dryness, yielding a brown, brittle material (9llB96). This material was partially (911B100) were obtained. The supernatant was evaporated to remove

Transcription

1 ANTIBIOTIC SUBSTANCES SEPARATED FROM SUMAC' H. J. CARLSON, H. G. DOUGLAS, AND H. D. BISSELL Department of Pediatrics, Western Reserve University School of Medicine, and the Babies and Childrens Division, University Hospitals of Cleveland, Cleveland, Ohio Received for publication January 19, 1948 It has become evident (Osborn, 1943; Lucas and Lewis, 1944) that higher plants are potential sources of antibiotic agents. Several investigators (Little and Grubaugh, 1946; Atkinson and Rainsford, 1946; Sanders, Weatherwax, and McClung, 1945) have reported antibiotic effects of juices and of crude extracts of plants on various microorganisms. The active materials can be separated in some degree of purity (Cavallito, Buck, and Suter, 1945; Cavallito, Bailey, and Kirchner, 1945; Heatley, 1944), and there is evidence that the agents so obtained can be introduced into the animal body and will control experimental infections (Carlson, Bissell, and Mueller, 1946). In screening several hundred plants (Carlson, Douglas, and Robertson, 1948) the authors observed that a species of sumac contained an antibiotic agent extractable in aqueous solutions. In later work this or similar agents were obtained from sumac by extraction with ethyl ether. It is the purpose of this paper to report the antibiotic activity of partially purified extracts from a species of sumac, Rhus hirta. METHODS Chemical procedures. Stems and leaves collected in the fresh state were finely chopped and sufficient ethyl ether was added to cover the material. Extraction was carried out for 24 to 48 hours at room temperature. The ether extraction (911B1) was separated from the plant residue by decanting. The plant residue was then allowed to stand at room temperature for 24 to 48 hours with an equal volume of distilled water (see flow chart, figure 1). The supernatant water extract (911B90) was decanted from the plant residue and was evaporated to dryness, yielding a brown, brittle material (9llB96). This material was partially soluble in distilled water as a red solution (911B97) with the persistence of some red-gray precipitate (911B98). Ten volumes of ethyl alcohol (95 per cent) were added to the red solution. A clear yellow supernatant and a gray precipitate (911B100) were obtained. The supernatant was evaporated to remove the alcohol. A dark brown solution remained (911B102). The ether was evaporated from the original extraction (911B3) leaving a green gum (911B15). The gum was placed in a Soxhlet extraction thimble and extracted with chloroform until colorless (approximately 5 hours). The chloroform was decanted and evaporated again leaving a thick green gum (911B50). The residue (911B47A), after chloroform extraction, was divided Supported in part by a U. S. Naval grant, Office of Naval Research. 607

2 608 H. J. CARLSON, H. G. DOUGLAS, AND H. D. BISSELL [VOL. 55 into two portions and re-extracted with ethyl ether and water (911B94). The residue (911B55) remaining in the extraction thimble was re-extracted with distilled water, yielding a clear brown solution (911B71). The ether extract was evaporated to dryness, yielding a greenish-brown gum (911B47). This gum ETHER FILTRATE EVAPORATED TO THICK GREEN GUM ACTIVE EXTRACTED IN SOXHLET WITH CHLOROFORM EVAPORATED TO THE 911B47A GUM INACTIVE ALIQNT PREPARATION FLOW SHEET CHOPPED 911 COVERED WITH ETHER PLANTrRESIDVE REDDI -BROWN HaO INl IACTIVE EXTRACT EXTRACTED WITH EXTRACTED REO-GRErY PECIPITATE OLUTION ETHYL ETHER WITH H2O INACTTIVE ACTIVE ACTIVE RESIDUE ETHER EVAPORATED TO 10 VOLUMES 95% EXTRACTED GREEN GUM 911B47 ALCOHOL ADDED WITH H20 I ~~~~~~~~~~~GREY PRIECPAE LR YELLOW BROWN SOLUTION ETHYL ETHER INACTIVVE 911B100 SUPERNATANT ACTIVE Bt9 AND SHAKEN WITH NORITA. FILTERED NORIT A. INACTIVE EVAPORA ED TO THICK AMBER GUM I BLTLY CLEAR YELLOW SOLUTION ACTIVE 911BTB DISSOLVED IN ACETONE PARTLY EVAPORATED AND COOLED GREEN PR IPITATE YELLOW SUPERNATANT INACTIVE 911BSO EVAPORATED TO THICK AQUEOUiS ESETRACT AMBER GUM.DRIED IN AIR ED SLOWLY EVAPORATED TO FINE ACTIVE 911B8188AORATE ~ ~ ~ ~ *TO GIVE I GRI:E'tPOWDER YELLOW WAXY POWDER SHOOK WITH HaO INDESSICCATOR ACTIVE GREEN PRECIPITATE CLEARYELLOW ACTIVE'E 91 5B8 / INACTIVE 911B104 FILTRATE EXTRACT WITH H1O ACTIVE YELLOW Pf'RECIPITATE YELLOW SOLUTION VE ACTIVE INACTI' FIG. 1. FLOW CHART RESIDUE COVERED WITH EVVAPORATED TO DRYNESS Ht0 BROWN- BRITTLE RESIDUE ACTIVE HtO ADDED EVAPORATED TO REMOVEIALCOHOL DARK BROWN SOLUTION ACTIVE SHAKE WITH ETHER was redissolved in ethyl ether (911B79) and shaken with 2 parts norit A. The resultant solution (911B78) was a clear, brilliant yellow. This was evaporated until a thick amber gum was obtained. The gum was dissolved in acetone and the solution evaporated to one-tenth its original volume. The green pre-

3 1948] ANTIBIOTICS SEPARATED FROM. SUMAC 609 cipitate (911B80) that formed was removed by filtration. The filtrate was evaporated to a thick green syrup (911B81), which was dried to a solid mass and pulverized. This powder (911B81) was shaken with distilled water and the suspension filtered. A green precipitate (911B104) was removed in the paper, and the filtrate (911B103) was a clear, yellow solution. The filtrate (911B103) was shaken for 15 minutes with ethyl ether. After being shaken, the ether was decanted and evaporated to dryness. A fine yellow, waxy powder (911B105) was obtained. This powder was partially soluble in distilled water, yielding a yellow solution (911B110), but there remained a yellow insoluble precipitate (911B109) that was removed by filtration. From the aqueous residue of the ether extraction of 911B103 there developed on slow evaporation an amber solution containing a whitish granular precipitate. This precipitate (911B108) became a gray powder on desiccation. Assay. Nutrient and enriched agar (Difco), 20 ml, favorable for the growth of the organisms on test, was seeded with 0.5 ml of an 18- to 24-hour broth culture of the organisms. Fungi and mold cultures were thoroughly agitated to break up mycelium formations before addition to the agar. After mixing, the agar was poured into sterile petri plates. The surface of the seeded agar plates was allowed to dry in air for 30 to 60 minutes. Several species of bacteria were grown on blood agar. One-tenth ml of a broth culture was placed on the surface and spread evenly with a curved sterile glass rod. These plates were allowed to dry for 30 to 60 minutes. Porcelain cylinders were placed on the surface of the seeded plates and were filled with the aqueous solutions to be tested for antibiotic activity. In a second method small blocks of agar were removed from the plate surface by vacuum, and the wells so formed were filled with the aqueous solutions. Dry powders and gums were placed directly on the surface of the seeded agar. Ether extracts were tested as previously described (Carlson, Douglas, and Robertson, 1948). The areas of inhibition were measured in mm whenever possible. In those areas where the diffusion of the active agent was uneven, inhibition was noted as negative (N), slight-8 to 11 mm (S), fair-12 to 14 mm (F), good-15 to 19 mm (G), or excellent-20+ mm (E). The letter "C" preceding any number or abbreviation denotes complete inhibition and "P" denotes partial inhibition. Toxicity. Extracts 911B94, 911B102, and 911B108 were prepared in 10 per cent solutions and passed through a Seitz filter. The sterile extracts were injected in 0.25, 0.5, and 1 ml amounts by the intraperitoneal route. Adult white Swiss mice were used as test animals. Toxicity was observed at intervals of 15 and 45 minutes and at the end of 1 and 24 hours. RESULTS The antibiotic activity of the extracts and fractions prepared during partial purification is shown in table 1 (also see flow chart). The different extracts appeared to be equally effective against the gram-positive coccus, Staphylococcus aureus, and the gram-negative organism, Escherichia coli. One extract, 911B15,

4 610 H. J. CARLSON, H. G. DOUGLAS, AND H. D. 13ISSELL a gum obtained from ether evaporation, stimulated the test organisms, as indicated by a more dense growth immediately surrounding the inhibited zones. The effective agents appeared to be soluble equally in ethyl ether and water. As observed in the preparation flow sheet, two original extracts, water-soluble and ether-soluble, were prepared. These extracts were prepared to yield extract 911B102, a dark-brown aqueous solution from the water-soluble fraction, and aqueous extracts 911B108 and 911B110 derived from the original ether fraction. The effective agent obtained in the aqueous fraction after ether extraction appeared to have been bound in some manner as it had not been apparent before EXTRACT TABLE 1 Antibiotic activity of extracts* SOLVENT nhbimon MM S. aurcus E. coli 91lB90 Water C15P25 C15P25 911B96 -(a) C15P22 CPED 911B97 Water C14P20 C18P24 91B102 Water C14P20 C18P24 911B3 Ether CFD CPFD 911B15 -(b) CGDS CEDS 911B94 Water C16P24 C16P30 911B71 Water C20P30 C20P25 911B78 Ether C15P25 C0OP30 911B81 -(a) C17P28 C18P28 911B103 Water C13 C17 911B108 Water C13P20 C14P18 911B105 -(a) CGD CGD 911BllO Water C17 C19 [VOL. 55 Legend: (a)-residue or powder; (b)-gum; 0complete; P-partial; FD-fair diffusion, mm; GD-good diffusion, mm; ED-excellent diffusion, 20 plus mm; S-stimulation outer area of inhibition. * See flow chart (figure 1). this procedure. Aqueous extraction of the plant alone did not demonstrate the water-soluble agent when tested on the seeded agar plate. The antibiotic activity of three extracts of Rhus hirta against 58 strains and species of bacteria, molds, and fungi is shown in table 2. Extracts 911B102, 911B94, and 911B108 (see flow chart) were used. Extract 911B94 is a step fraction of the partially purified fraction 911B108. The general activity of these three extracts is quite similar in that the effective agents inhibited equally well the growth of the microorganisms tested. Bacteria were observed to be more susceptible to the agent than were the various molds and fungi. The extracts were observed to exhibit marked bacteriostatic activity on the growth of gram-negative bacteria. Among those tested were several human pathogens, Shigella paradysenteriae (Flexner), ShigeUa dysenteriae, Shigells

5 19481 ANTIBIOTICS SEPARATED FROM SUMAC 611 TABLE 2 Bacterial, mold, and fungus spectrum ORGANISM INHIBMON mm 911B B94 911B108 CAE Shigella paradysenteriae.c15 C17 Shigella dysenteriae C20 C16 CFD Shigella sonne... C18P22 C15 C16 Eberthella typhosa. C20P26 C17 C15P22 Pseudomonas fluorescens.c18p22 C15P17 CPGD Pseudomonas aeruginoa. C15P22 C14P18 CPGD Proteus sp... C20P32 C22P33 C17P27 Proteus vulgaris C20P28 C18P22 C16P20 Bacillus subtilis.c20p23 C21 C23 Bacillus megatherium.c20p28 C24P30 C17P20 "Zoogleal"sp.0C22 C24 Achromobacter lacticum.c16p25 C15P18 C16P20 Serratia marcescens.c20p22 C18P28 PAE Bacillus circulans... C30 C27 C24 Escherichia coli 0.C15P24 C15X18 CAE Escherichia coli1. C16P22 C16P24 C17P29 Escherichia coli S.A.W. C14P20 C15 CAE Staphylococcws aureus0. C20P22 C16P20 C9P18 Staphylococcus aureus1. C18P24 C16P18 C12P18 Staphylococcus aureus2. C16P25 C16P20 ClOP20 Streptococcus viridans... C17 Cll(b) C15P18 Streptococcus viridans Ċ13P15 C15(b) C12P15 Streptococcus pyogenes. N N C14 Streptococcus faecali.. C20 C14P20 Diplococcus pneumonae..n N(a) Clostridium perfringens.n* N* N* Clostridium putrificum. P* P* P* Clostridium histolyticum.n* N* N* Clostridium botulinum. P* N* N* Clostridium sporogenes.n* N* P* Micrococcus tetragenu. C15S24 C14 C15 "Mucosus capsulatus... C25 C22 C20 Corynebacterium diphtheriae... C18P25 C18P25 CPGD Mycobacterium phlei.. C20 C18 Mycobacterium smegma...0c25 C35X40 Neisseria gonorrhoeae... C30 C20 C20 Neisseria intracellularis... C20 C18 C20 Hemophilus influenzae... C20 C16 P24 Agrobacterium sp.. C18P23 C20P25 Mycoderma lactis... C15 C18P20 CAE Mycoderma sp... N C18 CAE Penicillium (Waksman)... N N N Aspergillusterreus.N N N C-complete; P-partial; S-stimulation; FD-fair diffusion; GD-good diffusion; AE-area exposed; N-negative; a-partial inhibition in broth; b-complete inhibition in broth. * Broth.

6 612 H. J. CARLSON, H. G. DOUGJA5, AND H. D. BISSELL [VOL. 55 TABLE 2-Continued ORGANISM EBMION MM 911B B94 911B108 Aspergillus niger... N N N Trychophyton sp... N CFD CFD Trychophyton suiphereum C IO C15P18 C15 Candida albicans N C20 CAE Microsporum trichoderma P12 C12 CAE Penicillium cyclopium... C23 C24 C15 Pythium debaryanum... C24 C17 C12P18 Fusarium culmorum... C24 C20 C17 Fusarium sp... N C17 C20 Rhizoctonia solani... N N N Rhizoctonia oryzae... N N N Pestallozia funera... C19P25 C20 Mucor sylvaticus C17P27 C16 sonne, and Eberthella typhosa of the enteric group. Pseudomonas fluorescens. Pseudomonas aeruginosa, Proteus vulgaris, Proteus sp., Bacillus circulans, and 3 strains of Escherichia coli were tested, and these nonpathogens were equally susceptible. Neisseria gonorrhoeae, Neisseria intracellularis, and Hemophilus influenzae were inhibited by the three extracts in moderate zones of diffusion. The gram-positive bacteria tested did not consistently exhibit susceptibility to the effective agents in these extracts. Bacillus subtilis, Bacillus megatherium, Serratia marcescens, Staphylococcus aureus, Streptococcus viridans, Streptococcus faecalis, Micrococcus tetragenes, and "Mucosus capsulatus" were completely inhibited in varying zones of diffusion. Streptococcus pyogenes was only susceptible to 911B108. Diplococcus pneuumoniae was not inhibited, when grown on blood agar, by extracts 911B94 and 911B108, though an apparent reduction of growth was noticed in broth cultures of this microorganism in the presence of 911B108. Of the five strains of Clostridium tested, only one, C. putrificum, was susceptible to the three extracts, and it was only partially inhibited; C. botulinum was partially inhibited by extract 911B102; and C. sporogenes was partially inhibited by extract 911B108. Two strains of nonpathogenic Mycobacterium, M. phlei and M. smegmatis, were very susceptible to the effective agents in extracts 911B94 and 911B108. Extract 911B102 was ineffective against the growth of three human dermatophytes, Mycoderma sp., Trychophyton sp., and Candida albicans. These organisms were sensitive to extracts 911B94 and 911B108. The extracts appeared to be without significant action against the molds, Aspergillus terreus, Aspergillus niger, and Penicillium (Waksman), but they exhibited marked fungistatic activity toward Penicillium cyclopium. Rhozoctonia solani and Rhozoctonia oryzae were not susceptible to the antibiotic activity of the three extracts. Extract 911B102 was without significant effect upon a plant-wilt pathogen, Fusarium sp., though extracts 911B94 and 911B108 were observed to

7 1948] ANTIBIOTICS SEPARATED FROM SUMAC 613 elicit definite inhibitory activity against this micoorganism. Fusarium culmorum was inhibited equally well by the three extracts. Human dermatophytes, Mycoderma lactis, Trychophyton sulphereum, and Microsporum trichoderma, and phytopathogens, Pythium debaryanum, Pestallozia funera, and Mucor sylvaticus, were found to be susceptible to the fungistatic action of the extracts. The extracts were partially inactivated after being subjected to a temperature of 123 C for 10 minutes. Only surface growth was completely inhibited in the diffused area against the gram-positive organism, Staphylococcus aureus. No activity against gram-negative organisms was observed with the heat-treated solutions. Extract 911B108 lysed red blood cells, but 911B94 and 911B102 had no apparent effect on the red blood cells. Toxicity tests. A 10 per cent solution of extract 911B102 when injected intraperitoneally into mice in 1-, 0.5-, and 0.25-ml amounts did not appear to produce toxic symptoms. Extract 911B94 in 1-ml amounts caused death of the animals in 45 minutes, whereas 0.5- and 0.25-ml amounts caused death in 18 to 20 hours. Extract 911B108 was found to be toxic, causing death in 18 to 20 hours when 1-, 0.5-, and 0.25-ml amounts were injected intraperitoneally. DISCUSSION The cause of the whitish opacity when extracts of this plant were placed on agar is not known. Tests conducted to determine the presence of alkaloids have proved negative. The second area of discoloration observed in the presence of many of the bacteria tested was deemed to be caused by by-products of the organisms reacting with the diffused agent in a manner similar to an indicator. In the original screening and later experiments the authors were not able to demonstrate sufficient activity in water or saline extracts to justify further work on this fraction. This did not hold true for extractions with ethyl ether. The aqueous residue after ether extraction showed definite antibiotic activity that was diffusible and measurable on the seeded agar plate. This watersoluble fraction appeared to be released by extraction with ether or possibly was changed chemically to become soluble in water by the treatment. The two partially purified derivatives, 911B102 and 911B108, appear to be rather closely related agents because of their observed effectiveness against the bacteria tested, although their reaction in the presence of molds and fungi places more emphasis on the probability that they are different substances derived from the same plant. Sufficient chemical analysis has not been completed to state of what these effective agents are composed. Tests have shown that the gums and watersoluble fractions give no positive tests for alkaloids. SUMMARY The antibiotic activities of three extracts obtained from Rhus hirta are presented. These extracts were tested against 58 different strains and species of

8 614 H. J. CARLSON, H. G. DOUGLAS, AND H. D. BISSELL [VOL. 55 bacteria, molds, and fungi. The activity against these microorganisms indicates that this plant contains two separate antibiotic substances. REFERENCES ATKINSON, N., AND RAINSFORD, K. M Antibacterial substances produced by plants. I. Preliminary survey. Australian J. Exptl. Biol. Med. Sci., 24, CARLSON, H. J., BISSELL, H. D., AND MUELLER, M. G Antimalarial and antibacterial substances separated from higher plants. J. Bact., 52, CARLSON, H. J., DOUGLAS, H. G., AND ROBERTSON, J Antibacterial substances separated from plants. J. Bact., 55, CAvALLITO, C. J., BAILEY, J. H., AND KIRCHNER, F. K The antibacterial principle of Arctium minus. I. Isolation, physical properties and antibacterial action. J. Am. Chem. Soc., 67, CAVALLITO, C. J., BUCK, J. S., AND SUTER, C. M Allicin, the antibacterial principle of Allium sativum. II. Determination of the chemical structure. J. Am. Chem. Soc., 6, HEATLEY, N. G An antibiotic from Crepis taraxacifolia. Brit. J. Exptl. Path., 25, LITTLE, J. E., AND GRUBAUGH, K. K Antibiotic activity of some crude plant juices. J. Bact., 52, LUCAS, E. H., AiD LEWIS, R. W Antibacterial substances in organs of higher plants. Science, 100, OSBORN, E. M On the occurrence of antibacterial substances in green plants. Brit. J. Exptl. Path., 24, SANDERS, D. W., WEATHERWAX, P., AND MCCLUNG, L. S Antibacterial substances from plants collected in Indiana. J. Bact., 49,