PL243408B1 - Method of obtaining a bioactive mixture, especially a sugar-glycoprotein mixture, bioactive mixture and use of bioactive mixture - Google Patents

Abstract

The subject of the application is a method of obtaining a bioactive mixture, especially a protein-glycoprotein mixture in the form of secretion from the larvae of medical flies from the Calipphoridae, (or) Sarcophagidae and/or Muscidae families, using a substrate for the multiplication of larvae and their incubation, which consists in the fact that which stimulates the growth of the larvae using only non-sterile medium, preferably based on beef liver extract, on which the larvae of medical flies from the Calipphoridae, Sarcophagidae and/or Muscidae families are hung and incubated until the production of larval secretions, and then the larvae after incubation and rinsing, preferably with physiological saline, transferred to a shaking incubator in a medium with physiological saline and incubated, preferably at 36°C for a period of three hours, after which the larvae are removed from the medium, in which secretions from the digestive system of the larvae with a protein concentration remain about 0.20 mg/mL, and the bioactive mixture obtained in this way - the preparation is sterilized, preferably by membrane or cellulose filter with centrifugation, and preferably frozen for further medical use. The subject of the application is also a bioactive mixture, especially a protein-glycoprotein mixture, in the form separated from the secretory fluid from the secretion of medical fly larvae, characterized by the fact that it is a mixture of bioactive agents containing: (i) a mixture of peptides, glycoproteins and high-molecular proteins, enzymes, and low molecular weight compounds (ii), proteins produced by fly larvae from the families Callipphoridae, Sarcophagidae and Muscidae; where therapeutic properties were possessed by proteins with enzymatic activity, mainly serine proteases, trypsin and chymotrypsin, as well as DNAses and endopeptidases, and in this fraction - mixture, a number of antimicrobial peptides can also be observed, such as diptericin, sapecin-B (a- defensin), lysozyme, lipases, trypsin-like serine proteases, cysteine proteases and a number of other proteases. This application also covers the use of the bioactive mixture obtained above. a method for producing a carrier-preparation with antibacterial and regenerative effects, including supporting the treatment of symptoms of a viral infection, preferably SARS-CoV-2 Covid 19, showing highly effective (up to 100%) cytoprotective activity on living cells, towards anti-apoptotic protection of cells.

Description

Description of the invention

The subject of the invention is a method of obtaining a bioactive mixture, especially a protein-glycoprotein mixture in the form of a secretion from the larvae of medical flies of the Calipphoridae Sarcophagidae and/or Muscidae families, and the use of the bioactive mixture.

Previously known methods used a sterile substrate to obtain an extract - a mixture of medical fly secretions.

Medicinal flies (Lucilia sericata) have been successfully used for therapeutic purposes in the treatment of hard-to-heal wounds. Proteins exhibiting enzymatic activity, mainly serine proteases, trypsin and chymotrypsin, as well as DNAses and endopeptidases, had known therapeutic properties. In this fraction, a number of antimicrobial peptides can also be observed, such as diptericin, sapecin-B (a-defensin), lysozyme, lipases, nine trypsin-like serine proteases, two cysteine proteases and a number of other proteases.

From the Slovak patent description No. SK288639B6 it is known to use larval secretions for the production of a hydrogel that has an antibacterial effect. Described herein is a biopolymer hydrogel containing xanthan gum and carubine in a weight ratio of 1:1.6 to 1.6:1, a combined content of both components in water from 0.5 to 2.6% by weight, and a drug component based on extracts of salivary glands and intestines of Lucilia sericata larvae. An ingredient that creates extracts from the salivary glands and intestines prepared from the larvae. II. and III. larval stages of Lucilia sericata flies high proteolytic and antibacterial activity of extracts 10 mg of extract / 1 ml of gel. A formulation for the treatment of chronic wounds comprising a hydrogel is also described.

From the Slovak patent description No. SK632015 there is known a device for mass breeding of larvae, separation of larvae and obtaining extracts from the salivary glands and intestines of larvae (Lucilia sericata).

The American patent description No. US2005260183 describes the treatment of surfaces inhabited by bacteria with an extract from Lucilia sericata. A surface inhabited by bacteria capable of producing a biofilm is preferably treated by contacting it with a substance having N-acylhomoserine lactone degrading activity obtained from the secretions and/or excretions of the larval form of the bottle fly, Lucilia sericata. In addition, a synergistic effect is obtained when larval secretions/excrements are used in combination with an antibiotic, e.g. tetracycline. Such a substance having an N-acylhomoserine lactone degrading activity obtained from secretions and/or excretions of Lucilia sericata larvae is also part of the invention.

A larval enzyme is known from Chinese Patent No. CN101541829A. These enzymes can be used for wound healing, wound cleansing, and cell regeneration. Chymotrypsin from larval secretions, destroying biofilm and healing chronic wounds (including bacterial ones).

From Chinese Patent No. CN102106316, a method for cultivating and sterilizing Lucilia sericata is known, which relates to the acquisition of sterile larvae, in particular including training in disinfection, disinfection, and worm ovum thread. The sterilization method of the worm egg given in this invention is to immerse the egg mass in 0.5-1% sodium sulfite rocking solution for 3-5 minutes, make the sticky egg mass separate, immerse the egg mass in 5-6% chlorine bleach, sterilization liquid for 3-5 minutes after filtering. The rivet sterilization method uses 0.3-0.5% iodophor solutions and 0.1% bromogeramine solution to separate the two-step sterilization of the thread. The effect of the method of the invention on the intrabiotic of housefly eggs is low, the mortality rate is 0%, the thread disinfection is fast and durable, the toxicity is low, and the organic matter is little affected. Not only can maintain the effective passage of the worm body, but also can make Lucilia sericata after sterilization to keep vigor and simple operation, easy to learn, popularization and application suitable for wide.

The Japanese patent description No. JP2011155974 relates to the use of one of the enzymes obtained from larval secretions for the treatment of chronic wounds by cleansing them. It is also antibacterial. Providing wound healing chymotripsin for tissue regeneration and wound healing.

The aim of the invention is to develop a new method of obtaining a bioactive mixture, especially a protein-glycoprotein mixture, from larval secretions stimulated with a non-sterile substrate, especially based on an extract and beef liver with cytoprotective and antibacterial properties.

Unexpectedly, it was found that medical fly larvae whose growth was stimulated only on a non-sterile substrate produce a new secretion - a bioactive mixture with other enhanced features (compared to known medical fly larva secretions on sterile substrates), which secretion is characterized by high cytoprotective activity in towards anti-apoptotic protection of cells. In vitro, 100% viability of infected cells was observed compared to control groups, where viability oscillated around 5-7%,

The strength of the method according to the invention is that the growth of the larvae is stimulated using only a non-sterile substrate, based on beef liver extract, on which the larvae of medical flies from the families Calipphoridae, Sarcophagidae and/or Muscidae are suspended and incubated until secretions are produced. larvae, and then the larvae, after incubation and rinsing with saline, are transferred to an incubator with shaking in a medium with saline and incubated at 36°C for three hours, after which time the larvae are removed from the medium in which they remain secretion from the digestive system of the larvae with a protein concentration of 0.20 mg/mL, and the thus obtained bioactive mixture - the preparation is subjected to sterilization, membrane or cellulose filter with centrifugation, and frozen for further medical use.

As a non-sterile medium, a medium infected with at least one microorganism is used. As a non-sterile medium, a medium based on 3-5 day old beef liver extract is used. As a non-sterile substrate, it is additionally subjected to an initial exposure to microorganisms for 3-5 days until decay and disintegration occur.

The non-sterile medium is additionally exposed to at least one strain of bacteria, fungi or the SARS-CoV-2 virus type or a mixture of microorganisms and/or viruses and/or fungi, Listeria monocytes medium 0.5% to 2.5% by weight.

The non-sterile medium additionally exposes, and thus activates, to at least one factor that is neutralized - killed bacteria and/or disintegrated viruses, being whole or their genetic fragments, i.e. DNA and/or RNA, or protein/sugar/lipid fragments or combinations thereof.

The bioactive mixture is used as a medical preparation.

Bioactive mixture as a carrier-preparation for use in the treatment of symptoms of viral infection, including SARS-CoV-2 Covid 19.

The carrier-formulation is used as an antifungal, antibacterial and/or antiviral preparation.

The carrier-preparation is used cytoprotectively on cells after viral, fungal and bacterial infection.

The carrier-preparation in the form of a lyophilized fraction is administered by injection and/or inhalation and/or aerosol.

The carrier-preparation is placed as a medical active substance in composite materials, hydrogels, polymers, and lipid monocrystals.

Bioactive mixture as a carrier-preparation for use in the treatment of disease for the reconstruction of blood vessels and accelerating tissue regeneration.

Technical effect

The test fraction - the bioactive mixture according to the invention shows high cytoprotective activity towards anti-apoptotic protection of cells. In vitro, 100% viability of infected cells was observed in comparison to control groups, where viability oscillated around 5-7%.

Stimulation of the non-sterile medium for the production of larval secretions also increased the effectiveness of the secretions by over 200% (antibacterial effect on pathogenic strains) against the main pathogens: E. coliATCC25922, Staphylococcus aureus ATCC 29213, Salmonella enterica ATCC 13314, Pseudomonas aeruginosa ATCC 27853 and Listeria mon cytogenes ATCC 7644.

An example of a cytoprotective application is shown on lung cells from the A549_ACE2 cell line attacked with the SARS-CoV-2 virus (Munchen-1.2 2020/984) - Fig. 1 and in Example 2.

Advantages

Bioactive mixture:

1. has a cytoprotective and antimicrobial effect on cells after infection,

2. will be used in antibacterial and regenerative actions and in supporting the treatment of symptoms of viral infection, including Covid-19,

3. shows high cytoprotective activity towards anti-apoptotic protection of cells

4. gives increased antimicrobial, angiogenic, biocompatible, non-toxic and bioactive properties of the material, to accelerate wound regeneration.

During the research, significantly lower (about two-three times lower) required average minimum inhibitory concentration-MIC (mg/l) for larval secretions stimulated according to the invention (stimulation) and sterile (absence) was observed for various pathogenic bacterial strains (Fig. 1 ).

(Definition) - Minimum inhibitory concentration, MIC (minimum inhibitory concentration) is the lowest concentration of a biocide (antibiotic or chemotherapeutic agent) that inhibits the growth of microorganisms (usually refers to bacteria and fungi), usually expressed in mg/l.

Thus, the obtained bioactive mixture according to the invention was at least 2-3 times stronger than secretion from a sterile medium (it could be used 2-3 times less in volume to obtain a similar MIC effect).

Usage

The invention will be used especially as a medical preparation:

a) it is used as an antifungal, antibacterial and/or antiviral preparation,

b) is used cytoprotectively on cells after viral, fungal and bacterial infection,

c) the preparation in the form of a lyophilized fraction is administered in the form of injection and/or inhalation and/or aerosol,

d) the preparation is placed as a medical active substance in composite materials, hydrogels, polymers, lipid monocrystals,

e) the preparation is placed as a medical active substance for direct application to the surface of internal or external wounds in order to activate in the tissue environment, restore blood vessels and accelerate tissue regeneration.

The subject of the invention is explained in an embodiment and in the drawings, in which:

Figure 1 shows the mean minimum inhibitory concentration-MIC (mg/L) of stimulated (stimulated) and sterile (absent) larval secretions for various pathogenic bacterial strains. Fig. 2 shows the study of the effect of the secretion - bioactive mixture according to the invention on the replication of SARS-CoV-2, Fig. 3 shows the study of the effect of the secretion - bioactive mixture according to the invention on the replication of SARS-CoV-2 - observations under the microscope of the cytopathic effect.

Example d 1 (non-sterile medium)

The growth of the larvae is stimulated only with a non-sterile medium, based on beef liver extract, on which the larvae of medical flies of the families Calipphoridae Sarcophagidae and/or Muscidae are suspended and incubated until the production of larval secretions, and then the larvae after incubation and washing with saline, is transferred to an incubator with shaking in a medium with saline and incubated at a temperature of 36°C for three hours, after which time the larvae are removed from the medium in which secretion from the digestive system of the larvae with a protein concentration of approximately 0.20 mg/mL, and the thus obtained bioactive mixture - preparation is subjected to sterilization, membrane or cellulose filter with centrifugation, and frozen for further medical use.

As a non-sterile medium, a medium infected with at least one microorganism is used.

(Description of induction and extraction of the extract)

Cages containing mature males and females of L. sericata (approximately 4 weeks after emergence of the pupa) were placed in black plastic tubes with a diameter of 35 mm. The inner walls of the tubes were lined with filter paper saturated with 0.9% saline solution. As an attractant, a 3-5 day-old bovine liver extract was placed in the lower part of the tube, separated by a mesh diaphragm. After two hours, the tube was taken to the lab for chemical sterilization of the eggs.

The eggs were transferred to sterile plastic containers which were then filled with 0.9% saline. Since the eggs were glued together, the extract was homogenized in an automated pipetting chamber (500 µl) for three minutes. The egg fraction after homogenization was filtered from the saline fraction and replaced with 5% chloramine. The resulting fractions were mixed for seven minutes in two cycles of 3.5 minutes. Sterile eggs were transferred to sterile saline to remove chloramine. The sterile eggs were then transferred to bovine liver medium that had been previously exposed to microbes for several days. This type of substrate was characterized by a significant degree of organic decomposition, with a suitable smell and consistency indicating putrefactive processes. The reference medium was an agar medium with 5% protein lyophilisate, sterilized in an autoclave. Both larval lines were incubated on two types of media for 72 h at 36°C.

The third instar larvae obtained after 72 hours were washed five times with 0.9% saline for 3 hours. After complete decolorization of the saline solution and removal of metabolites from liver decomposition, the larvae (2000 pcs.) were transferred to sterile saline and incubated in at 30°C in a shaking incubator for 4 hours. The resulting extract was subjected to membrane sterilization. The precipitate was filtered and centrifuged for 10 min at 10,000 rpm. The obtained extract was frozen at -30°C for further medical use.

Example 2 (SARS-COV-2 substrate infection and SARS-COV-2 inhibitor)

The growth of the larvae is stimulated only with a non-sterile medium, based on beef liver extract, on which the larvae of medical flies of the families Calipphoridae Sarcophagidae and/or Muscidae are suspended and incubated until the production of larval secretions, and then the larvae after incubation and washing with saline, is transferred to an incubator with shaking in a medium with saline and incubated at a temperature of 36°C for three hours, after which time the larvae are removed from the medium in which secretion from the digestive system of the larvae with a protein concentration of approximately 0.20 mg/mL, and the thus obtained bioactive mixture - preparation is subjected to sterilization, membrane or cellulose filter with centrifugation, and frozen for further medical use.

The medium infected with the virus - SARS-COV-2 (Munchen-1.2 2020/984) is used as a non-sterile medium.

Based on the results of the cytotoxicity test, the highest non-toxic concentration range was selected for each compound and used for further testing (Figure 2).

The experiment with SARS-CoV-2 was performed in triplicate biological replicates, in which individual concentrations of each compound were tested in triplicate. A543_ACE2 cells seeded 2 days earlier were incubated with compounds at 37°C for 1 hour. Then, the cells were infected with SARS-CoV-2 at TCID50 = 3200, the infection process was carried out at 37°C in the presence of a fresh batch of inhibitors. After 2 hours the virus was washed away by washing it three times with PBS and finally the cells were left in a medium containing inhibitors for 3 days at 37°C.

After this time, the cytopathic effect was observed and the media was collected in the lysis buffer. The controls in the graphs are cells infected with SARS-CoV-2 in the absence of inhibitors. In the case of secretion, a very pronounced inhibition of the cytopathic effect was observed (Fig. 3). RNA was isolated from the remaining samples and the number of copies of viral RNA derived from viral particles released from infected cells into the medium was determined by RT-qPCR. RT-qPCR results showed no inhibition of SARS-CoV-2 replication (Fig. 2)

An analysis of the substance secreted by medical flies (on a non-sterile medium) as potential inhibitors of SARS-CoV-2 was carried out: - a clear inhibition of the cytopathic effect caused by virus infection by medical fly secretion was observed. However, no reduction in viral RNA copies was observed in the samples.

The observed inhibition may indicate a cytoprotective mechanism in which the substance protects cells against the adverse effects of infection (cf. Fig. 3).

Example 3 (multi-pathogen-exposed medium)

The growth of the larvae is stimulated only with a non-sterile medium, based on beef liver extract, on which the larvae of the medical flies and families Calipphoridae Sarcophagidae and/or Muscidae are suspended and incubated until the production of larval secretions, and then the larvae after incubation and washing with saline, is transferred to an incubator with shaking in a medium with saline and incubated at a temperature of 36°C for three hours, after which time the larvae are removed from the medium in which secretion from the digestive system of the larvae with a protein concentration of approximately 0.20 mg/mL, and the thus obtained bioactive mixture - preparation is subjected to sterilization, membrane or cellulose filter with centrifugation, and frozen for further medical use.

The non-sterile medium is further exposed to a mixture of microbes and/or viruses and/or fungi, preferably Listeria monocytes 0.5% to 2.5% by weight.

Example 4 (medium exposed to multiple neutralized pathogens that are genetically whole or fragments)

The growth of the larvae is stimulated only with a non-sterile medium, based on beef liver extract, on which the larvae of medical flies of the families Calipphoridae Sarcophagidae and/or Muscidae are suspended and incubated until the production of larval secretions, and then the larvae after incubation and washing with saline, is transferred to an incubator with shaking in a medium with saline and incubated at a temperature of 36°C for three hours, after which time the larvae are removed from the medium in which secretion from the digestive system of the larvae with a protein concentration of approximately 0.20 mg/mL, and the thus obtained bioactive mixture - preparation is subjected to sterilization, membrane or cellulose filter with centrifugation, and frozen for further medical use. The non-sterile medium is additionally exposed, and thus activated, to at least one factor that is neutralized - killed bacteria and/or disintegrated viruses, being whole or their genetic fragments, i.e. DNA and/or RNA, or protein/sugar fragments/ lipids or combinations thereof.

Claims (13)

1. A method of obtaining a bioactive mixture, especially a protein-glycoprotein mixture in the form of a secretion from the larvae of medical flies and the Calipphoridae Sarcophagidae and/or Muscidae families, using a medium for multiplication of the larvae and their incubation, characterized in that the growth of the larvae is stimulated only with a non-sterile medium, based on an extract from beef liver, on which the larvae of medical flies from the families Calipphoridae, Sarcophagidae and/or Muscidae are hung and incubated until the production of larval secretions, and then the larvae, after incubation and rinsing with saline, are transferred to an incubator with shaking in the medium with saline and incubated at 36°C for three hours, after which time the larvae are removed from the medium in which secretion from the digestive system of the larvae with a protein concentration of 0.20 mg/mL remains, and the thus obtained bioactive mixture - the preparation is sterilized, either on a membrane or on a cellulose filter, with centrifugation, and frozen for further medical use. 2. The method of claim The method of claim 1, wherein the non-sterile medium is an infected medium with at least one microorganism. 3. The method of claim The method according to claim 1, characterized in that the medium based on beef liver extract, 3-5 days old, is used as the non-sterile medium. 4. The method of claim 1, characterized in that, as a non-sterile substrate, it is additionally subjected to an initial exposure to microorganisms for several days, 3-5 days, until putrefaction and disintegration occur. 5. The method of claim 1, characterized in that the non-sterile medium is additionally exposed to at least one strain of bacteria, fungi or the SARS-CoV-2 virus type or a mixture of microorganisms and/or viruses and/or fungi, Listeria monocytes 0.5% to 2.5% by weight. 6. The method of claim 1, characterized in that the non-sterile medium is additionally subjected to exposure, and thus activation, to at least one factor, i.e. killed bacteria and/or disintegrated viruses, being whole or their genetic fragments, i.e. DNA and/or RNA, or protein/sugar/lipid fragments or combinations thereof. 7. The method of claim The method of claim 1, characterized in that the resulting bioactive mixture is used as a medical preparation. 8. Bioactive mixture as a carrier-preparation for the treatment of symptoms of viral infection, including SARS-CoV-2 Covid 19. 9. The bioactive mixture according to claim The method according to claim 8, wherein the carrier-formulation is used as an anti-fungal, anti-bacterial and/or anti-viral preparation. 10. The bioactive mixture according to claim The method according to claim 8, characterized in that the carrier-preparation is used cytoprotectively on cells after viral, fungal and bacterial infection. 11. The bioactive mixture according to claim The method according to claim 8, characterized in that the carrier-preparation in the form of a lyophilized fraction is administered in the form of injection and/or inhalation and/or aerosol. 12. The bioactive mixture according to claim 1. 8, characterized in that the carrier-preparation is placed as a medical active substance in composite materials, hydrogels, polymers, lipid monocrystals. 13. A bioactive carrier-preparation mixture for use in the treatment of a disease to restore blood vessels and accelerate tissue regeneration.