View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector THE ISOLATION OF A UNIQUE STEROL FROM THE MYCELIUM OF A STRAIN OF TRICHOPHYTON RUBRUM* JOHN C. WIRTH, PH.D., THOMAS BEESLEY, B.S. AND WILLIAM MILLER, B.S. Little is known concerning the lipid contentknowledge, this represents the first positive of any of the dermatophytes. Okazaki andidentification of brassieasterol as a constituent Tamemasa (1) reported the presence of ergosterolof any fungus. This finding was deemed to be so in a strain of Trichophyton asteroides. Wirth,extraordinary that the scope of the investigation O'Brien, Schmitt and Sohier (2) isolated ergos-was broadened to include four additional strains terol from a strain (C-46) of T. rubrum. Merkelof T. rubrum. (3, 4, 5) and Zamieehowska-Miazgowa (6, 7) reported the presence (detected by paper chro- EXPERIMENTAL matographie methods) of palmitic, stearie and 1) Preparation of the Mycelium oleic acids in strains of T. mentagrophytes Stock cultures of the various strains of T. (gypseum), T. sulphureum, T. plicatile, Achorionrubrum were maintained in the refrigerator on schoenleini and A. guincheanum. However, itslants of Sabouraud's Conservation Agar and should be noted that the latter two authorssubcultured every three months. claimed lipid contents which are abnormally To obtain substantial quantities of the myce- high (41 to 75%). Prince (5) presented a dis-hum, a large number (from 20 to 150) of 2.5 1. tribution of the type of lipid present in a strainFernbach flasks were filled with 500 ml. of of lipid present in a strain of T. menta-Sabouraud's Dextrose Broth. These were then autoclaved and inoculated with a piece of myee- grophytes although he did not positively identifyhum cut from a Petri dish culture. After incuba- any of the component compounds. tion for 4 to 6 weeks at room temperature, the Previous work had strongly indicated, as wascultures were killed by tbe addition of 2.5 g. of almost to be expected, that the nonsaponifiablemercuric chloride to each flask. After inter- fraction (NSF) is likely to be a highly complexmittent shaking for 5 days, the mycelium was mixture. The one compound, ergosterol, whichharvested by filtering off the mats with an had been isolated in pure form was obtained inordinary household colander; washed with running that state only after repeated recrystallizations.water until free of mercuric ions; then dried Accordingly, it was decided to re-investigate the (first in air and finally in a vacuum desiccator over NSF. Unfortunately, in the intervening years,aluminum oxide); and ground to a fine powder. that particular strain had been lost and theThe yield of the dried and powdered myeeliuin work was continued with a new strain, (C-129).amounted to 2.5 to 3.5 g. per flask. The usual sterol in the fungi imperfecti is 2) Preparation of the Non-Saponifiable Fraction ergosterol. In strain C-129 ergosterol, at first, seemed to be totally absent; at least, it could The dried and powdered mycelium was first extracted with Skellysolve Bf (approximately not be positively isolated even in trace quanti-400 ml. of solvent per 100 g. of mycelium) at room ties. The sterol which was found to be thetemperature for a period of two weeks, during principal one in this strain is brassicasterolwhich the container was shaken periodically. which had been previously reported (9, 10, 11, 12)The mycelium was filtered from the solvent and in the literature as a constituent of higher plantsthe entire procedure repeated twice. After the and marine invertebrates. To the best of ourthird extraction with this solvent, the mycelium was air dried and then extracted three times with *Fromthe Graduate School of Arts and Sciences, St. John's University, Jamaica 32, N. Y. a one to one mixture of ethyl alcohol and ether. Abstracted in part from the Dissertations ofThe first extract gave rise to the so-called "free" Thomas Beesley and William Miller, submitted inlipids; the second, the so-called "bound" lipids. partial fulfillment of the requirements for theAfter thorough removal of the solvents by dis- MS. degree. This investigation was supported by a research t The various Skellysolves are commercial, grant, E-1761, from the National Institutes ofsaturated hydrocarbon fractions available from Health, U. S. Public Health Service, Bethesda,the Skelly Oil Company, Kansas City 10, Mo. Md. Skellysolve B has a boiling range of 60—71°; F, a Received for publication November 5, 1960. boiling range of 35—60°. 153 154 THEJOURNAL OF INVESTIGATIVE DERMATOLOGY TABLEI Lipid content of various strains of 7'. rubrum g. Lipid % Lipid' g. NSF % NSF2 g. Mycelium St tam Extracted Free Bound Free Bound Free Bound Free Bound C-129 343 19.0 16.5 5.5 4.8 1.7 1.6 8.9 9.7 M-40-58 88 2.32 2.63 2.6 3.0 0.20 0.29 8.6 11.0 M-1260-59 54 2.15 2.16 4.0 4.0 0.18 0.23 8.4 10.6 M-1558-59 67 2.70 3.69 4.0 5.5 0.17 0.22 6.3 5.9 M-722-59 52 1.30 1.44 2.5 2.8 0.22 0.15 16.9 10.4 1 Basedon weight of mycelium. Based on weight of lipid. tillation in vacuo, the ethyl alcohol-ether extractsmicrograms per lambda). To prevent possible were combined and then re-extracted with severaloxidation of the squalene all developments were portions of warm Skellysolve B until the insolublealways carried out in an atmosphere of nitrogen. portion (mainly pigments) was free of lipid. TheThe position of the spots on the paper was detected hydrocarbon soluble portion of the alcohol-etherwith iodine vapor using the procedure of Pan extract was considered to consist entirely of bound(15). While the Rf's of the known and unknown lipid though it was still heavily pigmented. on a single chromatogram never deviated from The NSF of both the free lipids and of the bound ech other by more than 0.01 of an Rf unit, never- lipids were then prepared in the usual way withtheless the Rf's of the known and unknown was alcoholic potassium hydroxide. Results of typical0.84 (average of 34 determinations) with an A.D. experiments with all of the strains used in thisof less than in contrast to the value reported investigation are presented in Table I. in the literature of 0.71. Repeated attempts to isolate and unequivocally 3) Separation of the NSF of Strain C-12,9 identify the squalene as the hexahydrochloride As preliminary experiments indicated that bothwere fruitless due probably to its low concentra- the free and bound lipid fractions contain sterol,tion. It should be noted that only one spot ap- the NSF's of both were usually combined andpeared in the case of the unknown on the chromatographed on alumina which was foundchromatogram. This could be attributed to the to be Grade II on the Brockmann (13) scale. Theabsence of other unsaturated hydrocarbons, at details of a typical chromatogram follow. Aleast in significant concentrations. Also, the column 19 mm. in diameter was packed with 40 g.entire fraction is devoid of fluorescent material, of the dry adsorbent (Merck's, C. P., Suitable forand since it did not absorb above 220 mji in the Chromatography) and prewetted with about 50ultraviolet, the absence of compounds containing ml of Skellysolve F. Then a solution of 1.0 g. ofa conjugated diene system is indicated. As was to the NSF dissolved in the same solvent was added.be expected, a test for sterols (precipitation with The column was then developed with solvents ofdigitonin) was negative. increasing eluting strength. The results of a Frcction II. This fraction was a yellow, semi- typical chromatogram are presented in Table II. solid. It gave a positive test with digitonin. In the Salkowski test the chloroform layer was blue and 4)FurtherInvestigation of the the sulphuric acid layer was light red. The Lieber- Individual Fractions mann-B urchard reaction was positive. These Fraction I. Repeated paper chromatography ofresults would indicate the presence of a sterol in the residue from the Skellysolve F eluate (Frac-at least trace quantities. Attempts to isolate tion I, Table II) was run according to the pro-crystalline material failed. cedure of Dauben et ai. (14). Two 1 lambda spots Fraction III. This fraction was a yellow, gummy of the residue (concentration of approximately 90solid. It gave a positive test with digitonin. In micrograms per lambda)t of the unknown was runthe Salkowski test the chloroform layer was color- simultaneously with a 1 lambda spot of authenticless and the sulphuric acid layer was cherry red. squalene (concentration of approximately 20In the Rosenheim test, a brilliant green fluo- tThislarge quantity was used to give a detect-rescence was obtained. These results would indi- able spot in the case of the unknown. cate the presence of a sterol in at least trace STEROLS OF TRICIIOPHYTON RUBRUM 155 quantities. Attempts to isolatecrystallineneedles with the above melting point were material failed. obtained. Fraction IV. This fraction was found to be a To obtain material of greater purity, the sterol highly pigmented, gummy solid. Preliminarywas converted to the acetate, then to the tetra- tests always indicated the presence of a sterolbromaeetate, then back to the acetate, and finally which was not ergosterol. The fraction gave ato the free sterol following the procedures of strongly positive test with digitonin, a non-Bergmann and Ottke (21) and of Dory and Geri reverse Salkowski (only a red interphase), absorp-(22). By so doing a sterol with a m.p.