that an antibiotic substance crystallized out in large amounts from this crude

Transcription

1 TOXICITY AND ANTIBIOTIC ACTIVITY OF KOJIC ACID PRODUCED BY ASPERGILLUS LUTEO-VIRESCENS HARRY E. MORTON, WALTER KOCHOLATY, RENATE JUNOWICZ- KOCHOLATY, AND ALBERT KELNER The William Pepper Laboratory of Clinical Medicine and the Departments of Bacteriology and Physiological Chemistry, School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania Received for publication July 9, 95 In a systematic investigation for new antibiotics it was observed by one of us (A. K.) that the crude culture filtrate of Aspergillus luteo-virescens was found to be antagonistic to gram-positive and gram-negative bacteria. In the chemical studies conducted by two of us (W. K., R. K.) it was noted that an antibiotic substance crystallized out in large amounts from this crude culture upon standing in the refrigerator, sometimes even at room temperature. The recrystallized material had a melting point of 5 C and gave a deep red color with FeCl. It was suggested by Dr. Charles Thom that this material resembled kojic acid, 5-hydroxy--hydroxymethyl-8-pyrone, C6H60, isolated some thirty years ago (Saito, 907; Yabuta, 9, 96). Subsequent study confirmed this identification. Kojic acid is a weak bacterial antagonist, its range of activity being in a dilution of :,000 to,000 against the common test bacteria. EXPERIMENTAL Aspergillus luteo-virescens forms kojic acid from a great variety of carbohydrates. However, glucose seems to be the best carbohydrate under the conditions employed. The medium adopted was the Raulin-Thom medium (Raistrick et al., 9), containing 8 times the normal amount of glucose (00 g per liter). For the production of kojic acid the strain of Aspergillus luteo-virescens was grown on this medium for 8 to 0 days at 8 C, the filtered crude culture placed in the refrigerator, and the crystals of kojic acid (which at times precipitated at room temperature during the removal of the pellicle) removed after standing for days at 0 C. Recrystallization from hot water or vacuum sublimation yields a colorless product. From liter of crude culture 50 g of pure kojic acid will precipitate out at 0 C and another 0 to 50 g will remain in the mother liquor. Colorimetric determination of the kojic acid in the mother liquid with FeCl or isolation of the kojic acid as the copper salt showed that all antibiotic activity of the mold filtrate was due to kojic acid. The melting point of the product was 5 C. Dr. George H. Connitt of the Smith, Kline, and French Laboratories obtained a mixed melting point with I This project has been supported by the Thos. H. Dougherty, Jr., Fund, and by grants from Smith, Kline and French Laboratories and the Department of Agriculture, Commonwealth of Pennsylvania. 579 Downloaded from on May, 08 by guest

2 580 MORTON, KOCHOLATY, JUNOWICZ-KOCHOLATY, AND KELNER this product and commercial kojic acid which showed no depression. He also prepared the diacetyl derivative and found it to have a melting point of 0 C, coinciding with the melting point of the kojic acid derivative reported (Yabuta, 96). Kojic acid gives a strong red color with FeCIl and reduces Fehling's solution. Prescott and Dunn (90) have reviewed the work on kojic acid and have compiled a large bibliography. They report that the microorganisms producing kojic acid are: A8pergillus glaucus, Aspergillus oryzae, Aspergillus flavus, Aspergillus gymnosardae, Aspergillus awamori, Aspergillus candidus, AspergiUua clavatus, Aspergillus fumigatus, Aspergillus giganteus, Aspergillus albus, Aspergillus effusus, Aspergillus nidulans, Aspergillus parasiticus, Penicillium dalae, and several species of Acetobacter. Jennings and Williams (95) reported the production of kojic acid by Aspergilu effusus Tiraboshi. To this list of orgaisms which produce kojic acid can now be added Aspergillus luteo-virescens. TABLE Toxicity of kojic acid for mice Nu "BR ONMCZ INECTED XOIC ACM NMZR OF ICE DYING NUMBER 0 MICE SUER. NUMBER 0 MICE INJECTED VIVIG AT 5 DAYS m8g per mou 6(0 * 0 50 * 0 0 * 0 0 * * All deaths took place in less than hours. The crude filtrate of a culture of Aspergillus luteo-virescens, grown for 5 days in Raulin-Thom medium containing 0 per cent glucose, inhibited Staphylococcus aureus at :5, Escherichia coli at :5, Eberthella typhosa at :5, and Pseudomonas aeruginosa at :0. In some experiments E. typhosa was more sensitive than E. coli. Large-scale production of kojic acid is usually based on the studies of May, Herrick, Moyer, and Wells (95) on its production by oxidative fermentation of glucose by such fungi as Aspergillus flavus and Penicillium chrysogenum. II Toxicity of Kojic Acid for Mice White mice weighing about 7 g each were used. Solutions of kojic acid were prepared in sterile 0.85 per cent NaCl solution. Final ph of 5 per cent solution of kojic acid in 0.85 per cent NaCl was.7 by brom cresol green. All injections were made intraperitoneally. In another experiment the kojic acid was dissolved in sterile distilled water and adjusted to ph 7. with sterile N NaOH. The results following the intra- Downloaded from on May, 08 by guest

3 TOXICITY AND ANTIBIOTIC ACTIVITY OF KOJIC ACID 58 TABLE Antibacterial activity of kojic acid ORGANISMt NUMB R CULTURE CONCENTRATION OF KOJIC ACD STRAINS.~EDIUM~ Complete inhibition Aerobacter aerogenes... Alcaligenes faecalis... Bacillus anthracis... Bacillus megatherium... Bacillus mesentericus... Bacillus mycoides... Bacillus novus... Bacillus subtilis... Brucella abortus... Brucella melitensis "R"... Brucella suis... Chromobacterium indicum... Chromobacterium violaceum... Clostridium botulinum... Clostridium novyi. Clostridium histolyticum. Clostridium perfringens... Clostridium sporogenes... Clostridium tetani... Clostridium putrificum... Corynebacterium diphtheriae.. Corynebacterium xerosis... Diplococcus pneumoniae, type and "S"... Diplococcus pneumoniae, types,, 5,... Diplococcus pneumoniae, types 6, 7,... Diplococcus pneumoniae, type... Eberthella typhosa... Escherichia coli... Escherichia 8p... Gaffkya tetragena... Klebsiella pneumoniae... Leptospira canicola... Leptospira icterohemorrhagiae... Micrococcus aurantiacus... Micrococcus roseus... Neisseria gonorrhoeae... Neisseria intracellularis... Neisseria perfiava... Neisseria sicca... 5 () () () () () No inhibition :,000 :,000 :,000 :,000 :,000 :,000 :,000 :,000 :0,000 :,000,000 :0,000 :,000 t<:,000 :,000-:,000 :,000 :500 :500 :500 :500 :500 :,000 :,000 :,000 :,000 :,000-:,000 <:,000 :,000 :,000-:,000 :,000-:,000 :0,000,000 :,000,000 :,000 :,000 :,000 :0,000 :,000 :,000 :,000 :,000 :.,000 :500-:,000 :500 :500-:,000 :,000 >:500 :500 :,000 >:500 :,000 :,000 :,000 :500 :,000 :,000-> :500 :500 >:500 >:500 :,000 :500 :,000 >:500 :500-:,000 :,000 :500->:500 :500-:,000 :500-:,000 :,000,000 :00,000 >:500 :500 :,000 :,000 :,000 Downloaded from on May, 08 by guest

4 58 MORTON, KOCHOLATY, JUNOWICZ-KOCHOLATY, AND KELNER Pasteurella caviae... Pasteurella pestis... Pasteurella pestis... Pasteurella avicida... Pasteurella boviseptica... Pasteurella equiseptica... Pasteurella oviseptica... Proteus mirabilis... Proteus vulgaris... Pseudomonas aeruginosa... Pseudomonas fluorescens... Salmonella aertrycke... Salmonella cholerae-suis.. Salmonella enteritidis... Salmonella paratyphi... Salmonella pullorum... Salmonella schottmuelleri... Sarcina lutea... Shigella dysenteriae... Shigella paradysenteriae... Serratia marcescens... Spirillum rubrum... Staphylococcus albus... Staphylococcus aureus... Staphylococcus citreus... Streptococcus, alpha... Streptococcus, alpha... Streptococcus, beta... Streptococcus, beta... Streptococcus faecalis... Vibrio comma... Vibrio proteus... Vibrio metchnikovii... Vibrio schuylkilliensis... TABLE -Continued NUMBER CULTURE CONCENTRATION OF KOJIC ACD STRANS DIU No inhibition Complete inhibition (6) :,000 :0,000 :,000 :,000 :,000 :,000 :,000 :,000 :,000 :,000 <:,000 :,000 :,000 :,000 :,000 :,000 :,000 :,000 <:,000-:,000 :,000-:,000 :,000 :0,000 :,000 :,000-:,000 :,000 :500 :,000 :500 :,000 :500 :0,000 :00,000 :0,000 :0,000 :500 :,000 :500 :500 :500-> :500 :,000 :,000 :500 :500 :500-:,000 :,000 :500 :,000 :500-:,000 :500 :500 :,000 >:500 :500->:500 >:500 :500 :500 :,000 :,000 :,000 :,000 Media used: Extract agar containing Bacto peptone, per cent; Bacto beef extract, 0. per cent; sodium chloride, 0.85 per cent; and agar, per cent. Reaction, ph 7.. () Bacto tryptose agar to which was added 0 per cent sterile, normal, defibrinated horse blood. Blood extract agar. One per cent sterile, normal, defibrinated horse blood added to extract agar. () SchUiffner's medium (Meyer, Stewart-Anderson, and Eddie, 99). Bacto proteose no. chocolate agar. (6) Tryptone glucose yeast extract broth (Morton and Engley, 95). t < The least concentration of kojic acid tested showed some inhibition. t > The greatest concentration of kojic acid tested did not completely inhibit growth of the test organism. Tests made in the liquid medium. Downloaded from on May, 08 by guest

5 TOXICITY AND ANTIBIOTIC ACTIVITY OF KOJIC ACID peritoneal injection of this solution into mice were essentially the same as for the unneutralized solution described above. Antibacterial Activity of Kojic Acid The proper amount of kojic acid was weighed out and added to 00 ml of appropriate nutrient agar, melted, and cooled to about 50 C. After thorough mixing 0-ml amounts were transferred to sterile petri dishes. After the medium had solidified in the dishes, broth cultures, usually hours old, of the test organisms were streaked over an area of the medium by means of a bent wire. The wire spreader was constructed by bending at a right angle the terminal -cm portion of the B. and S. gauge nickel-chromium wire which was used for inoculating needles. DISCUSSION There are only a few reports in the literature concerning the antibiotic and physiological properties of kojic acid. Yabuta (9, 96) reported that it has a weak antiseptic power upon fungi and yeasts, although certain fungi are able to use it as a source of nutriment. Oxford (9) stated that its antibacterial activity was of low order, failing to inhibit the growth of Staphylococcus aureus in a dilution of :6,000. Foster and Karow (95) reported briefly the action on a few bacteria of kojic acid produced by Aspergillus oryzea. In some cases our results are of the same order. It was found by Kramer, Greer, and Szobel (9) to possess no significant or appreciable prophylactic or therapeutic action in Swiss mice against the viruses of poliomyelitis and of St. Louis encephalitis. The toxic action of kojic acid in dogs, rabbits, and rats has been reported by Friedemann (9). One fact, persistently reported by all who have worked with molds producing kojic aid, is that the enzymatic system of the mold is capable of producing kojic acid with many sugars or even with organic acids as the initial substrate. SUMMARY The isolation of kojic acid from a culture of Aspergillus luteo-virescens is reported. The M.L.D. of kojic acid for 7-g mice injected intraperitoneally is about 0 mg per mouse. The inhibitory concentration of kojic acid when dissolved in nutrient agar was tested against 66 strains of bacteria. The results are shown in table. Two species of Leptospira were especially susceptible to the action of kojic acid. In view of the susceptibility of these organisms to the action of other antibiotics, in vivo tests were not made. REFERENCES FOSTER, J. W., AND KARow, E Microbiological aspects of penicillin. VIII. Penicillin from different fungi. J. Bact., 9, 9-9. FRIEDEMANN, T. E. 9 Chemical and physiological properties of kojic acid. Science, 80,. 58 Downloaded from on May, 08 by guest

6 58 MORTON, KOCHOLATY, JUNOWICZ-KOCHOLATY, AND KELNER JENNINGS, M. A., MM WILLIAMS, T. I. 95 Production of kojic acid by Aspergilue effusw Tiraboshi. Nature, 55, 0. KRAm i, S. D., GREER, H. A., AND SZOBEL, D. A. 9 The chemo-prophylactic and therapeutic action of a wide variety of chemical compounds on two neurotropic virus infections in mice. J. Immunol., 9, 7-. MAY, 0. E., HERRCK, H. T., MOYER, A. J., AD WELLS, P. A. 95 Oxidative fermentations of glucose. U. S. Patent,006,086. Chem. Abstracts, 95,9,659. MNzyzR, K. F., STEWART-ANDERSON, B., AND EDDIE, B. 99 Canine leptospirosis in the United States. J. Am. Vet. Med. Assoc., 95, MORTON, H. E., AND ENGLEY, F. B., JR. 95 The protective action of dysentery bacteriophage in experimental infections in mice. J. Bact., 9, OxPORD, A. E. 9 Antibacterial substances from moulds. Bact. III. Some observations on the bacteriostatic powers of the mould products citrinin and penicillic acid. J. Soc. Chem. Ind., 6, 8-5. PRESCOTT, S. C., AND DUNN, C. G. 90 Industrial microbiology. McGraw-Hill, New York. RAISTCK, H., BIRKINSHAW, J. H., MICHAEL, S. E., BRACKEN, A., GyE, W. E., HOPKINS W. A., AND GREMNWOOD. M. 9 Patulin in the common cold; collaborative research on a derivative of Penicillium patulin Bainier. Lancet, II, SMTO, K. 907 tyber die Saurebildung bei Aspergilus oryzae. Japan. J. Botany,,, 7-. YABuITA, T. 9 Kojic acid, a new organic acid formed by A8pergillus oryzae. 8th Intern. Congr. Pure Applied Chem. (Appendix), 5, 55. Chem. Abstracts, 9, 7, 9-9. YADuTA, T. 96 A new organic acid (kojic acid) formed by Aspergillus oryza. J. Chem Soc. Japan, 7, 85-, -69. Chem. Abstracts, 9, 7, Downloaded from on May, 08 by guest