Biological antibacterial peptide and traditional Chinese medicine pharmacodynamic element nano-carrier anticancer preparation and application thereof

Abstract

The invention discloses a biological antibacterial peptide and traditional Chinese medicine pharmacodynamic element nano-carrier anti-cancer preparation and application thereof.According to the purification technological process, extraction and purification are conducted through a series of physical process technological procedures which do not damage antibacterial peptide cardinal number protein substances and amino acid sequencing number in a specific negative-pressure vacuumizing environment, and the activity of antibacterial peptide is completely protected; an amino acid chain based on protein is not damaged, an acidic solvent cannot be added in the antibacterial peptide purification and extraction process, the acidic solvent added later can be combined with acidity in amino acid, basic protein is denatured, the acidity in the amino acid is recombined to be denatured, the risk that the original antibacterial peptide is denatured due to amino acid sequencing is reduced, and the antibacterial peptide purification and extraction efficiency is improved. On the premise of not destroying the activity of biological antibacterial peptides and pharmacodynamics elements, physical and chemical working procedures are integrated to extract the pure maggot antibacterial peptides. Super-strong biological antibacterial peptides extracted from organisms and animals and pharmacodynamics elements purified from Chinese herbal medicines are synthesized into the nano-carrier pure natural biological preparation which is antibacterial, anticancer, cancer tissue killing, antiviral and the like and has no side effect.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a biological antimicrobial peptide and a traditional Chinese medicine pharmacodynamic nanocarrier anticancer preparation and application thereof. Background Art

[0002] Antimicrobial peptides are a class of polypeptides produced by the immune system to protect against infection by external pathogens. In addition to their nonspecific protection against pathogens such as bacteria, fungi, and viruses, they also have anti-tumor effects, particularly against multidrug-resistant bacteria and tumor cells, without damaging normal human cells. They represent a new class of antimicrobial drugs with enormous potential for development. These active peptides possess advantages such as low immunogenicity, excellent thermal stability, good water solubility, and a broad antimicrobial spectrum. Studies have shown that adding antimicrobial peptides to animal diets can improve production performance and feed utilization, enhance the intestinal microecological environment, promote cellular and humoral immune responses, and enhance immune function and disease resistance. Furthermore, the addition of antimicrobial peptides to feed can replace antibiotics, eliminating the problem of antibiotic resistance in pathogens caused by overuse and antibiotic residues in agricultural products.

[0003] Flies and their larvae primarily thrive in harsh environments such as decaying matter, feces, and garbage, which are rich in bacteria. They carry numerous pathogens on their bodies, capable of transmitting a variety of diseases, yet they themselves do not die from these infections. This suggests that flies possess a robust immunity to pathogenic microorganisms and produce potent antimicrobial substances. These substances are known as fly maggot antimicrobial peptides. These peptides are small polypeptide chains composed of 7-60 amino acids, primarily protein, with a density of approximately 1.15 and a pH range of 6-7.5 in their puree. To protect the intact antimicrobial peptide activity and prevent the protein-based amino acid chain from being destroyed, acidic solvents must be avoided during the purification and extraction process. Added acidic solvents would combine with the acidity of the amino acids, denaturing the underlying protein and causing the acidity within the amino acids to recombine and denature, severely reducing the amino acid sequence and altering the pre-existing antimicrobial peptide.

[0004] Therefore, how to maximize the efficacy of fly maggot antimicrobial peptides is one of the technical problems that those skilled in the art need to solve urgently. Summary of the Invention

[0005] In response to the shortcomings of the existing technology, the present invention provides a nano-carrier anticancer preparation of a biological antimicrobial peptide and a traditional Chinese medicine pharmacodynamics and its application. In order to achieve the purpose of the present invention, the following technical solutions are proposed:

[0006] The first aspect of the present invention provides a preparation containing a biological antimicrobial peptide, wherein the biological antimicrobial peptide is obtained by the following method:

[0007] (1) Adding distilled water and then adding a gas without residual gas such as ozone to clean and disinfect the maggots in vitro to reduce the self-consumption of antimicrobial peptides:

[0008] (2) Grinding the fly maggots with sterile water in a weight ratio of 1:2 using a grinder to prepare a slurry;

[0009] (3) The dark raw pulp is vacuum-frozen and denatured, and the adsorbed waste is precipitated and separated, and the clear liquid in the middle is taken;

[0010] (4) centrifugation and negative pressure filtration to obtain the crude antimicrobial peptide solution;

[0011] (5) Add stainless steel mesh balls and separate the distilled water by negative pressure distillation;

[0012] (6) Heating to 75°C-85°C for 40-60 minutes, then immediately reducing the pressure, and immediately manually intervening to cool down to room temperature outside, immediately turning on the refrigeration, setting the temperature to 0°C to -4°C, and leaving it for 10-15 hours;

[0013] (7) Take out the stainless steel ball covered with flocculent matter and centrifuge to remove the residue;

[0014] (8) Freeze-drying to obtain active antimicrobial peptide freeze-dried powder.

[0015] In a preferred embodiment of the present invention, it is characterized in that step (3) is to open the refrigeration of the stainless steel kettle and simultaneously evacuate the vacuum, adjust the temperature to 0°C to -10°C, and then stir continuously, stop stirring after continuous stirring for 6-12 hours, perform vacuum freezing for 24 hours, and take the intermediate clear liquid;

[0016] In a preferred embodiment of the present invention, it is characterized in that step (4) is to freeze the intermediate clear night for 24 hours, take out the intermediate layer mother liquor, pour it into a 1400 mesh centrifuge for separation, and obtain the crude liquid of biological antimicrobial peptide after centrifugation.

[0017] In a preferred embodiment of the present invention, it is characterized in that step (5) is to pour the centrifugal separation in the high-frequency centrifuge into the stainless steel multifunctional reactor again, and then put a number of 316 stainless steel mesh balls into the reactor, and start vacuuming the reactor to make it negative pressure, the pressure is -55 to -95Kpa, and heating distillation is started to separate excess water.

[0018] In a preferred embodiment of the present invention, the biological antimicrobial peptide-containing preparation further comprises a traditional Chinese medicine plant pharmacodynamic element.

[0019] In a preferred embodiment of the present invention, the antimicrobial peptide essence and water-soluble Chinese herbal medicine powder are poured into a stainless steel stirred tank, stirred, vacuumed, and heated at 70°C to 80°C. After continuous operation for 3-5 hours, the mixture is cooled to room temperature and poured into a high-frequency centrifuge for high-frequency centrifugal filtration using 1400 mesh to obtain a nanocarrier anticancer agent synthesized from the biological antimicrobial peptide and Chinese herbal medicine.

[0020] In another aspect of the present invention, it also relates to the use of the above-mentioned biological antimicrobial peptide preparation in the preparation of antibacterial, antiviral and anticancer preparations; the preparations include medicines, health products and beauty products.

[0021] The purification process of the present invention performs extraction and purification through a series of physical process steps under a specific negative pressure vacuum environment without destroying the base protein substance and amino acid sequence of the antimicrobial peptide, thereby completely protecting the activity of the antimicrobial peptide and preventing the protein-based amino acid chain from being destroyed. Acidic solvents cannot be added during the purification and extraction of the antimicrobial peptide. The added acidic solvents will combine with the acidity in the amino acids, denaturing the base protein and reorganizing the acidity in the amino acids to denature them, thereby reducing the risk of denaturation of the existing antimicrobial peptides due to the amino acid sequence. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 : Maggot puree.

[0023] Figure 2 : Layered antimicrobial peptide crude solution.

[0024] Figure 3 : Unstable proteins, pigments, fats and other waste materials adsorb each other under negative pressure.

[0025] Figure 4 : Pigments, unstable proteins, fats, low-density waste, and calcifications turn into flocs under negative pressure.

[0026] Figure 5 : Antimicrobial peptide extract, the left side is not illuminated by a gem lamp, while the right side is illuminated by a gem lamp, and fluorescent substances appear. DETAILED DESCRIPTION

[0027] To further illustrate the technical means and effects employed by the present invention to achieve its intended objectives, the following detailed description of the specific implementation methods, technical solutions, features, and effects of the present invention is provided below, using preferred embodiments as an example. The specific features, structures, or characteristics of the various embodiments described below may be combined in any suitable manner.

[0028] Example 1:

[0029] ① In vitro cleaning and disinfection of maggots reduces the self-consumption of antimicrobial peptides:

[0030] The maggots should be cleaned with distilled water and disinfected with residual gas (alcohol and other disinfectants have residues, which will seriously damage the activity of antimicrobial peptides and seriously affect the efficacy, so they cannot be used).

[0031] ②Making the original pulp (do not use a beating tool with a blade):

[0032] Use a grinder to grind the maggots into a slurry by adding sterile water in a weight ratio of 1:2. Pour the slurry into a stainless steel vacuum stirring reactor, and then pour clay into it to absorb unstable proteins and waste oils, so that they can be quickly precipitated and separated from the antimicrobial peptides. Remove the air in the reactor and turn on the high-speed blender in a vacuum state. Stir thoroughly. Under a specific negative pressure (vacuum) environment, high-speed stirring and centrifugation will make most of the fat and impurities with a density below 0.9 and unstable proteins float up and separate quickly, wrapped in clay and mixed in the foam. At the same time, some animal pigments are adsorbed on the clay. Stir for 20-50 minutes each time, stay for 5-10 minutes, repeat 10-40 times, stop stirring, and then... Figure 1 As shown, a dark puree was obtained.

[0033] ③ Dark pulp vacuum freezing denaturation adsorption waste sedimentation separation method: After the above work is completed, the stainless steel kettle is opened for refrigeration and vacuumed at the same time. After the temperature is adjusted to 0℃ to -10℃, it is stirred continuously. After stirring for 6-12 hours, the stirring is stopped and the vacuum is frozen for 24 hours to achieve the denaturation of unstable proteins and other impurities and the stable adsorption of fat and some natural pigments on the synthetic clay; the unstable protein and waste oil are quickly adsorbed to accelerate the precipitation and rapid separation of antimicrobial peptides. After precipitation, it is divided into three layers: upper, middle and lower. The middle clear liquid is taken, such as Figure 2 shown

[0034] ④ Negative pressure suction filtration to obtain crude antimicrobial peptide liquid: After freezing for 24 hours, the middle layer mother liquor is taken out and poured into a 1400 mesh centrifuge for separation. A portion of synthetic clay that is invisible to the naked eye adsorbs unstable proteins and dark pigments and is centrifuged in a 1400 mesh high-frequency centrifuge to obtain a crude biological antimicrobial peptide liquid. The crude liquid is a yellow transparent liquid.

[0035] ⑤Micron unstable substance reactor negative pressure adsorption separation process:

[0036] After the high-frequency centrifuge is completed, it is poured into the stainless steel multi-functional reactor again. Several 316 stainless steel mesh balls are placed in the reactor to cooperate with the residual clay adsorption to adsorb unstable proteins, fats and residual impurities. The reactor begins to evacuate to make it negative pressure, and the pressure reaches -55 to -95Kpa. It begins to heat and distill to separate excess water. At the same time, stirring is turned on. In order to achieve rapid absorption by the human body while protecting the activity of antimicrobial peptides and proteins, and separating and removing proteins that can only be decomposed at high temperatures, the temperature is set to 30-50 degrees Celsius (the separation temperature for impurities with a density of more than 1.0). The initial temperature cannot exceed 60 degrees. Under the action of the corresponding negative pressure, the boiling point of water begins to boil at 35℃-40℃ at the earliest, and the distilled water is separated. At the same time, the continuous boiling inside causes the unstable denatured proteins, fats and impurities that have been frozen to roll and form filaments. After boiling, they are bundled and wrapped on the stainless steel wire balls, like loose fibers. Some are even twisted into "long ropes" and hung at the condenser tube mouth (see Figure 3 、 4 The initial color of the filtered clear pulp and the unstable impurities and waste such as protein, fat, pigment, calcification, etc. become flocculent under negative pressure and are adsorbed together).

[0037] ⑥ Extraction process of antimicrobial peptide initial solution: Continue to distill and separate excess water in a specific negative pressure environment until there is no foam in the reactor. At this time, the density of the liquid retained in the reactor is above 1.0. In order to extract high-purity antimicrobial peptide essence, the impurities in the initial solution are completely solidified, and at the same time, the antimicrobial peptides with strong activity are protected and selected. Then heat it to 75℃-85℃ for 40 minutes-60 minutes (depending on the indoor temperature), then immediately reduce the pressure and manually intervene to cool it down to room temperature outdoors. Immediately turn on the refrigeration and set the temperature to 0℃ to -4℃. Leave it for about 12 hours (depending on the indoor temperature). The impurities inside are all flocculent and larger than 77 microns, and the extraction and separation process is completed.

[0038] ⑦ The fly maggot antimicrobial peptide essence is cleaned to obtain the "fly maggot antimicrobial peptide essence": After the above process is completed, the stainless steel ball covered with flocculent objects is removed and the initial liquid of fly maggot antimicrobial peptide essence is poured into a 1400 mesh high-frequency centrifuge for residue separation. All particles smaller than 10 microns are removed to obtain the fly maggot antimicrobial peptide essence with a pH value of about 6-7.5. Because the biological antimicrobial peptide is not affected by external acidic reactions or high temperatures exceeding 86°C during the processing and extraction process, all pH values remain unchanged. The biological antimicrobial peptide extracted using this process has the strongest activity and the strongest bactericidal, antiviral and anticancer effects (see Figure 5 ).

[0039] ⑧ Obtaining soluble antimicrobial peptide powder: Place the above fly maggot antimicrobial peptide essence in a freeze dryer for freeze drying (the moisture content must be retained at 30%) to obtain active antimicrobial peptide freeze-dried powder.

[0040] Example 2: Antimicrobial peptide activity experiment

[0041] 1.1 Antimicrobial activity test

[0042] Inhibition zone method: Different concentrations of antimicrobial peptides (0.1-1.0 mg / mL) were added dropwise to agar plates containing bacteria, and the diameter of the inhibition zone was measured after incubation at 37°C for 24 hours.

[0043] 1.2 Antiviral experiments

[0044] Viral load detection: qPCR quantification of viral nucleic acid.

[0045] 1.3 Evaluation of anticancer activity

[0046] Cell proliferation inhibition (MTT assay): A549 and MCF-7 cells were co-cultured with different concentrations of MAPs for 48 hours, and the cell survival rate was detected.

[0047] Cell apoptosis detection: Annexin V / PI double staining combined with flow cytometry.

[0048] Animal model: Five different subcutaneous transplant tumor models were established in hamsters. Different concentrations of antimicrobial peptides (5 mg / kg / d) of 1:10, 1:20, 1:30, 1:40, and 1:50 were injected intraperitoneally, and tumor volume and survival were monitored.

[0049] 1.4 Hemolysis test

[0050] Hemolysis test: detect the hemolytic activity of antimicrobial peptides on red blood cells.

[0051] Acute toxicity experiment: observe the physiological state of hamsters after injection of high doses of antimicrobial peptides.

[0052] Experimental results

[0053] 1. Antimicrobial activity

[0054] The antimicrobial peptides showed significant inhibition against both Gram-positive bacteria (such as Staphylococcus aureus) and Gram-negative bacteria (such as Escherichia coli), with MIC values of 0.2-0.5 mg / mL.

[0055] The diameter of the inhibition zone increased with the increase of antimicrobial peptide concentration (up to 15±2 mm).

[0056] 2. Antiviral activity

[0057] Yersinia pestis was injected into three hamsters at different doses. Two days later, obvious pneumonia symptoms appeared. Then, three different doses of antimicrobial peptides, 1:30, 1:40, and 1:50, were injected intravenously twice a day. The cough and fever symptoms disappeared on the second day, and the hamsters were almost completely recovered on the third day.

[0058] 3. Anticancer activity

[0059] In the hamster experiment, the tumor volume of the MAPs-treated group was reduced by 50% (vs. the control group) and the survival period was extended by 20%.

[0060] Example 3:

[0061] ① Extraction of Chinese Herbal Pharmacopoeia Essence: Wash medicinal plants and crush them into small particles about 3 mm. Soak them in purified water at a ratio of 1:1 until they are completely saturated. Grind the pulp (do not use a beating tool with a blade). Using the same purification process for fly maggot antimicrobial peptides, heat the mixture to 80°C to produce a puree to obtain a transparent, slightly yellowish Chinese Herbal Pharmacopoeia Essence.

[0062] ② Obtaining water-soluble Chinese herbal pharmacological essence: Place the above-mentioned transparent and light-colored Chinese herbal pharmacological essence essence in a freeze dryer, adjust the freeze dryer temperature to -35°C to -65°C (depending on the type of Chinese medicine), open it and quickly vacuum, and at the same time heat the pharmacological essence tray to quickly sublimate its moisture to obtain water-soluble Chinese herbal pharmacological essence powder (for easy storage).

[0063] Example 4:

[0064] Synthesis of bio-antimicrobial peptides and Chinese herbal medicine pharmacodynamics as nanocarrier anticancer agents, synthesis of bio-antimicrobial peptides and effective pharmacodynamics:

[0065] Pour the antimicrobial peptide essence and the appropriate amount of water-soluble Chinese herbal medicine powder purified above into a stainless steel stirring kettle, turn on stirring, turn on vacuum, and heat at the same time, controlling the temperature between 70°C and 80°C (because Chinese medicine cannot exceed 80°C, and excessive temperature will also affect the efficacy). After continuous operation for 4 hours, cool it to room temperature. Because it is a biological preparation, a small amount of unstable protein will denature and solidify invisibly during the operation process, which will affect the efficacy when used by patients. It must be poured into a high-frequency centrifuge again and filtered by high-frequency centrifugation with 1400 mesh. The biological antimicrobial peptides and Chinese herbal medicines are absorbed into the Chinese herbal medicine under a specific vacuum environment. It can be observed under a 2000x microscope. It contains the composition of active antimicrobial peptides, that is, the synthesized nanocarrier anticancer agent.

[0066] The above disclosure is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any modifications or substitutions that can be easily conceived by a person skilled in the art within the technical scope disclosed in this invention should be included in the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be based on the scope of protection of the above claims.

Claims

1. Anticancer preparations with nanocarriers of biological antimicrobial peptides and Chinese herbal pharmacodynamics and their applications: extracting biological antimicrobial peptides from organisms such as maggots or insects; extracting Chinese herbal plant pharmacodynamics under a vacuum environment; synthesizing nanocarrier anticancer agents with biological antimicrobial peptides and Chinese herbal pharmacodynamics under a vacuum environment.

2. A preparation containing a biological antimicrobial peptide, wherein the biological antimicrobial peptide is obtained by the following method: (1) Adding distilled water and then adding a gas without residual gas such as ozone to clean and disinfect the maggots in vitro to reduce the self-consumption of antimicrobial peptides: (2) Grinding the fly maggots with sterile water in a weight ratio of 1:2 using a grinder to prepare a slurry; (3) The dark raw pulp is vacuum-frozen and denatured, and the adsorbed waste is precipitated and separated, and the clear liquid in the middle is taken; (4) centrifugation and negative pressure filtration to obtain the crude antimicrobial peptide solution; (5) Adding a stainless steel mesh ball and separating the distilled water by negative pressure distillation; (6) Heating to 75°C-85°C for 40-60 minutes, then immediately reducing the pressure, and immediately manually intervening to cool down to room temperature outside, immediately turning on the refrigeration, setting the temperature to 0°C to -4°C, and leaving it for 10-15 hours; (7) Remove the stainless steel ball covered with flocculent matter and centrifuge to remove the residue; (8) Freeze-drying to obtain active antimicrobial peptide freeze-dried powder.

3. The biological antimicrobial peptide preparation according to claim 2, characterized in that: Step (3) is to open the refrigeration of the stainless steel kettle and evacuate the vacuum at the same time, adjust the temperature to 0°C to -10°C, and stir continuously. After stirring continuously for 6-12 hours, stop stirring, perform vacuum freezing for 24 hours, and take the intermediate clear liquid.

4. The biological antimicrobial peptide preparation according to claim 2, characterized in that: Step (4) is to freeze the middle clear night for 24 hours, take out the middle layer mother liquor, pour it into a 1400 mesh centrifuge for separation, and obtain the crude liquid of biological antimicrobial peptide after centrifugation.

5. The biological antimicrobial peptide preparation according to claim 2, characterized in that: Step (5) is to pour the mixture into a stainless steel multifunctional reactor again after centrifugal separation by a high-frequency centrifuge, and then place a number of 316 stainless steel mesh balls in the reactor. The reactor is evacuated to a negative pressure of -55 to -95 kPa, and heating and distillation are started to separate excess water.

6. The bio-antimicrobial peptide-containing preparation according to claim 2, further comprising a Chinese herbal medicine plant pharmacodynamics.

7. The biological antimicrobial peptide preparation according to claim 6, wherein the antimicrobial peptide essence and water-soluble Chinese herbal medicine powder are poured into a stainless steel stirred tank, stirred, vacuumed, and heated at 70° C. to 80° C. for 3-5 hours, then cooled to room temperature, poured into a high-frequency centrifuge and filtered at high frequency using 1400 mesh to obtain a nanocarrier anticancer agent synthesized from the biological antimicrobial peptide and Chinese herbal medicine.

8. The bio-antimicrobial peptide preparation according to any one of claims 2 to 7, and its use in the preparation of antibacterial, antiviral, and anticancer preparations; the preparations include medicines, health products, and beauty products.

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