An effective site of animal medicine small molecule peptide and a preparation method and broad-spectrum anti-tumor application thereof

Abstract

The application discloses an effective part of animal medicine small-molecule peptide and a preparation method and broad-spectrum antitumor application thereof. The preparation method comprises the following steps: taking one or more of geckos, centipedes, scorpions, earthworms, leeches, turtle shells, deer horns, silkworms, cicadas, earthworms, and nine insects, and the like, performing water extraction, and performing ultrafiltration through a 2.5-3.5 kD ultrafiltration membrane, collecting an ultrafiltration permeate with a molecular weight less than 3 kD, and concentrating to obtain the effective part. The effective part is not limited to the animal medicine source, and also includes products with the same characteristics obtained by expression of a genetic engineering host cell (such as a plant cell or a microbial cell). The effective part has a high impurity and pyrogen removal rate, a small-molecule peptide enrichment rate of greater than or equal to 40%, a significant inhibitory effect on human lung cancer, liver cancer, gastric cancer, breast cancer and various solid tumor cells, an induction of tumor cell apoptosis, low toxicity to normal cells and good safety. The application has the advantages of simple process, green environmental protection, industrial production, and the like, is suitable for various sources, and provides a new material basis and production platform for development of broad-spectrum antitumor drugs.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to an effective fraction of a small molecule peptide with a molecular weight <3kD, isolated and purified from animal drugs, its preparation method, and its application in the preparation of broad-spectrum anti-tumor drugs. The effective fraction described in this invention includes, but is not limited to, animal drug extracts, as well as products with the same characteristics obtained through expression in genetically engineered host cells. Background Technology

[0002] Animal-based medicines are an important component of traditional Chinese medicine, characterized by their high activity and significant therapeutic effects. Centipedes, scorpions, earthworms, leeches, turtle shells, deer antlers, silkworm pupae, cicada molts, ground beetles, and nine-fragrant insects are commonly used in traditional medicine to treat ailments such as abdominal masses, arthralgia, and stroke. Modern research indicates that they contain abundant protein and polypeptide active ingredients, exhibiting various pharmacological activities including anti-tumor, anticoagulant, and immunomodulatory effects.

[0003] However, most existing animal drug extracts are crude extracts, with complex compositions, many impurities, and high pyrogens, making it difficult to meet the quality requirements of modern formulations, especially injections. Meanwhile, the active ingredients of different animal drugs are often concentrated in specific molecular weight ranges, especially small molecule peptides, which have characteristics such as well-defined activity, easy absorption, and low toxicity.

[0004] Ultrafiltration is a physical separation method based on molecular weight. Ultrafiltration membranes with a molecular weight cutoff of 3kD can accurately separate small molecule peptides with a molecular weight of <3kD, while removing impurities such as large molecule proteins, polysaccharides, and pyrogens. However, there are currently no reports on the application of 3kD ultrafiltration technology systems to the separation and purification of small molecule peptides from various animal drugs, and on verifying their broad-spectrum antitumor activity.

[0005] In addition, with the rapid development of genetic engineering technology, it has become possible to produce bioactive small molecule peptides through host cell expression systems such as plants and microorganisms. However, there are currently no reports of expressing small molecule peptides from animal drugs through genetic engineering and obtaining products with equivalent anti-tumor activity. Summary of the Invention

[0006] I. Purpose of the Invention

[0007] The present invention aims to provide a simple, reproducible, and industrially scalable method for preparing effective fractions of small molecule peptides for animal drugs. The resulting effective fractions have a molecular weight of <3kD, relatively concentrated components, high removal rates of impurities and pyrogens, significant inhibitory effects on various tumor cells, and low toxicity to normal cells. They can be used to prepare broad-spectrum antitumor drugs.

[0008] Another object of the present invention is to provide an effective fraction of small molecule peptides for animal drugs that is not limited by source, including products with the same characteristics obtained by expression in genetically engineered host cells.

[0009] II. Technical Solution

[0010] The "effective fraction of small molecule peptides in animal drugs" as described in this invention refers to a small molecule peptide composition with a molecular weight <3kD, a small molecule active peptide content ≥40%, a macromolecular impurity and pyrogen removal rate ≥70%, and broad-spectrum antitumor activity. The effective fraction includes not only products directly extracted from animal drugs, but also products obtained through expression in genetically engineered host cells (including but not limited to plant cells, microbial cells, and animal cells) and subsequent separation and purification, possessing the same or equivalent molecular weight range (<3kD) and active peptide components. This invention covers the effective fractions from all sources, without limitation on their production method or species of origin.

[0011] This invention provides a method for preparing the effective fraction of small molecule peptides in animal drugs, comprising the following steps:

[0012] (1) Take the dried animal medicine, pulverize it and pass it through a 20-60 mesh sieve. The animal medicine is selected from one or more of the following: gecko, centipede, scorpion, earthworm, leech, turtle shell, deer antler, silkworm, cicada molting, ground beetle, nine-fragrant insect, black-tailed snake, Agkistrodon halys, gecko, seahorse, sea dragon, oyster, and pearl; or small molecule peptide extracts produced by a genetic engineering host cell expression system that have the same molecular weight range and antitumor activity as the small molecule peptides from the animal medicine source.

[0013] (2) Add 8-12 times the amount of purified water or water for injection, soak for 1-2 hours, heat and decoct to extract 1-3 times, 1-2 hours each time, combine the extracts and filter;

[0014] (3) The extract was subjected to room temperature ultrafiltration through an ultrafiltration membrane with a molecular weight cutoff of 2.5 kDa to 3.5 kDa. The ultrafiltration pressure was 0.12 to 0.18 MPa and the temperature was 25 to 40°C. The ultrafiltration permeate with a molecular weight of <3 kDa was collected.

[0015] (4) The ultrafiltration permeate is concentrated under reduced pressure to a clear extract with a relative density of 1.05 to 1.25 at 60°C, or further dried to obtain the effective fraction of the small molecule peptide of the animal drug.

[0016] Preferably, step (2) involves two extractions, the first lasting 1.5-2 hours and the second lasting 1 hour.

[0017] Preferably, the molecular weight cutoff of the ultrafiltration membrane in step (3) is 3kD.

[0018] The effective components were tested and found to have a macromolecular impurity and pyrogen removal rate of ≥70% and a small molecule active peptide content of ≥40% with a molecular weight <3kD.

[0019] III. Pharmacological Activity

[0020] In vitro experiments showed that the effective components of this invention had significant inhibitory effects on the proliferation of human lung cancer A549 cells, human liver cancer HepG2 cells, human gastric cancer MGC-803 cells, human colorectal cancer HT-29 cells, human breast cancer MCF-7 cells, human esophageal cancer EC-109 cells, human pancreatic cancer PANC-1 cells, human cervical cancer HeLa cells, human ovarian cancer SK-OV-3 cells, and human prostate cancer PC-3 cells, with IC50 values ​​in the range of 20-200 μg / mL. Flow cytometry analysis showed that it could induce tumor cell apoptosis, and the apoptosis rate was concentration-dependent. It also showed low toxicity to normal human liver cells LO2 and normal human gastric mucosal cells GES-1, with a selectivity index >3.

[0021] Further experiments showed that the effective fractions of small molecule peptides obtained through expression in genetically engineered host cells (such as plant cells) have the same antitumor activity as products obtained by direct extraction from animal drugs, proving that the activity of the effective fractions described in this invention is determined by their own structure and is not limited by their source.

[0022] IV. Beneficial Effects

[0023] Broad applicability: The method of this invention is applicable to a variety of animal drugs and can systematically prepare effective fractions of small molecule peptides from different animal drug sources, thus possessing universality.

[0024] Source not limited: This invention explicitly covers the effective parts of small molecule peptides obtained through expression in genetically engineered host cells, and is not limited by source, thus plugging the loophole of circumventing patents by using alternative sources such as plants and microorganisms.

[0025] The process is green: it uses water extraction and physical ultrafiltration throughout, without the use of organic solvents, eliminating the risk of solvent residue and conforming to the concept of green pharmaceuticals.

[0026] Quality controllable: The ultrafiltration process precisely controls the molecular weight cutoff, resulting in a relatively concentrated effective component and a high removal rate of impurities and pyrogens, which can meet the requirements of oral and injectable preparations.

[0027] Clear activity: It has a significant inhibitory effect on more than 10 types of solid tumor cells, low toxicity to normal cells, and good selectivity.

[0028] Sustainable raw materials: All of the animal drugs mentioned can be artificially bred, ensuring a stable source of raw materials; genetically engineered sources can also be sustainably produced, making them suitable for industrial development. Detailed Implementation

[0029] Example 1: Preparation of the effective fraction of gecko small molecule peptides

[0030] Take 100g of dried gecko powder, soak it in 10 times its volume of purified water for 1 hour, and extract it twice by heating and decocting, the first time for 1.5 hours and the second time for 1 hour. Combine the decoctions and filter. The filtrate is then ultrafiltered through a 3kD ultrafiltration membrane (pressure 0.15MPa, temperature 30℃). The permeate is collected and concentrated under reduced pressure to a clear extract with a relative density of 1.10-1.15 at 60℃, thus obtaining the effective fraction of gecko small molecule peptides. Testing showed that the small molecule peptide content was 45.2%, and the pyrogen test was satisfactory.

[0031] Example 2: Preparation of the effective fraction of centipede small molecule peptides

[0032] Take 100g of dried centipede coarse powder, soak it in 10 times the amount of purified water for 1 hour, and extract it twice by heating and decocting, the first time for 1.5 hours and the second time for 1 hour. Combine the decoctions and filter. The filtrate is ultrafiltered through a 3kD ultrafiltration membrane (pressure 0.15MPa, temperature 30℃). The ultrafiltration permeate is collected and concentrated under reduced pressure to a clear extract with a relative density of 1.10-1.15 at 60℃, which is the effective fraction of centipede small molecule peptides. The content of small molecule peptides was found to be 43.8%, and the pyrogen test was qualified.

[0033] Example 3: Preparation of the effective fraction of small molecule peptides from whole scorpions

[0034] Take 100g of dried whole scorpion powder, soak it in 10 times the amount of purified water for 1 hour, and extract it twice by heating and decocting, the first time for 1.5 hours and the second time for 1 hour. Combine the decoctions and filter. The filtrate is ultrafiltered through a 3kD ultrafiltration membrane (pressure 0.15MPa, temperature 30℃). The ultrafiltration permeate is collected and concentrated under reduced pressure to a clear extract with a relative density of 1.10-1.15 at 60℃, which is the effective fraction of small molecule peptides from whole scorpions. The content of small molecule peptides was found to be 44.5%, and the pyrogen test was qualified.

[0035] Example 4: Preparation of the effective fraction of earthworm small molecule peptides

[0036] Take 100g of dried earthworm coarse powder, soak it in 10 times the amount of purified water for 1 hour, and extract it twice by heating and decocting, the first time for 1.5 hours and the second time for 1 hour. Combine the decoctions and filter. The filtrate is ultrafiltered through a 3kD ultrafiltration membrane (pressure 0.15MPa, temperature 30℃). The ultrafiltration permeate is collected and concentrated under reduced pressure to a clear extract with a relative density of 1.10-1.15 at 60℃, which is the effective fraction of earthworm small molecule peptides. The content of small molecule peptides was tested and found to be 42.3%, and the pyrogen test was qualified.

[0037] Example 5: Preparation of the effective fraction of leech small molecule peptides

[0038] Take 100g of dried leech powder, soak it in 10 times the amount of purified water for 1 hour, and extract it twice by heating and boiling, the first time for 1.5 hours and the second time for 1 hour. Combine the decoctions and filter. The filtrate is then ultrafiltered through a 3kD ultrafiltration membrane (pressure 0.15MPa, temperature 30℃). The permeate is collected and concentrated under reduced pressure to a clear extract with a relative density of 1.10-1.15 at 60℃, thus obtaining the effective fraction of leech small molecule peptides. Testing showed that the small molecule peptide content was 41.7%, and the pyrogen test was satisfactory.

[0039] Example 6: Preparation of the effective fraction of small molecule peptides from turtle shell

[0040] Take 100g of dried turtle shell powder, soak it in 10 times its volume of purified water for 2 hours, and extract it twice by heating and decocting, the first time for 2 hours and the second time for 1.5 hours. Combine the decoctions and filter. The filtrate is then ultrafiltered through a 3kD ultrafiltration membrane (pressure 0.15MPa, temperature 30℃). The permeate is collected and concentrated under reduced pressure to a clear extract with a relative density of 1.10-1.15 at 60℃, thus obtaining the effective fraction of small molecule peptides from turtle shell. Testing showed that the small molecule peptide content was 40.2%, and the pyrogen test was satisfactory.

[0041] Example 7: In vitro antitumor activity assay

[0042] The effective fractions of the small molecule peptides prepared in Examples 1-6 were used to detect their inhibitory effects on the proliferation of human lung cancer A549 cells, human liver cancer HepG2 cells, human gastric cancer MGC-803 cells, and human breast cancer MCF-7 cells using the CCK-8 assay. The results showed that each effective fraction exhibited significant inhibitory effects on the above-mentioned tumor cells within a concentration range of 50-200 μg / mL, with the inhibition rate being concentration-dependent and the IC50 values ​​ranging from 30-150 μg / mL.

[0043] Taking the effective components of gecko as an example, after 48 hours of treatment with 200 μg / mL, the inhibition rate of A549 cells reached 78.5%, the inhibition rate of HepG2 cells reached 72.3%, the inhibition rate of MGC-803 cells reached 68.9%, and the inhibition rate of MCF-7 cells reached 65.2%.

[0044] Example 8: Apoptosis Induction Assay

[0045] The effective fraction of the gecko small molecule peptide prepared in Example 1 was used to detect its effect on apoptosis of human lung cancer A549 cells by Annexin V-FITC / PI double staining flow cytometry. The results showed that after 24 hours of treatment with 100 μg / mL, the apoptosis rate was 28.6%; after 24 hours of treatment with 200 μg / mL, the apoptosis rate was 45.3%, indicating that this effective fraction can significantly induce tumor cell apoptosis.

[0046] Example 9: Normal Cytotoxicity Test

[0047] The effective fraction of the gecko small molecule peptide prepared in Example 1 was used to detect its toxicity to normal human hepatocytes (LO2) and normal human gastric mucosal cells (GES-1) using the CCK-8 assay. The results showed that at a concentration of 200 μg / mL, the inhibition rate against LO2 cells was only 15.3%, and against GES-1 cells only 12.8%. The selectivity index (IC50 of tumor cells / IC50 of normal cells) was >3, indicating that this effective fraction has low toxicity to normal cells and good safety.

[0048] Example 10: Preparation and Activity Verification of Effective Fragments of Genetically Engineered Small Molecule Peptides

[0049] Based on the component analysis results of the effective fraction of small molecule peptides from geckos disclosed in this invention, the amino acid sequence information of its main active peptides was obtained by mass spectrometry sequencing. The nucleic acid sequence encoding the peptide was chemically synthesized, a plant expression vector was constructed, and tobacco plants were transformed. Small molecule peptide components were extracted from plant tissues and purified by ultrafiltration using a 3kD ultrafiltration membrane to obtain the effective fraction of small molecule peptides derived from genetic engineering. The content of small molecule peptides was found to be 44.8%, and the pyrogen test was satisfactory.

[0050] The inhibitory effect of the genetically engineered active ingredient on human lung cancer A549 cells was detected using the CCK-8 assay. The results showed that at a concentration of 100 μg / mL, the inhibition rate reached 72.5%, comparable to the gecko extract in Example 1 (75.3%); at a concentration of 200 μg / mL, the inhibition rate reached 81.6%, with no significant difference compared to the gecko extract in Example 1 (78.5%). Flow cytometry analysis showed that after 24 hours of treatment at 100 μg / mL, the cell apoptosis rate was 27.8%, comparable to the gecko extract in Example 1 (28.6%).

[0051] This embodiment demonstrates that the effective fractions of small molecule peptides obtained through expression in genetically engineered host cells (such as plant cells) have the same antitumor activity as products obtained by direct extraction from animal drugs, indicating that the activity of the effective fractions described in this invention is determined by their own structure and is not limited by their source.

Claims

1. A method for preparing the effective fraction of a small molecule peptide from an animal drug, characterized in that... The process includes the following steps: (1) Take crude powder of animal medicine, wherein the animal medicine is selected from one or more of the following: gecko, centipede, scorpion, earthworm, leech, turtle shell, deer antler, silkworm, cicada molt, ground beetle, nine-fragrant insect, black-tailed snake, Agkistrodon halys, gecko, seahorse, sea dragon, oyster, pearl; or small molecule peptide extract produced by a genetic engineering host cell expression system, which has the same molecular weight range and antitumor activity as the small molecule peptide from the animal medicine source; (2) Add 8 to 12 times the amount of purified water or water for injection, heat and decoct to extract 1 to 3 times, each time for 1 to 2 hours, combine the extracts and filter; (3) The extract is subjected to room temperature ultrafiltration through an ultrafiltration membrane with a molecular weight cutoff of 2.5 kDa to 3.5 kDa, ultrafiltration pressure 0.12 to 0.18 MPa, temperature 25 to 40°C, and ultrafiltration permeate with a molecular weight <3 kDa is collected; (4) The ultrafiltration permeate is concentrated or dried under reduced pressure to obtain the effective part of the small molecule peptide of the animal medicine.

2. The preparation method according to claim 1, characterized in that, Step (2) involves two extractions, the first taking 1.5 to 2 hours and the second taking 1 hour.

3. The preparation method according to claim 1, characterized in that, Step (3) The ultrafiltration membrane has a molecular weight cutoff of 3 kDa.

4. The preparation method according to claim 1, characterized in that, Step (3) The removal rate of macromolecular impurities and pyrogens after ultrafiltration is ≥70%, and the content of small molecule active peptides with a molecular weight <3 kDa in the obtained effective fraction is ≥40%; the effective fraction is not limited to animal drug sources, but also includes small molecule peptide effective fractions with the same molecular weight range and antitumor activity expressed by genetically engineered host cells.

5. An effective fraction of a small molecule peptide used in animal medicine, characterized in that, Its molecular weight is <3 kDa, the content of small molecule active peptides is ≥40%, the removal rate of macromolecular impurities and pyrogens is ≥70%, and it has broad-spectrum anti-tumor activity; the effective part is not limited by its source, including but not limited to animal drug extraction source or genetic engineering host cell expression source.

6. The use of the effective fraction of the small molecule peptide from animal drugs according to claim 5 in the preparation of antitumor drugs.

7. The application according to claim 6, characterized in that, The antitumor activity includes inhibition of one or more of the following cancers: lung cancer, liver cancer, stomach cancer, colorectal cancer, breast cancer, esophageal cancer, pancreatic cancer, cervical cancer, ovarian cancer, and prostate cancer.

8. The application according to claim 6, characterized in that, The drug dosage form is any one of oral liquid, syrup, tablet, capsule, granule, powder, injection, lyophilized powder for injection, or transdermal preparation for external use.

9. A pharmaceutical composition, characterized in that... It contains the effective fraction of the small molecule peptide of the animal drug as described in claim 5 and pharmaceutically acceptable excipients.

10. The pharmaceutical composition according to claim 9, characterized in that, The pharmaceutical composition is an antitumor drug.