JPH07501328A - Fungicidal products, pharmaceutical compositions and food additives containing the same - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Bactericidal products, pharmaceutical compositions and food additives containing the same This invention relates to a novel bactericidal substance having a spectrum.

Among the known bactericidal substances, it has been possible to identify some antibiotics, especially preservatives. Wear.

Preservatives are chemical agents that inhibit the growth and destroy microorganisms. Examples of these are , chlorhexidine, halogens, heavy metal salts, quaternary ammonium salts, salicylic acid and derivatives thereof.

Antibiotics were first defined as chemicals produced by microorganisms and treated in dilute solutions. Having the power to inhibit or destroy bacteria or other microorganisms. Many of these molecules are Currently, it is produced by semi-synthesis or chemical synthesis.

These molecules have bacteriostatic or bactericidal action.

Several large families of antibiotics are identified based on their structure and mode of action. It is. The main ones are β-lactams (penicillins and cephalosporins). phosphorus), aminocytes, cyclins, phenicols, macrolides, poly Myxins and quinolones. Sulfamides are another group of bactericidal constitute a product.

However, strains that are resistant to known fungicides are constantly emerging, and this characteristic rapidly develops. The search for new active products continues forever.

In addition, antibiotics with a narrow spectrum of activity or aimed at specific applications are increasingly being used. This is especially true without disturbing the natural microbiological balance within the gastrointestinal tract. This is for a good reason.

One of the advantages of the product according to the invention is that it contains bacteria, especially C. It has substantial activity against the order 1 osHid.

Indeed, it has been discovered that porphyrin derivatives have fungicidal activity.

The subject of the invention is a fungicidal product characterized in that it contains a porphine ring.

This porphine ring consists of four pyrrole rings connected by four -CH= groups. The basis of porphyrin represented by the following formula (in the formula, R=R1-R2=H) It is a ring.

The present invention relates to substances with therapeutic activity, in particular polymers for application as fungicidal products. Regarding filin derivatives.

Among the fungicidal products according to the invention, in particular R=CH3, R'=CH0H-CH3 and List the products containing hematovolphyri molecules where R2=CH2CH2COOH be able to.

Other particularly suitable fungicidal products according to the invention are selected from hemin and hematin. The present invention also provides a product having at least one molecule with a low heme group. A small amount derived from chlorophyllins, which are salts of at least some spiders or various chlorophylls. It relates to a fungicidal product containing at least one molecule.

By bonding the metal groups in the form of a complex to the four nitrogen atoms of the basic skeleton, A series of prosthetic groups suitable for practicing the invention are obtained.

However, the presence of this metal group does not appear to be essential and the bactericidal activity of the derivatives It is possible to change its properties while maintaining its properties. These parameters are It will be adapted by those skilled in the art.

The fungicidal product according to the invention is synthetic, at least for its porphyrin part. This is both for safety reasons and for specificity of activity. It has advantages in terms of

According to another aspect of the invention, the fungicidal product is a hemoglobin-containing substrate. is incubated with an enzyme mixture containing at least one protease. (and then recovering the resulting product from solution) be able to.

This bactericidal product is compatible with various hemoglobin-containing substrates, especially whole blood, erythrocytes, and erythrocytes. It can be obtained from the contents or purified hemoglobin.

An enzyme mixture containing at least one protease, especially trypsin, is added to this base. For best results, trypsin is reacted with other enzymes, such as pancreatic juice. or obtained by combining with that found in the fecal supernatant of germ-free animals. Ru.

This bactericidal product also inks hemoglobin-containing substrates with proteases. It can also be obtained by cubating. In this case, preferably about 50°C. Hydrolysis is carried out at temperature.

Incubation conditions will vary depending on the activity of the enzyme mixture and the source of the substrate. Digestion is preferably carried out at 37°C for about 24 hours; however, lower temperatures down to 20°C and about It is also possible to use periods of 1 hour to 72 hours.

After incubation, the residual solid components are removed and the product is recovered in solution. Ru.

The product according to the invention can be produced specifically by the following steps.

a) centrifuging a sample of mammalian whole blood and collecting a pellet containing red blood cells; b) After washing (e.g. with isotonic buffer), expose the pellet to distilled water to remove red blood cells. perform hemolysis; C) centrifuge the mixture; d) incubating the supernatant obtained in step C) with pancreatic juice; e) Sterilize the germicidal product solution obtained by filtration (0° 45 μm).

This blood can be obtained from a variety of mammals. This is an anti-inflammatory drug like heparin. Add coagulant and collect.

The red blood cells are centrifuged for 10 minutes at, for example, 2000 + pm and +4°C. After removal, wash with the same volume of buffer, preferably PBS buffer, then wash with the same volume of buffer, preferably PBS buffer. Hemolyze by adding distilled water to remove cell debris from the red blood cell contents, e.g. ．． Separate by centrifugation for 10 minutes at 1 (IQ + pm, +4°C).

If the resulting extract is not immediately subjected to enzyme action, it should be stored at +4°C or It can be frozen at -20°C.

Incubation in the presence of an enzyme mixture more particularly involves incubating the substrate with the enzyme. After dilution in preparation, it is carried out under aerobic conditions at 37° C. for 24 hours.

According to another aspect of the invention, the bactericidal product is a purified human and veterinary product. can be obtained by incubating hemoglobin with pancreatic juice. can. This product can be sterilized by filtration.

If incubation is performed with pancreatic juice, use porcine pancreatic juice diluted to 2%. It is preferable to

Serum derived from coagulation, blood separated by centrifugation from heparinized blood Acting an enzyme mixture as defined above on plasma or red blood cell walls separated after hemolysis. A product without fungicidal activity is obtained. Similarly, add the enzyme mixture to globin Incubating with only This indicates that a program is required. When the enzyme mixture acts on whole blood, it produces bactericidal The activity of the product will be lower than that obtained from red blood cell contents.

Usually the bactericidal activity of this product is carried out by the water-insoluble porphyrin fraction. Ru.

Accordingly, preferred water-soluble products of the invention optionally have substituents and / or complexed with a metal and 3 to 10 amino acid residues, more particularly contains a porphine ring attached to amino acids derived from a globin chain.

Chromatographic analysis of crude hemoglobin hydrolyzate with protease K When done, the smallest water-soluble molecules with bactericidal activity can be isolated and purified. Ru.

This is a compound that has absorption at 214 nm and 380 nm, and has absorption at 600 nm. It has an apparent molecular weight of from 1000 Daltons.

These compounds have good bactericidal activity, which allows for good distribution into biological fluids. enhanced by the enabling solubility.

When this product is orally administered to germ-free mice, the activity of the product is preserved in the intestinal contents. It can be observed that Therefore, it is a product that remains in the lumen of the gastrointestinal tract without being absorbed. It is a substance that cannot be broken down by digestive enzymes.

If desired, the fungicidal product can be further improved by containing a larger number of amino acids. It can increase the solubility and have systemic activity beyond the barrier of digestion. Ru.

Active molecules have a porphyrin group as a common structure; The remaining amino acid residues are different.

These can be autoclaved.

The fungicidal product according to the invention consists essentially of peptides derived from globin. It is a tidal product. However, the resulting product naturally has a large number of amino acids. It consists of a mixture of different molecules.

This is a stable product that remains active even after treatment at 60° C. for 15 minutes.

This product can be lyophilized.

Furthermore, it is stable against the action of proteases for 24 hours at 50°C.

This product is clearly derived from hemoglobin, which contains all or part of heme. That's it. The fungicidal product according to the invention is suitable for obligately anaerobic Durham-positive bacteria, especially C1 Bacteria of the genus ost+idium, such as C1osj+idiumpe+l+ing ens and C1osl+idium bully+icum% and human bacteria Active against spore-forming bacteria isolated from flora.

Inactive against some strains of C1ost+idium dillicile . Other strains of the main bacterial flora for adults, such as bilides 5peplos Heptococci and Eubacje+ium have activity.

It also has activity against bacteria of the genus Bxillus.

Therefore, this product is of natural origin and is associated with particularly dangerous infectious diseases. It is particularly important because it has activity against microorganisms such as C1osl. +idiumbutY “icum is involved in neonatal necrotizing enterocolitis.

Therefore, this product represents a new generation of inhibitors that can be freeze-dried and filter-sterilized. It is something. This purified product can be used in particular as a food additive, e.g. Diseases seen in pigs during weaning can be prevented.

The bacterial product according to the invention can also be used as a preservative.

The invention therefore provides for the use of the products according to the invention as active ingredients, especially by the oral route. The present invention relates to pharmaceutical compositions containing excipients compatible with therapeutic use.

These include, inter alia, lozenges, tablets, powders or other forms that can be administered orally. It can be a composition of.

The invention relates to a tablet intended for nutrition, in particular for animal nutrition, comprising a product according to the invention. Contains concentrates and is intended to be added to actual feed or drinking water; It also relates to mixtures of animal feed containing the products of the invention.

Other features and advantages of the invention will become apparent from an understanding of the following examples. Will.

Figure 1. Illustration of the bactericidal effect of inhibitors produced by ES hydrolysates.

Example 1: Preparation of bactericidal substances from whole blood Whole blood or its fractions (washed red blood cells, red blood cell contents) are treated with digestive enzymes. .

a) Preparation of blood matrix Blood obtained from any mammal, human or animal, or by conventional blood fractionation methods. You can use the various amounts obtained by Serum derived from coagulation, Centrifuge plasma and heparin-treated blood at 2000+pm for 10 minutes at +4°C. The separated supernatant, Centrifuge pellets of red blood cells, heparinized blood, in 0.01 M PBS for 3 min. Washed twice, washed and centrifuged (13,000+pm, 10 minutes, 4°C) ) The red blood cell wall and red blood cell hemolysate obtained after hemolyzing the red blood cells in distilled water are centrifuged. Separated supernatant, red blood cell contents (E).

b) Enzyme preparation Use the following.

10x freshly collected feces from germ-free (microbe-free) rats or mice. The supernatant diluted to 01M PBS.

Purified bovine trypsin (Sigma Ta205) 5mg/ml P.B.S.

Pancreatic juice and trypsin are prepared immediately before use.

C) Incubation of substrate with enzyme preparation Blood substrate, freshly prepared in enzyme preparations or stored in portions at room temperature or at low temperature. It was diluted 0 times and 20 times and kept at 37° C. for 24 hours. The results are shown in Table 1.

Human whole blood, washed red blood cell pellets, or soluble fractions of hemolysed red blood cells were added to the pancreas. Incubation with visceral enzymes produces bactericidal activity in vitro.

The production of inhibitors is by enzymatic method, with control, blood substrate and enzyme alone. It has no effect.

This substrate is not a component of serum or plasma. This enzyme activity is activated against red blood cells. but against the red blood cell contents rather than its walls.

The bactericidal effect is weak in whole blood, probably related to the presence of trypsin inhibitors. There is. Bactericidal activity is the action of enzyme mixtures present in the feces or pancreatic juice of germ-free animals. The latter is higher than that with trypsin alone.

Example 3. Preparation of bactericidal substances from hemoglobin crude, preferably purified human Hemoglobin (H) derived from (Hl) or animal (H2) is combined with any type of protein. This does not necessarily have to be a digestive protease. refined Proteinases contain proteins, glycoproteins, peptides and amino acids. It is preferable to use one that hydrolyzes acid esters.

a) Preparation of hemoglobin solution Hl: Dilute human hemoglobin manufactured by Sigma (Sigma H73791°Hl) , used at the same concentration as that of adult human blood.

Dilute 12.5-15 g of hemoglobin in 100 ml of 0.01 M PBS do.

H2 crude or purified bovine hemoglobin (Sigma H2625-H2S [10). Dilute H2 10 times and give 1.2 to 1.5 g/P B 8100 m Purify in the same manner as Hl, except for using Hl.

b) Enzyme product A stock solution of 10 mg/ml of proteinase was added in Q, 01M Tris buffer. Prepare with H7,9. Add this solution Stabilize in the form of CaCl2 with 2mM Ca.

C) Hydrolysis conditions Using proteinase K at various concentrations, preferably the final concentration of hemoglobin Hydrolyze bottle 0.1 mg/ml. This hydrolysis is preferably carried out slowly at 50°C. Do this while stirring. This hydrolysis time varies between 1 hour and 24 hours. can be done. The hydrolyzate obtained after 2 hours of incubation is good. produces significant antibiotic activity.

Example 4: Inhibitory efficacy of various types of hydrolysates The obtained hydrolysates were 0.1 m g/m I proteinase-treated red blood cell contents or purified hemog Robin has been shown to produce a BFR inhibitor with efficacy up to I/128. There is. The amount of bactericidal substances produced depends on the concentration of hemoglobin that is hydrolyzed do. Hydrolysis of red blood cell contents by pancreatic juice is more diffusive and therefore Further soluble substances are produced.

The results are shown in Table 2.

1. Production of bactericidal substances from hemoglobin fraction a) From hemoglobin H1 Chemical Extraction of Heme From extraction of globin from heme proteins by removal of heme. Methods of F, ^5coli, etc. (Methods inenBmoloB, vol. ,, 76.5.1981) is used. This method uses hemoglobin under the conditions described. From mixing 2.5 ml of phosphorus with 100 ml of a solution of hydrochloric acid and acetone Become. The precipitate or centrifugation pellet contains globin, which can be solubilized in PBS. Including. The supernatant contains heme. To test the activity of this supernatant and alcohol The 5peed Evaporate it using Vac. This concentrate is insoluble in PBS, but It can be partially solubilized in 60% acetonitrile dissolved in % trifluoroacetic acid. At 0.1, it is inactive against BFR cells.

b) Bactericidal action of purified human hemoglobin fraction treated with HCI mixture The results shown in Table 3 show that the active ingredient of inhibition is the dark blood fraction, i.e. the hemin fraction. It is shown that it is localized in minutes. The globin fraction is inactive, but the hemin fraction is The active hydrolyzate already used to produce a bactericidal product insoluble in PBS. have different chemical structures.

Properties of (Fe or Mg) or chemically bonded (FeC1, Fe0H) is not significant. The ring is insoluble in water, but the protease used (e.g. pancreatic juice) and the degree of denaturation of the bound globin by the potency of protease K). chemically solubilized (in the case of chlorophyllin) or in a somewhat soluble form ( (in the case of hydrolyzate). All porphyrin-containing pigment proteins The protein produces inhibitory substances from its prosthetic groups. In the case of hemoglobin, Enzymatic hydrolysis is cheaper and more specific than chemical extraction; Active hemin molecules have different degrees of efficiency and water solubility depending on the type (e.g. ES and HK) are produced.

Proof of antibiotic activity against target bacterial cells 1, Principles of antibiotic activity test and implementation 108 viable viable cells of the bacterial strain used as target were added to 15 ml of Inoculation was carried out within the medium cap or on the surface.

Heart-plain medium (Dilco 0037) 37g/1 yeast extract ( Dilco 0127) 5 g/1B11ck agar (Dilco 013) 8) 14 g/1 well cut within that depth of agar in a Petri dish. , and 30 μl of test antibiotic. Antibiotics after incubation Quality activity is determined by measuring the radius of total lytic plaques formed around the wells in mm.

2. Reference target strain Strains of Bicillus Iυbtilis characterized by easy handling BFR strain, which is a type of An mBsophilic strain capable of generating a population was isolated from canned food. use This strain does not form spores in the medium described above. This strain has various types are highly sensitive to antibiotics, and various hydrolysates have been tested, while their on the other hand, to explore and evaluate the results of the active fraction in the purification process. It is used for

3. Effects of various physical and chemical treatments on the activity of ES hydrolyzate 4. Proof of the bactericidal effect of inhibitory substances produced by ES hydrolyzate Contains 9 ml of agar-free medium containing increasing dilutions of crude ES hydrolyzate. Inoculate 1 ml of viable BFR cells from a population of 3 x 107 into test tubes.

After 8 hours of contact at 37°C, the number of viable cells and half of the antibiotic zone were determined in each tube. Measure the diameter.

The results are shown in Figure 1.

Bacterial growth was observed using ES hydrolyzate as is and diluted up to 32 times. and kill the inoculated bacteria. A 32-fold dilution is the lowest inhibitory concentration at which the bactericidal effect begins. All strains, with the exception of highly collected strains or strains obtained from Belgium, were collected in the laboratory. were isolated from the main microbiota in the intestines of children and adults. The pediatric sample is A. obtained from the neonatal department of Becle+e Hospital, these premature children are At ~2 months, there was no intestinal pathology. Adult samples were collected from healthy, hospitalized patients. from an individual or patient (poucbitej or colitis).

Characterization of laboratory isolated strains These are classified by type of morphology. Some of these are 20 different types using biochemical reactions (API20A) and the final products of its fermentative metabolism (non-volatile It is characterized by using volatile acids and volatile fatty acids).

Test method for bactericidal activity against bacterial strains Any anaerobic strain is tested as control strain 11acillus FR.

Two techniques were used for obligate anaerobic strains:

A strain of bacteria that is highly sensitive to oxygen (EOS) is cultivated in an anaerobic chamber (Fret ed). These were inoculated onto the surface of agar and incubated in an anaerobic room at 37°C for 48 hours. tion.

Anaerobic strains, which are less sensitive to oxygen, are inoculated outside into agar caps. these was cultured in an anaerobic jar at 37°C for 48 hours (Ga+pack s7slem ).

Susceptibility testing of various bacterial strains to crude or 3-fold concentrated hydrolysis products: Freeze the anti-crude hydrolyzate of 82 to H2 in the gastrointestinal tract of germ-free (bacteria-free) mice. Dry, concentrate 10 times, and sterilize at 100℃ for 30 minutes. , administered to germ-free mice using a gastric tube using the following procedure.

Table 6 A 10-fold dilution of intestinal contents in PBS was evaluated for target cell BFR and the results The results are shown in Table 7.

Antibiotics contained in the crude hydrolyzate are not absorbed. This is a disinfection method for germ-free mice. 2 hours after initial uptake, all contents and feces are removed. It reappears in a form active against BFH.

In control mice, the appearance of a small antibiotic zone in IG3 can respond to the inhibitory effect of digestive proteases secreted by

Table 7 Purification and preliminary characterization of substances a) Purification by fractionation of water-soluble portion The crude hydrolysis product was treated with Frxclogel Butyl TSK 650.5 Elute on the column. Active fractions are identified using an antibiotic test against BFR. Ru. The results obtained from the chromatogram show that after both fractions (fraction I) are not retained, The first retained fraction (fraction 11) was shown to have an antibiotic effect. There is. The active fraction eluted with 20% ethanol was added to the insoluble part of the HIK antibiotic. It seems to be compatible.

b) Purification of water-soluble active fraction Crude hydrolysis product and F+acroHI BO171 series fractions ① and 1 Comparative CI8HP L C chromatogram for 1 with buffer 8100% Only the fractions eluted with absorption at 214 and 380 nm were found to be active. It shows.

This active fraction was purified from eluate 11, Fzc+ogel Bu+71. Obtained in a state.

C) Measurement of molecular weight 0 18HP LC purification investigated by 5ephadex LH2G gel filtration Fractions with absorptions at 2+4 and 380 nm and corresponding molecular weights between 600 and 10 Elutes as a single active fraction of 0.00 daltons.

International investigation report DrTln Q21n1nG1 front page continuation (51) Int, C1' Identification symbol Internal office reference number A23L 3/3544 502 7432-4BA61K 31/40 9454-4C35/18 7 431-4C 35/39 7431-4C 38100ADZ (72) Inventor: Revo, Vinyl France: Jouie-en-Jossa, Le, Pal, Darbien, Abounus, Georges Fremenceau, 38 I

Claims (18)

[Claims] 1. A fungicidal product characterized in that it contains a porphine ring. 2. The hemoglobin-containing substrate is treated with an enzyme mixture containing at least one protease. by collecting the resulting product solution after incubation with the compound. A fungicidal product characterized in that it can be produced by: 3. Claim No. 1, wherein the enzyme mixture comprises at least trypsin or an equivalent enzyme. A fungicidal product according to paragraph 1 or paragraph 2. 4. According to any one of claims 1 to 3, wherein the enzyme mixture is pancreatic juice. Products listed. 5. Any of claims 1 to 3, wherein the protease is proteinase K. The fungicidal product according to item 1. 6. According to any one of claims 1 to 5, wherein the substrate consists of red blood cell contents. Products as described. 7. Any one of claims 1 to 6, containing at least a part of heme group. A fungicidal product as described in. 8. 2. A fungicidal product according to claim 1, comprising hematoporphyrin molecules. 9. Claims comprising at least one molecule selected from hemin and hematin. 1. A fungicidal product according to item 1. 10. comprising at least one chlorophyllin molecule. Bactericidal products. 11. Claims further comprising at least three amino acids attached to the porphine ring. A fungicidal product according to any one of paragraphs 1 and 7 to 10. 12. A fungicidal product according to claim 11 having a molecular mass of about 1000 Daltons. . 13. a) Centrifuge the mammalian whole blood sample and collect the pellet containing red blood cells. , b) After washing, expose the pellet to distilled water to perform hemolysis of the red blood cells; c) centrifuging the mixture; d) incubating the supernatant obtained in step c) with pancreatic juice; e) recovering the bacterial product which can be filter sterilized; Claims 1 to 7, 11 and 11 can be manufactured by steps. and a fungicidal product according to any one of paragraphs 12 and 12. 14. produced by incubating hemoglobin with pancreatic juice 5. A fungicidal product according to any one of claims 1 to 4, which can be used as a disinfectant. 15. According to any one of claims 1 to 14, which is in a freeze-dried form. Products as described. 16. At least one of the products according to any one of claims 1 to 15 A crude pharmaceutical product comprising one. 17. 16. A pharmaceutical composition according to claim 15, formulated for oral administration. 18. comprising a product according to any one of claims 1 to 15. ,Food additive.