigma-Aldrich), polyethylene glycol 660-12-hydroxystearate (HS 15, BASF), cocamidopropyl betaine, linoleyl betaine, myristyl betaine, cetyl betaine, polyethoxylated castor oil (currently Kolliphor BASF), and lecithin.

[0024] As used herein, the term "tonic agent" refers to a substance used to adjust the osmotic pressure of a formulation. Tense generally refers to the osmotic pressure of a solution relative to human serum. Formulations can be hypotonic, isotonic, or hypertonic. Formulations are typically preferred to be isotonic. An isotonic formulation is a liquid or a liquid reconstituted from a solid form, such as a lyophilized form, and indicates a solution having the same osmotic pressure as some other solution in contrast, such as physiological saline and serum. Tense agents can help reduce pain and irritation during injection. Suitable tense agents include glucose, glycerol, hydroxyethyl starch, lactose, mannitol (e.g., D-mannitol), raffinose, sorbitol, sucrose, trehalose, sodium chloride, calcium chloride, magnesium chloride, and potassium chloride.

[0025] As used herein, the term "buffer" refers to an excipient that stabilizes the pH of a pharmaceutical composition. Suitable buffers are well known in the art and can be found in the literature. Examples of suitable buffers include histidine-buffers, citrate-buffers, succinate-buffers, acetate-buffers, and phosphate-buffers, or mixtures thereof. The most preferred buffer comprises citrate, L-histidine, or a mixture of L-histidine and L-histidine hydrochloride. Another preferred buffer is a citrate buffer. Independent of the buffer used, the pH can be adjusted using acids or bases known in the art, such as hydrochloric acid, acetic acid, phosphoric acid, sulfuric acid, citric acid, sodium hydroxide, and potassium hydroxide.

[0026] Ionic polymers suitable for use as additional excipients include ionic carboxymethyl cellulose (CMC), hyaluronic acid, poly(glutamic acid), poly(aspartic acid), poly(glutamic acid-co-glycine), poly(aspartic acid-co-glycine), poly(glutamic acid-co-alanine), poly(aspartic acid-co-alanine), sodium glycolate starch, polygalacturonic acid, poly(acrylic acid), carrageenan, and alginate.

[0027] Furthermore, the present invention also provides the use of ginseng oligopeptide and / or active anticancer peptide Rs-A-2 in the preparation of pharmaceutical compositions for the treatment of lung adenocarcinoma.

[0028] Specifically, the pharmaceutical composition also contains other therapeutic agents for combined use.

[0029] Specifically, the other therapeutic agents may be selumetinib, ifosfamide, chlorambucil, busulfan, melphalan, nitrogen mustard, uramustine, thiotepa, nitrosoureas, nitrogen mustards (e.g., nitrosoureas, nitrosoureas, nitrosoureas, nitroso, nitrogen mustard, cyclophosphamide, chlorambucil, melphalan), ethyleneimine and methylmelamine (e.g., hexamethylmelamine, thiotepa), alkyl sulfonates (e.g., busulfan), nitrosoureas (e.g., carmustine, lomomex). Stimulosin, semustine, streptozoline, triazine (ampicillin), antimetabolites (e.g., 5-thiazolinone, leucovorin, capecitabine, fludarabine, gemcitabine, pemetrexed, raltitrexed, folic acid analogs (e.g., methotrexate) or pyrimidine analogs (e.g., fluorouracil, fluorouracil, cytarabine), purine analogs (e.g., mercaptopurine, thioguanine, pentostatin), etc.), phytoalkaloids (e.g., vincristine, vinorelbine, vindesine, podophyllotoxin). Antibiotics include: paclitaxel, docetaxel, topoisomerase inhibitors (e.g., irinotecan, topotecan, acridine, etoposide (VP16), etoposide phosphate, teniposide, etc.), antitumor antibiotics (e.g., doxorubicin, daunorubicin, epirubicin, actinomycin, bleomycin, mitomycin, mitoxantrone, procainoxic acid, etc.), platinum-based compounds (e.g., cisplatin, oxaloplatin, carboplatin), anthraquinones (e.g., mitoxantrone), substituted ureas (e.g., hydroxyurea), and methyl... Hydrazine derivatives (e.g., procarbazine), adrenocortical inhibitors (e.g., mitotane, aminoglutethimide), epipodophyllotoxin (e.g., etoposide), antibiotics (e.g., daunorubicin, doxorubicin, bleomycin), enzymes (e.g., L-asparaginase), mitogen-activated protein kinase signaling inhibitors, 5-aza-2'-deoxycytidine, all-trans retinoic acid, doxorubicin, vincristine, etoposide, gemcitabine, imatinib (Gleevec.RTM.). Gerdemycin, 17-N-allylamino-17-demethoxygerdemycin (17-AAG), flavonoids, LY294002, bortezomib, trastuzumab, BAY 11-7082, PKC412, PD184352, 20-EPI-1, 25-dihydroxyvitamin D3; 5-ethynyluracil; Abiraterone; Ararubicin; Acylfullerene; Adecilpine; Adozelecin; Aldehyde-interleukin; ALL-TK antagonist; Hexamethylmelamine; Ambastine; Amidox; Amifostine; Aminolevulinic acid; Ararubicin; Acridine; Anagrelide; Anastrozole; Andrographolide; Angiogenesis inhibitor; Antagonist D; Antagonist G; Atalactone; Anti-dorsal morphogenetic protein-1; Anti-androgen, prostate cancer; Anti-estrogen; Anti-tumor ketone; Antisense oligonucleotide; Glycine aphthylglycerol ester; Apoptosis gene regulator; Apoptosis regulator; Depurine acid; ara-CDP-DL-PTBA; Arginine deaminase; Asolacline; Atamitan; Atramustine;Acilostatin 1; Acilostatin 2; Acilostatin 3; Azasetron; Azazotoxin; Azacyclobutane; Gibberellin III derivative; Barano; Palmastat; BCR / ABL antagonist; Benzodihydroporphyrin; Benzoyl astrosaponin; β-lactam derivative; β-alanine ethyl ester; Betamycin B; Betulinic acid; bFGF inhibitor; Bicalutamide; Bisantren; Diacrylylspermine; Diazepam; Distratene A; Breflate; brompirimidine; budesonide; sulfadiazine; calcipotriol; calmodulin C; camptothecin derivative; canarypox IL-2; capecitabine; formamide-amino-triazole; carboxamidotriazole; carboxylate M3; cahn 700; chondrodermal inhibitor; cazilexin; casein kinase inhibitor (ICOS); spermine; cecropin B; cetrolec; dihydroporphyrin; chloroquine sulfonamide; cisporin; cladribine; clomiphene analogue; clotrimazole; quasi-directed mycin A; cristatin B; cobustatin A4; cobustatin analogue; conagenin; crambescidin 816; cristatol; cryptophytin 8; cryptophytin A derivative; curacin A; cyclopentafenone; cycloplatin; cypemycin; ocfosfate; cytolysin; cytosolic kinase; daliximab; decitabine; dehydrodemnin B; desclorelin; dexamethasone; desiphosphamide; dextrozosen; dexverapamil; deazigonone; didemnin B; Didox; Diethylnorsemine; Dihydro-5-azacytidine; 9-Dioxomycin; Diphenylspiraquinone; Docosanol; Dolasenolide; Deoxyfluorouridine; Droloxifen; Drocannabinol; Becamycin SA; Ebusilon; Ecomustine; Edifosine; Edrecolomab; Eflunomide; Elimexene; Emitiflu; Epirubicin; Epristeride; Estrogen agonists; Estrogen antagonists; Ethamidazole; Etoposide phosphate; Exemestane; Fatrazol; Fazalabin; Fenivel Aamine; Filgrastin; Finasteride; Flavopiridol; Flezelastine; Flustarabone; Fludarabine; Fluoride hydrochloride Daunorubicin; Fofenimide; Formexostriecin; Formustine; Texacosafidazolium; Gallium nitrate; Galotetabine; Ganirelix; Gelatinase inhibitor; Gemcitabine; Glutathione inhibitor; Heptanylsulfamic acid; Heregulin; Hexamethylene diacetamide; Hypericin; Ibandronic acid; Idarubicin; Idroxifene: Idramantone; Imofosine; Ilostatin; Imidazolidine; Imiquimod; Immunostimulatory peptide; Insulin-like growth factor-1 receptor inhibitor; Interferon agonist; Interferon; Interleukin; Iobenguane; Iodomycin; Ipomitol, 4-; Iroplact; Isoprazole; Isohohalogenated hydrocarbon B;Itasetron; jasplakinolide; cahalalelide F; spirotin-N-triacetate; lanreotide; ranamycin; lenograstim; lentinan sulfate; letrostatin; letrozole; leukemia inhibitory factor; leukocyte alpha interferon; leuprorelin + estrogen + progesterone; leuprorelin; levamisole; liazole; linear polyamine analogs; lipophilic disaccharide peptides; lipophilic platinum compounds; lissoclinamide 7; Lobaplatin; Lombricin; Lometroxo; Clonidamine; Loxoanthraquinone; Lovastatin; Loxoribin; Letotken; Dixamethicone; Lysofylline; Lysolytic peptides: Matancin; Mannostatin A; Malimasitol; Masoprococcus; Maspine; Matrix lysozyme inhibitors; Matrix metalloproteinase inhibitors; Menogalil; Melbaron; Miterlin; Methioninase; Metoclopramide; MIF inhibitors; Mifepristone; Mitefocin; Mimo Stigmine; mismatched double-stranded RNA; mitotoxin; mitotoxin; mitomycin analogue; mitonafil; mitotic toxin fibroblast growth factor-saponin; mitotoxantrone; mofarotin; mogludec; monoclonal antibody, human chorionic gonadotropin; monophospholipid A+ Myobacterium cell wall SK; mupidamo; multidrug resistance gene inhibitor; therapies based on multiple tumor suppressor factor 1; mustard anticancer agent; mycaperoxide peroxide B; Mycobacterium cell wall extract; Myristyl alcohol; N-acetyldinalin; N-substituted benzamide; Nafarelin; Nagrestiprip; Naloxone + Pentazocine; Napaldavan; Naphthol; Natogstin; Nedaplatin; Nemorubicin; Neridronate; Neutral endopeptidase; Nilumet; Nissamycin; Nitric oxide regulator; Nitrogen oxide antioxidant; Nitrogstiprip; O6-benzylguanine: Octreotide; Oxylenone; Oligonucleotide; Ondansetron; Ondansetron; Oracin; Oral cytokine inducer; Omaplatin; Oxatazoline; Oxaliplatin; Oxanoxamycin; Palolamycin; Palmitoyl rhizobacterin; Pamidronate; Ginsenoside; Panomiphen; Parabastine; Pazliprin; Pegaspargase; Perdex; Sodium pentosan polysulfate; Pentostatin; Tebuconazole; Perflubr on; perforated cyclic amine; perillyl alcohol; phenazinamide; phenyl acetate; phosphatase inhibitor; picibanil; pilocarpine hydrochloride; pirarubicin; pyrithione; placebo A; placebo B; plasminogen activator inhibitor; platinum complex; platinum compound; platinum-triamine complex; porfiromycin sodium; prednisone; propyl bis-acridone; prostaglandin J2; proteasome inhibitor; protein A-based immunomodulator; protein kinase C inhibitor; protein kinase C inhibitor, microalgae; protein tyrosine phosphatase inhibitor; purine nucleoside phosphorylase inhibitor; purpuricin; pyrazoloacridin; pyridoxal-aldated hemoglobin polyoxyethylene conjugate; RAF antagonist; raltitrexed; ramosetron; ras formyl protein transferase inhibitor; ras inhibitor;Ras-GAP inhibitors; demethylated retiritin; rhenium Re186 etidronate; rhizobacterium; ribozyme; RII retinamide; rosiglitazone; roxithromycin; roxithromycin; roxithromycin; rubiginone Bl; ruboxyl; safmgol; ceintopin; sacinoxetine; inositol A; saxaglastine; Sdi 1 mimic; semustine; senescence-derived inhibitor 1; sense oligonucleotides; signal transduction inhibitors; signal transduction modulators; single-chain antigen-binding proteins; xizofuran; sobutol; sodium borooctanoate; sodium phenylacetate; solverol; phytostimulant-binding protein; sonamin; sparphosphamide; spicamycin D; spiromostatin; spongistatin 1; squalamine; stem cell inhibitors; stem cell division inhibitors; stepiramide; stromely Sin inhibitors; sulfinosin; potent vasoactive intestinal peptide antagonists; suradista; suramin; sorghum extract; synthetic glycosaminoglycans; tamustine; tamoxifen methyliodide; tauromustine; tazarotene; tecogalenol sodium; tegafur; tellurium; telomerase inhibitors; temopofol; temozolomide; teniposide; tetrachlorodecoxide; tetrazolomide; thaliblastine; thiocralidine; thrombopoietin; thrombopoietin mimics; thymosin; thymopoietin receptor agonists; thymosin; thyroid-stimulating hormone; Ethylxanthinetin; Tirazamine; Dichlorodicarboxylate; Topsentin; Toremifene; Totipotent stem cell factor; Translation inhibitor; Retinoic acid; Triacetyluridine; Triceribenzylbenzyl; Trimethoprim; Triptorelin; Tropanetron; Torost; Tyrosine kinase inhibitor; Tyrosine phosphorylase; UBC inhibitor; Ubenimex; Urogenital sinus-derived growth thromboplastin; Urokinase receptor antagonist; Vaportide; Mutant protein B; Vector system, erythrocyte gene therapy; Verlaresol; Vertrolidine; Fuldins; Vertebrate FM; Long Chunrebine; Vinpocetine; Vitaxin; Vorozolol; Zanotolone; Zeniplatin; Zirascob; Stimalamer (Nettostatin); Doxorubicin; Dermatomycin; Bleomycin; Vincristine; Cisplatin; Axivecin; Ararubicin; Acozole Hydrochloride; Adenosine; Adozelecin; Aldehyde Interleukin; Hexamethylmelamine; Ammoniac; Ametanzoline Acetate; Aminoglutamine; Acridil; Anastrozole; Anthramycin; Asparaginase; Aspergillus; Azacitidine; Azetepa; Nitrosaccharin; Bamasta; Benzodepa; Bicalutamide ; bisambroxol hydrochloride; diphenhydramine dimethsulfate; pyrazinamide; bleomycin sulfate; buquina sodium; brompirimidine; busulfan; cactinomycin; carotestosterone; caracemide; carbetastatin; carboplatin; carmustine; carrubicin hydrochloride; pyrazinamide; cefdifenol; chlorambucil; siromycin; cladribine; crissenasol mesylate; cyclophosphamide; cytarabine; dacarbazine; daunorubicin hydrochloride; decitabine; dextromethorphan; dezaguanine; dezaguanine mesylate; deaziquinone;Doxorubicin; Doxorubicin hydrochloride; Droloxifen; Droloxifen citrate; Dromustanone propionate; Bixazomycin; Edatraxa; Eflunomide hydrochloride; Elsamitrucin; Enloplatin; Enpromethazine; Epirubicin hydrochloride; Ibuprofen; Erubicin hydrochloride; Estrostimine; Estrostimine sodium phosphate; Ethamidazole; Etoposide; Etoposide phosphate; Etoprine; Fatrazole hydrochloride; Immunotoxins (e.g., anti-CD33 monoclonal antibody-gallic acid conjugate, anti-CD22 monoclonal antibody-pseudomonas exotoxin conjugate, etc.), Radioimmunotherapy (e.g., anti-CD20 monoclonal antibody conjugated to nickel, 90Y, or 131I, etc.), Tripterygium wilfordii, Homoharringtonine, Dermabramycin, Doxorubicin, Epirubicin, Topotecan, Itraconazole, Vinpocetine, Cerivastatin Tinidazole, vincristine, deoxyadenosine, sertraline, pitavastatin, irinotecan, clofazimine, 5-nonyloxyamine, vemurafenib, dabrafenib, erlotinib, gefitinib, EGFR inhibitors, epidermal growth factor receptor (EGFR) targeted therapy or therapeutic agents, cetuximab (Erbitux™), lapatinib (Tykerb™), panitumumab (Vectibix™), vandetanib (Caprelsa™), afatmib / BIBW2992, CI-1033 / canertinib, neratmib / HKI-272, CP-724714, TAK-285, AST-1306, ARRY334543, ARRY-380, AG-1478. Dacomitinib / PF299804, OSI-420 / desmethylerlotinib, sorafenib, imatinib, sunitinib, dasatinib, etc.

[0030] Beneficial effects

[0031] This invention provides a ginseng oligopeptide with anti-tumor effects and its preparation method. The ginseng oligopeptide is obtained by enzymatic hydrolysis of ginseng protein using a dual-enzyme approach. Further screening and identification of the oligopeptide yielded an Rs-A-2 active anticancer peptide, which exhibits good inhibitory effects on cancer cell proliferation. Animal experiments have confirmed that the oligopeptide and / or anticancer peptide can effectively reduce the expression of target epigenes and inhibit tumor growth in mice. The anticancer peptide also helps improve the immune function of tumor-bearing mice. The oligopeptide prepared into a drug according to this invention has good anti-tumor effects and broad application prospects. Attached Figure Description

[0032] Figure 1 Figure 1 shows the effect of ginseng oligopeptides on cancer cell survival.

[0033] Figure 2 Figure 1 shows the effect of each group on the expression level of EZH2 protein in tumor tissue. Detailed Implementation

[0034] Those skilled in the art can refer to the content of this document and appropriately improve the process parameters to achieve the desired results. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to implement and apply the technology of this invention. Unless otherwise specified, the methods, equipment, and materials in the following embodiments are all conventional methods, equipment, and materials in the art and are commercially available.

[0035] Example 1: Preparation of Ginseng Oligopeptides

[0036] Fresh Northeast ginseng was vacuum dried at below 50℃ until the moisture content was less than 5%, then pulverized and set aside. The dried ginseng powder was then subjected to supercritical CO2 extraction for defatting under conditions of 25 MPa pressure, 35℃ temperature, 100 min time, and 30 L / h CO2 flow rate. After extraction, the ginseng powder had a fat content of less than 0.1%. Subsequently, microwave treatment was performed at 600 W power for 10 min, a solid-liquid mass-to-volume ratio of 1:25, and a ginseng powder particle size of 0.125 mm to remove ginseng polysaccharides. The resulting crude protein residue was then dried and pulverized for later use.

[0037] The crude protein powder was adjusted to pH 1.6 with 1M hydrochloric acid, then placed in a constant temperature water bath and incubated at 37℃ for 8 min. Pepsin was then rapidly added at an enzyme-to-substrate ratio of 4000 U / g, and hydrolysis was carried out with shaking for 2.5 h. After hydrolysis, the protein was immediately inactivated at 100℃ for 10 min. After cooling to room temperature, the pH was adjusted to 8.5 with 1M sodium hydroxide solution, and trypsin was added at an enzyme-to-substrate ratio of 2000 U / g. Hydrolysis was carried out at 55℃ for 5 h. The hydrolysate was then inactivated in a 100℃ water bath for 10 min, centrifuged at 4000 rpm for 10 min, filtered through a 0.25 μm microfiltration membrane, and freeze-dried to obtain ginseng oligopeptides.

[0038] Example 2: Activity Verification of Ginseng Oligopeptides

[0039] Mouse Lewis lung cancer cells (catalog number Delf-10303, Wanwu Biotechnology) were routinely cultured in RPMI-1640 medium containing 10% fetal bovine serum and 1% penicillin and streptomycin in an incubator at 37°C with 5% CO2. Cells were passaged after digestion when they reached 80%–90% confluence with the bottom of the dish, and passaged every 2–3 days.

[0040] A549 cells in good growth condition were trypsinized and seeded into 96-well plates at a density of 5000 cells per well. After 12 hours, the cells were divided into a control group and groups treated with different concentrations of ginseng oligopeptides prepared in Example 1, at concentrations of 0, 50, 200, 500, and 1000 μg / mL, respectively. Each group had 5 replicates. After 48 hours of culture, 20 μL of MTT reagent (5 g / L) was added to each well, and the cells were cultured for another 4 hours. The precipitate was dissolved in DMSO, and the absorbance (A) of each well was measured at 490 nm using a microplate reader. The cell viability of each group was calculated. Cell viability = (Average A value of experimental wells / Average A value of control wells) × 100%. The results are as follows: Figure 1 As shown.

[0041] from Figure 1 The results showed that after 48 hours of treatment with different concentrations of ginseng oligopeptides, Lewis lung cancer cells showed varying degrees of inhibition of cell survival with increasing oligopeptide concentration, and the cell survival rate gradually decreased with increasing oligopeptide concentration (P<0.05). Compared with the control group, the cell survival rate of the 1000 μg / mL group was (52.95±2.54)%, showing the most significant decrease (P<0.01).

[0042] Example 2: Screening and Identification of Active Anticancer Peptides in Ginseng Oligopeptides

[0043] To more accurately identify the active anticancer peptides in ginseng oligopeptides, the ginseng oligopeptides prepared in Example 1 were separated and their structures identified using high-performance liquid chromatography-mass spectrometry (HPLC-MS / MS). The HPLC system used was a Thermo Fisher Scientific TMQ Exactive, with an Aquity UPLC BEH C18 column (1.7 μm particle size), and the mass spectrometer was a quadrupole Orbitrap mass spectrometer. The oligopeptides prepared in Example 1 were separated using HPLC-MS / MS, yielding mass spectra. The mass spectrometry data were imported into Peak Studio 8.5 software for de novo analysis, identifying 387 oligopeptide sequences with a confidence level of over 50%. The bioactivity of 17 oligopeptides with a confidence level greater than 95% was evaluated using the Peptide Ranker platform, yielding three oligopeptides with a score > 0.5: FHAQDM, CHKEHWE, and NVANPAG.

[0044] The inventors discovered that EZH2 is an effective target for the treatment of lung adenocarcinoma. Molecular docking was performed using HPEPDOCK. The human EZH2 protein sequence was first found in the PDB. Using the HPEPDOCK server, molecular docking was performed on three oligopeptides with a score > 0.5. The docking scores of the three oligopeptides are shown in Table 1.

[0045] ;

[0046] Table 1 shows that all three oligopeptides have high affinity for EZH2 protein (Docking Score < -100), with the Rs-A-2 peptide exhibiting the highest affinity (Docking Score = -168.4). The most active Rs-A-2 anticancer peptide was then synthesized artificially by Peptide Valley Biotechnology for future use.

[0047] Example 3 Animal experiments on Rs-A-2 active anticancer peptides

[0048] Lewis lung cancer cells in the logarithmic growth phase were digested and centrifuged. The serum-containing culture medium was discarded, and the cells were resuspended in PBS, adjusting the cell concentration to 5 × 10⁻⁶. 7 Cells / mL were prepared and placed on ice. The right side of the mice (Kunming SPF mice, female, 5-6 weeks old, weighing 18-22 g) was then shaved using electric clippers. The skin was disinfected with an alcohol swab and residual hair was removed. The injection point was selected at the subcutaneous site above the right buttock of the mouse. The syringe was advanced horizontally approximately 1 cm, and 150 μL of cell suspension was slowly injected. After rotating and withdrawing the needle, the injection point was pressed with a cotton swab for 2-3 seconds to prevent leakage. The mice were observed for changes in condition after inoculation. After 30 minutes, when all reactions were normal, they were returned to their cages for further experimentation. On day 6 after inoculation with Lewis lung cancer cells, when the subcutaneous xenograft volume was approximately 70-90 mm², the mice were grouped as follows for experiments: 2

[0049] Experimental animals were randomly divided into a normal control group, a tumor-bearing control group, a 25 mg / (kg∙d) cyclophosphamide positive control group, a 25 mg / kg∙d Example 1 oligopeptide group (oligopeptide group), a 10 mg / kg∙d Rs-A-2 active anticancer peptide group (anticancer peptide group), and a 25 mg / kg∙d Example 1 oligopeptide + 10 mg / kg∙d Rs-A-2 active anticancer peptide group (combination therapy group). Except for the normal control group, all other groups were vaccinated with the above-mentioned doses via gavage starting 6 days later. The normal control group was gavage with an equal volume of physiological saline for 9 consecutive days. On the 10th day, blood was collected from the orbital cavity, and the mice were euthanized by cervical dislocation. Tumors, thymus, and spleens were harvested, washed with physiological saline, blotted dry with filter paper, and weighed. The tumor inhibition rate, thymus index (mg thymus / g body weight), and spleen index (mg spleen weight / g body weight) were calculated. Tumor inhibition rate (%) = (average tumor weight of control group - average tumor weight of dosage group) / average tumor weight of control group × 100. The results are shown in Table 2:

[0050] ;

[0051] The results in Table 2 show that ginseng oligopeptides have a certain inhibitory effect on tumor growth, while the Rs-A-2 active anticancer peptide screened from the oligopeptides has a better inhibitory effect on cancer cell proliferation. Using the anticancer peptides and oligopeptides together can synergistically increase the inhibitory effect on cancer cell proliferation. Furthermore, the results based on the thymus index and spleen index indicate that the oligopeptides of this invention and the screened anticancer peptides are beneficial in improving the immune function of tumor-bearing mice.

[0052] Tumor tissues from mice in each group were extracted, homogenized in equal volumes, and total protein was extracted from the homogenates. Protein quantification was performed using the BCA method. Equal volumes of protein were separated by electrophoresis on a 10% SDS-PAGE gel (70V 30min, 110V 1h), transferred to a PVDF membrane (200mA, 2h), blocked with 5% industrial skim milk powder at room temperature for 2h, incubated overnight at 4℃ with primary antibody (GAPDH 1:50000, EZH2 1:1000), washed with TBST for 30min, and incubated at room temperature for 1h with HRP-labeled secondary antibody (HRP-goat anti-rabbit IgG 1:5000). The bands were then exposed and developed using enhanced chemiluminescence. Results are as follows: Figure 2 As shown.

[0053] from Figure 2 The results of Western blotting also showed that EZH2 in tumor tissue could be significantly downregulated in both the anticancer peptide and the combination therapy, thereby achieving the effect of treating tumors.

[0054] Although embodiments of the present invention have been described above in conjunction with the accompanying drawings, the present invention is not limited to the specific embodiments and application fields described above. The specific embodiments described above are merely illustrative and instructive, and not restrictive. Those skilled in the art can make many other forms based on the guidance of this specification and without departing from the scope of protection of the claims of the present invention, and all of these are within the scope of protection of the present invention.

Claims

1. A ginseng active anticancer peptide Rs-A-2, characterized in that... The amino acid sequence is FHAQDM.

2. Use of the ginseng active anticancer peptide Rs-A-2 as described in claim 1 in the preparation of a pharmaceutical composition for inhibiting the proliferation of lung adenocarcinoma cells.

3. The use as described in claim 2, wherein the pharmaceutical composition further contains ginseng oligopeptide.

4. The use as described in claim 3, wherein the ginseng oligopeptide is obtained by treating total ginseng protein with pepsin and trypsin.

5. The use as described in any one of claims 2-4, characterized in that... The pharmaceutical composition further contains a pharmaceutically acceptable carrier.

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