DE1211754B - Preservative to combat bacterial growth - Google Patents

Description

FEDERAL REPUBLIC OF GERMANY

GERMAN

PATENT OFFICE

EDITORIAL

Int. Cl .:

A 611

German class: 30 i - 3

Number: 1211754

File number: S 79683IV a / 30 i

Filing date: May 29, 1962

Opening day: March 3, 1966

R. H. Ha 11 and G. O. G a 1 e (Proc. Soc. Exper. Biology and Medicine, 103, pp. 234 to 238 [1960]) have shown that kinetin, that is 6-furfurylamine purine, as well as some other purines which are substituted in the 6-position amino group increase the effectiveness of the antibiotic compound chlorotetracylin against Staphylococcus albus, although they themselves are not toxic to the strain of Staphylococcus albus, the are an antibiotic-resistant strain. Kinetin causes a minor but statistically significant one Increase in the effectiveness of chlorotetracycline as well as the related antibiotic compound Tetracycline, against an antibiotic-sensitive strain of Staphylococcus aureus.

It has now been found, surprisingly, that N ^ -benzyl-adenine has a strong increase the effectiveness of antibiotics against antibiotic sensitive strains of many different organisms evokes.

The r \ ^{T | i} -benzyladenine is an amphoteric compound and forms with both acids and bases with readily salts. In order to change the solubility of the adenine compound, it can be advantageous to use the N ⁽ⁱ -benzyladenine as a salt.

So salts of inorganic acids, such as hydrohalic acids, in particular of Hydrochloric and hydrobromic acids, as well as those of sulfuric acid, Phosphoric acid and boric acid. Both complete and partial salts can be formed. Organic acid salts can also be used. Examples of suitable organic Acids are the aliphatic mono- and polycarboxylic acids, of which the alkane mono- and dicarboxylic acids having up to 10 carbon atoms are preferably used. This also includes the substituted, for example the halogenated acids, the hydroxyl-substituted acids and similar, alkane and arylsulfonic acids, phosphonic acids. Phosphinic acids, phosphorous acid, their partial esters and the like.

The salts of bases include both those of the inorganic and those of organic bases. The alkali and alkaline earth salts and the salts of others are particularly suitable inorganic bases, such as the ammonium salts and the salts of polyvalent metals. Are suitable likewise the quaternary ammonium salts, the sulphonium salts and the phosphonium salts.

The use of the N "-benzyladenine as a synergistic on antibiotics, which is the subject of the present invention, is outside the preservative for combating
Bacterial growth

Applicant:

Shell Internationale Research Maatschappij N.V., The Hague

Representative:

Dr.-Ing. F. Wuesthoff, Dipl.-Ing. G. Pulse and
Dipl.-Chem. Dr. rer. nat. E. Frhr. v. Pechmann, patent attorneys, Munich 9, Schweigerstr. 2

Named as inventor:

Marvin Legator, Modesto, Calif. (V. St. A.)

Claimed priority:

V. Si. ν. America May 31, 1961 (113 598)

medical application. Because the commonly used antibiotics are not only effective inhibitors against the reproduction of pathogenic microorganisms, but also against the a large number of other bacteria that attack human and animal food. One himself constantly expanding range of application of antibiotics in the field of »non-medical Use «is offered to the animal nutritionist, the industrial bacteriologist and the Plant pathologists. One of the common "non-medical uses" of antibiotics their use as feed additives, feed preservatives and pesticides. They also have animal growth-promoting properties.

A large amount of food goes off every year as a result of the action of microorganisms. Indeed, the microorganisms infest the food! of humans and animals. Various antibiotics, such as chloramphenicol, Chlortetracycline and oxytetracycline. are used extensively for the preservation of Allow beef, fish and poultry meat. Plant diseases limit crop yields of fruits, vegetables and field crops considerably. The use of antibiotics, like for example that of streptomycin, for the control of plant pathogens has in the increased sharply in recent years.

609 510/377

Due to the widespread use of antibiotics as supplementary feed, as feed preservatives and in the fight against plant pathogens the problem of antibiotic arises Leftovers in human food. Remaining amounts of it can be found in meat or milk or eggs come from animals whose feed either contained antibiotics, or which had been treated with therapeutic amounts of antibiotics. Leftovers from antibiotics can also occur in feed and in foods that have been treated with antibiotics to protect you Spoilage of the same or to prevent plant diseases.

Could the amount of antibiotics required for "non-medical use" go even further are reduced than is usually the case, but their effectiveness can be increased, for example about four to six times, so there could be various disadvantages and setbacks of the "non-medical Use «the antibiotics can be overcome, because then a smaller amount of the same for the achievement of the desired Purpose would be needed. The present invention provides a solution to this problem.

The invention is concerned with antibiotic preparations which, in addition to an antibiotic, have a synergistic amount of N ⁶ -benzyladenine. Preferred antibiotic preparations according to the invention contain an antibiotic with a broad spectrum of activity, ie with a spectrum against a large variety of organisms. In particular, they contain an antibiotic produced by representatives of the Streptomyces strain and by synthetic derivatives thereof, hereinafter referred to as Streptomyces-type antibiotics.

The synergistic effect of the N ^e -benzyladenine is a completely unexpected one, because until now it has been held that the individual mode of action of the antibiotic and the synergistic substance are completely opposite. It is well known that antibiotics such as chloramphenicol inhibit protein synthesis. The N ^e -benzyladenine, on the other hand, increases protein synthesis or inhibits the normal rate of degradation of proteins.

The antibiotics that can be used according to the invention include, for example, the penicillins, aspergillic acid, Clavacin, penicillic acid, citrinin, gliotoxin, fumigacin, chaetomin, penatin, tyrothricin, gramicidin, Tyrocidin, Gramicidin S, Bacitracin, Subtilin, Colistatin, Eumycin, Licheniformin, Aerosporin, Polymyxin and chlorellin. As noted above, eminent representatives are preferred Antibiotics with a wide spectrum of activity, especially those used by representatives of the tribe Streptomyces and synthetic derivatives thereof, such as streptomycin, Dihydrostreptomycin, chlortetracycline, chloramphenicol, oxytetracycline, tetracycline, neomycin, erythromycin, Carbomycin and viomycin. Of the Streptomyces-type antibiotics, those are most preferably those belonging to the group of chloramphenicol and tetracycline. To this Group includes such well-known antibiotics as chloramphenicol, chlortetracycline, oxytetracycline and tetracycline.

example 1

The synergistic effect of N ⁶ -benzyladenine on chloramphenicol was demonstrated by carrying out the standard test by means of serial dilution.

Two sets of twenty-one test tubes each, each of which has 5 ml of nutrient solution Emerson were inoculated with B. subtilis, that had grown for 24 hours. Chloramphenicol was added to the test jars of the first set given in different doses: nothing was added to the first three test glasses; in 1 part per 1,000,000 by weight was added to each of the following three glasses. In a 2 parts per 1,000,000 were added to each of the following three glasses; and the remaining twelve test glasses were divided into four groups of three, each with a dose of 5, 10, and 20 40 parts by weight to 1,000,000.

The second set of test tubes was charged with different amounts of chloramphenicol in the same way. In addition, however, 5 ^{parts by weight per 1000,000 N ß} -benzyladenine in the form of a solution of 1% by weight of N ^e -benzyladenine, 10% by weight of acetic acid and 89% by weight of water were added to each test tube.

The two sets of test tubes were incubated for 16 hours at 37 ° C. In the following Table shows the growth of B. subtilis as it is visually in each group of three test glasses treated in the same way could be observed. A minus sign means that there was no discernible growth and a plus sign means that there was clearly discernible growth Growth was present.

Parts by weight

1 million in inflicted

Chloramphenicol

Growth of

B. subtilis without

N ° -Benzyladenine

Growth of

B. subtilis with

N ⁶ benzyladenine

10

20th

40

From this table it can be seen that N ^G -benzyladenine almost fivefold the inhibitory effect of chloramphenicol. This could essentially be confirmed with the help of the standard agar diffusion test (standard agar diffusion test). The effect of N ^ß -benzyladenine alone was also investigated using this method. Six dishes, each containing approximately the same amount of agar, were each inoculated with B. subtilis that had grown for 24 hours. Seven antibiotic test discs impregnated with chloramphenicol in a concentration of 0, 10, 20, 40, 60, 80, 100 parts by weight to 1000,000 were placed in two of the test dishes. Seven antibiotic test disks impregnated with chloramphenicol in the same concentrations were placed in two other test dishes, with each disk additionally being used

50 parts by weight to 1,000,000 of N «-benzyladenine was impregnated. In each of the other two test ^{dishes, seven antibiotic test disks were placed, each of which was impregnated with N u} -benzyladenine in a concentration of 0, 10, 20, 40, 60, 50 and 100 parts by weight to 1,000,000. All experimental dishes were incubated for 6 hours at 37 ° C. The following table shows the results of the experiments in which chloramphenicol was added in various concentrations, expressed by the width of the zones of growth inhibition observed, measured in millimeters:

size size Divide up to 1 million
at added of growth
inhibition zone of growth
inhibition zone Chloramphenicol without
N ^B benzyladenine with
N ' ^: -Benzyladenine mm mm 0 0-0 0-0 10 0-0 9-9 20th 0-0 12-12 40 9-9 14-15 60 10-13 18-17 80 12-13 20-19 100 16-17 22-20

Divide up to 1 million
added N ^ -Benzyladenine Size of growth
inhibition zone mm 0 0-0 10 0-0 20th 0-0 40 O-O 60 0-0 80 0-0 100 0-0

Parts by weight
to 1 million Parts by weight
to 1 million size
of growth Ν '' - benzyladenine Chloramphenicol inhibition zone
mm 0 5 11 0 5 10 0 5 10 1 5 11 1 5 11 1 5 10 10 5 17th 10 5 17th 10 5 17th

15 The experiments were repeated using streptomycin as an antibiotic. The corresponding Results are given in the table below.

25th

The following table summarizes the results of the experiments which were carried out with various concentrations of N ⁶ -benzyladenine, but without the addition of chloramphenicol.

35

Parts by weight
to 1 million Parts by weight
to 1 million size
the growth N'v Benzyladenin Streptomycin mm 0 2 19th 0 2 19th 0 2 19th 1 2 19th 1 2 19th 1 2 20th 10 2 20th 10 2 20th 10 2 20th

The experiment was also repeated using erythromycin as an antibiotic. the the following table contains the results obtained.

40

45

It is clear from this that N "-benzyladenine approximated the inhibitory effect of chloramphenicol four to six times, whereas this compound alone has no inhibitory effect shows.

Example 2

N -benzyladenine ^ß was mixed in a concentration of 0.1 to 10 parts by weight of 1 000 000 for Emerson agar, which was then used for the fitting-out of nine test shells. Each experimental dish was inoculated with 1.5 ml of a solution containing S. aureus grown for 24 hours. Then, in the same manner as in Example 1, experimental antibiotic disks each containing 5 parts by weight per 1,000,000 of chloramphenicol were placed in the dishes. The dishes were ring inoculated at 37 ° C for 24 hours. The results obtained are shown in the table below.

Parts by weight Parts by weight size
fler AVacTistums- to 1 million to 1 million inVn Hit i nn sznn e N ^e -benzyladenine Erythromycin 1111 Il L * J. Il UU öaAWAW
mm 0 2 18th 0 2 19th 0 2 17th 1 -> 19th 1 2 19th 1 2 19th 10 2 19th 10 2 19th 10 2 20th

Claims (1)

Claim: Preservative for combating bacterial growth, containing an antibiotic compound and a synergistic purine compound which contains a substituted amino group in the 6-position, characterized in that the purine compound present is N ^G -benzyladenine, optionally in the form of one of its salts. Considered publications:
U.S. Patent No. 3,126,318. 609 510/377 2.66 © Bundesdruckerei Berlin