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Metabolites of Pathogenic Fungi. II
METABOLITES OF PATHOGENIC FUNGI. II THE ISOLATION OF XANTHOMEGNIN FROM TRIUHOPHYTON MEGNINI BLANCHARD
1896* F. BLANK, DR.SC.NAT.,t DR.SC.TECHN., W. C. DAY, PH.D. AND G. JUST, PH.D.
Since the publication of Sabouraud's book "Les
pigments A—C, described as bright yellow, orange-
Teignes" (1) in 1910, a very large number of red and bluish-red, respectively and obtained B papers on dermatophytes and diseases caused by
and C in a crystalline form. He thought that the
them have been published. They deal almost ultraviolet and visible spectra of these pigments exclusively with the structure and classification of dermatophytes and describe the clinical and epidemiological aspects of these infections. Little knowledge or insight into the biochemistry and chemistry of these organisms has been gained which, for instance, could support the taxonomy of these organisms, hitherto exclusively based on
morphological features. In order to try to fill some of the gaps in a field in which, according to Rivalier (2), "la fantaisie s'est donnd libre cours",
were similar to those of anthraquinones. In 1957, Wirth et at. (8) described the isolation
of three crystalline pigments in a very small yield from T. rubrum. He referred to these pigments as red needles, orange plates and purple needles and listed their analyses, physical properties and some color reactions.
This report deals with the isolation of several pigments of Trichophyton megnini Blanchard, 1896.
we have started a series of investigations of the ISOLATION OF THE PIGMENTS chemical constituents of pathogenic fungi, in The powdered mycelium of T. megnini had a particular, dermatophytes. The pigments of the genus Trichophyton have pale rose tint. Extraction with petroleum ether gave a nearly received mention in a number of articles. Tate colorless product, shown to consist mainly of (3) stated that the pigments of T. rubrum and T. mentagrophytes behaved as acid-base indi- fatty material and brassicasterol (9). Since concators, being yellow in acidic and red in basic ventional solvents did not seem to free colored media. The pigments could be reduced with material from the mycelium, it was extracted sodium dithionite in alkaline solution and re- with cold acetic acid. The chloroform soluble oxidized by air. The influence of media on the portion of the acetic acid extract was then production of pigments by T. purpureum (4), defatted with ether. Attempts to isolate any pure pigment from T. gallinae (5) and T. megnini (5) has also been reported, but the investigation of the pigments this defatted substance by crystallization, of these dermatophytes apparently had not been chromatography, sublimation or additional expursued beyond the exploratory stage. It had traction procedures were unsuccessful. Moreover, zinc dust distillation failed to give any detectable been suggested (3, 6) that the pigments might be anthracene derivatives, but no experimental work aromatic hydrocarbon. An elemental analysis indicated 2.5% nitrogen was reported in support of this view. Mier (7) and 8.2% incombustible residue. Emission ran a paper chromatogram of a colored extract spectroscopic analysis of the inorganic constitof T. rubrum and reported the Rf values of his
uents in the ash showed the metals to be
*
From the Departments of Bacteriology and
Immunology and Chemistry, McGill University, Montreal, Canada. This investigation was supported by the Brown Hazen Fund and by a PHS research grant E-4050
from the National Institute of Allergy and Infectious Diseases, Public Health Service.
t Present address: Skin and Cancer Hospital of Philadelphia, Departments of Dermatology and Microbiology, Temple University School of
Medicine, Philadelphia, Pa. Received for publication May 5, 1962.
primarily calcium and magnesium in ratios from 1:1 to 10:1*.
Deionization was effected by passing a 70% acetic acid solution of the crude pigment extract
over a cation exchange resin. A considerable amount of substance was absorbed on the resin and was removed by elution with glacial acetic * Analysis by Dr. G. C. B. Cave, Chemistry Dept., McGill University.
133
134
THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
acid oniy. Its color was brown-black and it was not considered as a source of our main pigment. From the deionized material, two yellow fractions could be obtained by Soxhlet extraction for
one hour and several days respectively. The latter was further purified by chromatography on silica gel. Elution with chloroform gave an orange material. Chromatography on cellulose powder of this orange material separated its
The insoluble residue recovered from the Soxhlet is most probably rich in chitin. The structure of xanthomegnin (11) seems to indicate a close relationship, if not identity, with the orange platelets isolated by Wirth et al. (8) from T. rubrum. EXPERIMENTAL
Preparation of Mycelium The fungus was grown in 500 ml. Erlenmeyer passing a benzene solution of the orange fraction flasks containing 250—300 ml. medium at room on cellulose powder. Elution with benzene and temperature for 6—8 weeks. The medium conchloroform gave a yellow fraction and a very sisted of 4% crude glucose (cerelose), 1 % neopeptone Difco, 0.005% inositol, 0,001% thiamine small amount of red material. Crystallization from benzene, and then chioro- and 2% agar. The cultures were autoclaved, and form-benzene, furnished orange platelets. The the harvested mycelium washed free of adhering purity of the product was confirmed by paper medium by boiling three times in water. After chromatograms in which several solvent systems pressing, the mycelium was freeze-dried, and failed to show more than one component. This milled in a ball-mill. pigment was named xanthomegnin. Lsolation of Xanthomegnin Xanthomegnin exhibited no melting point but Two kilograms of powdered mycelium of cindered at 264°. It was insoluble in hydroxylic solvent, only slightly soluble in acetone, ethyl Trichophyton megnini were placed in a large acetate or benzene, and moderately soluble in Soxhlet extractor and defatted with petroleum
main constituents from a red contaminant. Further fractionization could be effected by
chloroform.
ether (b.p. 30—60°) for one week.
The residual mycelium was divided into three The mother liquors from the crystallization of xanthomegnin yielded another yellow pigment portions, each of which was extracted five times which possessed an aldehyde function, and hence with glacial acetic acid by adding the solvent, was named megninal. Megninal has not yet been stirring the slurry occasionally, and then allowing the mixture to stand overnight before filtering. purified. The Soxhlet extraction of the residual crude A total of 45 lbs. of acetic acid was used. The pigment was continued using benzene, and then bulk of the solvent was evaporated at 40° under chloroform. A silica gel chromatogram of these vacuum with a bubbler and a stream of nitrogen, extracts furnished the various pigment fractions and the remainder was removed in vacuo with a as shown in the separation scheme.* Since an rotating evaporator. The residue was freed of analysis of the dark brown flakes (0) showed traces of the solvent by azeotroping with benzene. The residue (ca. 227 gm.) was dissolved in 20 3.14% nitrogen, this fraction is suspected of lbs. of chloroform and the solution was filtered being chitinous material (10) chemically unrethrough celite. The residue was 101 gm. of a lated to the megnin pigments. The red pigment, dark, gummy gelatinous substance (A). The a very small amount of and purpuromegnin (Q), solvent was evaporated from the red-violet filtrate a yellow pigment (P) were eluted together and and the residue (126 gm.) was then defatted with were closely bound to less definable dark confive extractions with ether. Some red pigment taminants. Although purpuromegnin has not yet dissolved into the ether. The 71 gm. of etherbeen obtained completely free of inorganic im- insoluble material was a black vitreous solid (B) purities, the content has been reduced to such which was collected by filtration. trace amounts that we are assured the red color The pigment concentrate (B) was dissolved in is inherent to the organic chromophore and is 4.2 litres of acetic acid and then water was not due to metal chelation. added until the volume of solution was six litres (70% solution). The mixture was filtered through * letters on the following pages refer Capital glass wool to remove insoluble materials, and the to fractions listed in the separation scheme.
135
METABOLITES OF PATHOGENIC FUNGI FLOW-SHEET OF ISOL&TION PROCEDURE
Driedjmycelium
LExtd._with petroleum ether
Soluble fats
Residual mycelium Extd. with acetic acid
r
Solution Residual mycelium Evap. off solvent at 400 Residue Dissolved in chloroform
Red-violet solution Insoluble gummy residue (A) Evap. off solvent Residue Washed with ether Soluble fats (some red pigment)
Black glass (B) Dissolved in 70% acetic acid and passed through Amberlite IR-120
70% acetic acid eluate Glacial acetic Washed with pet. ether acid elution Evap. off solvent Brown sludge (C) Yellow-brown residue (D) (Soxhiet Extractor) I Ether extraction (one hour) Yellow fatty residue (E) Ether extraction (two days) Yellow solid (F) F Chromatographed on silica gel
[enzene Oily material (G)
r
Yellow band (J)
Chloroform Orange eluate (H) Mixed pigments (I) Rechromatographed benzene soln. through cellulose column I
Trailing material
Red band (E)
Mother liquor
Orange platelets
I
(Xanthomegnin) (M)
Megninal (L) Benzene extraction (one week)
r
Dark brown flakes (0)
Yellow-brown solid (N) Chromatographed on silica gel I
r1• Xanthomegnin
2. Megninal
Chloroform extraction (one week)
-
1. Yellow pigment (P) 2. Purpuromegnin (Q) Red-brown solid (R)
Insoluble residue (5)
filtrate was divided into four portions. Each bined 70% acetic acid eluates were extracted portion was deionized by passing the solution twice with petroleum ether to remove fatty conthrough a column (4 cm. x 60 cm.) of Amberlite stituents and the solvent was then removed IR-120 strong acid ion-exchange resin. The corn- under vacuum. The last traces of the solvent
136
THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
were removed by azeotropic drying with benzene megnin) represented 0.06% of the dried myand ethanol on a rotating evaporator in vacuo. celium. Chromatograms of xanthomegnin on The yield was 38.5 gm. of a yellow-brown solid Whatman No. 1 paper with the following solvent
(D). A brown sludge (C) was recovered from the systems showed a single spot: column by deionizing the ion-exchange resin with glacial acetic acid. A subsequent deionization and Sat'd aqueous benzene elution with glacial acetic acid containing a small Chloroform-hexane (7:3) amount of concentrated hydrochloric acid did Pyridine-fusel oil-water (6:3:1) not furnish any additional adsorbed material. Residue (D) was placed in a Soxhlet and after Anal. C H 0 extracting five times with ether the extraction
Rf
0.26 0.96
0.10 OCH,
furnished 5.93 gm. of yellow fatty material (E) which had been extracted. The Soxhlet extraction was then continued for two days with ether. The extraction yielded 12—13 gm. of yellow solid material (F). Extract (F) was dissolved in 400 ml of benzene and chromatographed on a column (3 cm. x 90 cm.) of dehydrated silica gel (B.D.H.). An oily substance (388 mg.) passed through the column unadsorbed. The eluting solvent was changed to chloroform and a vivid orange fraction which moved slowly with this solvent was collected. The total weight of the red-orange glass (H) obtained from this band was 6.30 gm. After elution of the orange band, the eluent was changed to chloroform-ethyl acetate (1:1 v/v) and additional pigments were eluted from the column. Ethyl acetate elution gave further pigmented material (trailing). The column was then washed with ethyl acetate-methanol (9:1 v/v) followed by ethyl acetate-acetic acid (9:1 v/v). All the fractions after the orange band (H) were combined (I). The orange glass (H) was dissolved in approximately 100 ml. of benzene and passed onto a dry-packed cellulose column (4.5 cm. x 43 cm.). Elution was carried out with benzene. An intense yellow band (J) which moved close to the solvent front was collected and furnished 5.41 gm. of material. The trailing substance in the benzene
I C.)
was interrupted. Evaporation of the solvent Found: Calc'd. for
62.46* 3.99t 32.52 31.57 62.50 4.20 33.30
10.87
3.01
11.45
5.22
3.86
10.80
5.23
12.40
C1 5H1 20 :
Calc'd. for
62.72
33.42
C301122012
* Average of 62.33, 62.58, 62.47, 62.48, 62.46.
f Average of 4.09, 3.76, 3.98, 4.01, 4.12.
The work-up of the mother liquors from the crystallization of xanthomegnin furnished another yellow pigment which contained an aidehyde function as indicated by the n.m.r. spectrum. This compound, megninal, has not yet been obtained in a pure state. The Soxhlet extraction of (D) was continued with benzene. Extraction for one week yielded 7.2 gm. of material (N). The extract was dissolved in 300 ml. of chloroform and chromatographed on 400 ml. of silica gel (B.D.H.). A dark brown material (729 mg.) (0) passed through the column unadsorbed. An analysis of a sample of this substance precipitated from benzene solution showed 3.14% nitrogen and 4.3% ash. It gave no melting point. An orange band followed slowly in the chloroform solvent which, upon combining the fractions, yielded 2.96 gm. or orange residue. Recrystallization from benzene gave 1890 mg. of crystalline material. Two recrystallizations from chloroform-benzene furnished 850 mg. of pure
eluates was kept separate, as was also a red xanthomegnin with the remainder apparently pigment (K) which was removed from the uppermost part of the column by changing to chloroform as the eluting solvent. Crystallization of (J) from benzene furnished
mixed with megninal. Further elution with the chloroform-ethyl acetate gave pigment mixtures similar to those obtained upon silica gel chroma-
chloroform-benzene yielded 1200 mg. of orange platelets, decomp. at 264° by cindering without
tected but could not be separated. The Soxhlet extraction of (D) was continued
melting. The pure orange platelets (xantho-
with chloroform for one week and 5.8 gm. of
tography of the ether extract (F). Two com2.86 gm. of orange crystals in two crops. Re- pounds, an extremely insoluble yellow pigment crystallization of this product three times from and a red pigment (purpuromegnin) were de-
METABOLITES OF PATHOGENIC FUNGI
137
extract (R) was obtained. A chromatograph of this extract on silica gel produced fractionation in the manner described above for the benzene extract. Some dark brown material (327 mg.) passed through unadsorbed, 1347 mg. was re-
2. RIvALIER, E: Mycologie médicale in Histoire
covered from the orange band, and the remainder consisted of red and brown material which could
development of the pigment of Trichophytort.
not be purified in the ethyl acetate elution
5. SILYA, M. AND BENHAM, R. W.: Nutritional
sequence.
Attempts to purify purpuromegnin by fractional crystallization or by rechromatographing on Davidson silica gel were unsuccessful. A sample gave the analysis: C 64.52; H 4.54; ash 2.4. After passing the sample through a strong acid ion-exchange with 70% acetic acid it gave the new analysis: C 63.51; H 4.19; ash. 1.2. SUMMARY
A method for the separation and isolation of several pigments from the mycelium of Trichophylom megnini was devised. The principal pig-
de Ia Botanique en France. 8e Congr. Intern. Botaniquc Paris-Nice 307—312, 1954.
3. TATE, P.: The dermatophytes or ringworm fungi. Biol. Rev., 4: 41—75, 1929.
4. GREENBERG, E. AN0 TIT5wORTH, E.: The
effect of animal and plant peptones on the
purpureum. J. Invest. Derm., 20: 197—200, 1953.
studies of the dermatophytes with special reference to Trichophyton megnini Blanchard
1896 and Trichophyton gallin.ce (Megnin
1881) comb. nov. J. Invest. Dcrm., 18: 453—472, 1952.
6. GEORO, L. K. AND MAECHLING, E. H.: Tnchophyton mentagrophytes (variety nodular).
A mutant with brilliant orange-red pigment
isolated in nine cases of ringworm of the skin and nails. J. Invest. Derm., 13: 339—350, 1949.
7. MIRE, P. D. : Pigments in Tnichophyton rubrum. Nature, 179: 1084—1085, 1957. 8. WIaTH, J. G., O'BRJEN, P. J., SCHMITT, F. L. AND SOHLER, A.: The isolation in crystalline form of some of the pigments of Trichophyton rubrum. J. Invest. Derm., 29: 47—53, 1957. 9. BLANK, F., SHORTLAND, F. S. AND JUsT, G.:
The free sterols of dermatophytes. J.
ment, xanthomegnin, was obtained in a pure Invest. Derm., 39: 91—94, 1962. 10. BLANK, F.: The chemical composition of the state. cell-walls of dcrmatophytes. Biochim. Bio-
REFERENCES 1. SABOURAUD, R.: Maladies du cuir chevelu.
III. Lcs maladies cryptogamiques. Les teignes. Paris, Masson et Cie, 1910.
phys. Acta, 10: 110—113, 1953. 11. JusT, G., DAY, W. AND BLANK, F.: Metabolites
of pathogenic fungi. III. The structure of Xanthomegnin. Can. J. Chem., 41: 74—79,
1963.
THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
94
linolenic acid extract. Arch. This pdf is a scanned copy UV of irradiated a printed document.
24. Wynn, C. H. and Iqbal, M.: Isolation of rat
skin lysosomes and a comparison with liver Path., 80: 91, 1965. and spleen lysosomes. Biochem. J., 98: lOP, 37. Nicolaides, N.: Lipids, membranes, and the 1966.
human epidermis, p. 511, The Epidermis
Eds., Montagna, W. and Lobitz, W. C. Acascopic localization of acid phosphatase in demic Press, New York. human epidermis. J. Invest. Derm., 46: 431, 38. Wills, E. D. and Wilkinson, A. E.: Release of 1966. enzymes from lysosomes by irradiation and 26. Rowden, C.: Ultrastructural studies of kerathe relation of lipid peroxide formation to tinized epithelia of the mouse. I. Combined enzyme release. Biochem. J., 99: 657, 1966. electron microscope and cytochemical study 39. Lane, N. I. and Novikoff, A. B.: Effects of of lysosomes in mouse epidermis and esoarginine deprivation, ultraviolet radiation and X-radiation on cultured KB cells. J. phageal epithelium. J. Invest. Derm., 49: 181, 25. Olson, R. L. and Nordquist, R. E.: Ultramicro-
No warranty is given about the accuracy of the copy.
Users should refer to the original published dermal cells. Nature, 216: 1031, 1967. version of1965. the material. vest. Derm., 45: 448, 28. Hall, J. H., Smith, J. G., Jr. and Burnett, S. 41. Daniels, F., Jr. and Johnson, B. E.: In prepa1967.
Cell Biol., 27: 603, 1965.
27. Prose, P. H., Sedlis, E. and Bigelow, M.: The 40. Fukuyama, K., Epstein, W. L. and Epstein, demonstration of lysosomes in the diseased J. H.: Effect of ultraviolet light on RNA skin of infants with infantile eczema. J. Inand protein synthesis in differentiated epi-
C.: The lysosome in contact dermatitis: A ration. histochemical study. J. Invest. Derm., 49: 42. Ito, M.: Histochemical investigations of Unna's oxygen and reduction areas by means of 590, 1967. 29. Pearse, A. C. E.: p. 882, Histochemistry Theoultraviolet irradiation, Studies on Melanin, retical and Applied, 2nd ed., Churchill, London, 1960.
30. Pearse, A. C. E.: p. 910, Histacheini.stry Thearetscal and Applied, 2nd ed., Churchill, London, 1960.
31. Daniels, F., Jr., Brophy, D. and Lobitz, W. C.: Histochemical responses of human skin fol-
lowing ultraviolet irradiation. J. Invest. Derm.,37: 351, 1961.
32. Bitensky, L.: The demonstration of lysosomes by the controlled temperature freezing section method. Quart. J. Micr. Sci., 103: 205, 1952.
33. Diengdoh, J. V.: The demonstration of lysosomes in mouse skin. Quart. J. Micr. Sci., 105: 73, 1964.
34. Jarret, A., Spearman, R. I. C. and Hardy, J. A.:
Tohoku, J. Exp. Med., 65: Supplement V, 10, 1957.
43. Bitcnsky, L.: Lysosomes in normal and pathological cells, pp. 362—375, Lysasames Eds., de Reuck, A. V. S. and Cameron, M. Churchill, London, 1953.
44. Janoff, A. and Zweifach, B. W.: Production of inflammatory changes in the microcirculation by cationic proteins extracted from lysosomes. J. Exp. Med., 120: 747, 1964.
45. Herion, J. C., Spitznagel, J. K., Walker, R. I. and Zeya, H. I.: Pyrogenicity of granulocyte lysosomes. Amer. J. Physiol., 211: 693, 1966.
46. Baden, H. P. and Pearlman, C.: The effect of ultraviolet light on protein and nucleic acid synthesis in the epidermis. J. Invest. Derm.,
Histochemistry of keratinization. Brit. J. 43: 71, 1964. Derm., 71: 277, 1959. 35. De Duve, C. and Wattiaux, R.: Functions of 47. Bullough, W. S. and Laurence, E. B.: Mitotic control by internal secretion: the role of lysosomes. Ann. Rev. Physiol., 28: 435, 1966. the chalone-adrenalin complex. Exp. Cell. 36. Waravdekar, V. S., Saclaw, L. D., Jones, W. A. and Kuhns, J. C.: Skin changes induced by
Res., 33: 176, 1964.
1896* F. BLANK, DR.SC.NAT.,t DR.SC.TECHN., W. C. DAY, PH.D. AND G. JUST, PH.D.
Since the publication of Sabouraud's book "Les
pigments A—C, described as bright yellow, orange-
Teignes" (1) in 1910, a very large number of red and bluish-red, respectively and obtained B papers on dermatophytes and diseases caused by
and C in a crystalline form. He thought that the
them have been published. They deal almost ultraviolet and visible spectra of these pigments exclusively with the structure and classification of dermatophytes and describe the clinical and epidemiological aspects of these infections. Little knowledge or insight into the biochemistry and chemistry of these organisms has been gained which, for instance, could support the taxonomy of these organisms, hitherto exclusively based on
morphological features. In order to try to fill some of the gaps in a field in which, according to Rivalier (2), "la fantaisie s'est donnd libre cours",
were similar to those of anthraquinones. In 1957, Wirth et at. (8) described the isolation
of three crystalline pigments in a very small yield from T. rubrum. He referred to these pigments as red needles, orange plates and purple needles and listed their analyses, physical properties and some color reactions.
This report deals with the isolation of several pigments of Trichophyton megnini Blanchard, 1896.
we have started a series of investigations of the ISOLATION OF THE PIGMENTS chemical constituents of pathogenic fungi, in The powdered mycelium of T. megnini had a particular, dermatophytes. The pigments of the genus Trichophyton have pale rose tint. Extraction with petroleum ether gave a nearly received mention in a number of articles. Tate colorless product, shown to consist mainly of (3) stated that the pigments of T. rubrum and T. mentagrophytes behaved as acid-base indi- fatty material and brassicasterol (9). Since concators, being yellow in acidic and red in basic ventional solvents did not seem to free colored media. The pigments could be reduced with material from the mycelium, it was extracted sodium dithionite in alkaline solution and re- with cold acetic acid. The chloroform soluble oxidized by air. The influence of media on the portion of the acetic acid extract was then production of pigments by T. purpureum (4), defatted with ether. Attempts to isolate any pure pigment from T. gallinae (5) and T. megnini (5) has also been reported, but the investigation of the pigments this defatted substance by crystallization, of these dermatophytes apparently had not been chromatography, sublimation or additional expursued beyond the exploratory stage. It had traction procedures were unsuccessful. Moreover, zinc dust distillation failed to give any detectable been suggested (3, 6) that the pigments might be anthracene derivatives, but no experimental work aromatic hydrocarbon. An elemental analysis indicated 2.5% nitrogen was reported in support of this view. Mier (7) and 8.2% incombustible residue. Emission ran a paper chromatogram of a colored extract spectroscopic analysis of the inorganic constitof T. rubrum and reported the Rf values of his
uents in the ash showed the metals to be
*
From the Departments of Bacteriology and
Immunology and Chemistry, McGill University, Montreal, Canada. This investigation was supported by the Brown Hazen Fund and by a PHS research grant E-4050
from the National Institute of Allergy and Infectious Diseases, Public Health Service.
t Present address: Skin and Cancer Hospital of Philadelphia, Departments of Dermatology and Microbiology, Temple University School of
Medicine, Philadelphia, Pa. Received for publication May 5, 1962.
primarily calcium and magnesium in ratios from 1:1 to 10:1*.
Deionization was effected by passing a 70% acetic acid solution of the crude pigment extract
over a cation exchange resin. A considerable amount of substance was absorbed on the resin and was removed by elution with glacial acetic * Analysis by Dr. G. C. B. Cave, Chemistry Dept., McGill University.
133
134
THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
acid oniy. Its color was brown-black and it was not considered as a source of our main pigment. From the deionized material, two yellow fractions could be obtained by Soxhlet extraction for
one hour and several days respectively. The latter was further purified by chromatography on silica gel. Elution with chloroform gave an orange material. Chromatography on cellulose powder of this orange material separated its
The insoluble residue recovered from the Soxhlet is most probably rich in chitin. The structure of xanthomegnin (11) seems to indicate a close relationship, if not identity, with the orange platelets isolated by Wirth et al. (8) from T. rubrum. EXPERIMENTAL
Preparation of Mycelium The fungus was grown in 500 ml. Erlenmeyer passing a benzene solution of the orange fraction flasks containing 250—300 ml. medium at room on cellulose powder. Elution with benzene and temperature for 6—8 weeks. The medium conchloroform gave a yellow fraction and a very sisted of 4% crude glucose (cerelose), 1 % neopeptone Difco, 0.005% inositol, 0,001% thiamine small amount of red material. Crystallization from benzene, and then chioro- and 2% agar. The cultures were autoclaved, and form-benzene, furnished orange platelets. The the harvested mycelium washed free of adhering purity of the product was confirmed by paper medium by boiling three times in water. After chromatograms in which several solvent systems pressing, the mycelium was freeze-dried, and failed to show more than one component. This milled in a ball-mill. pigment was named xanthomegnin. Lsolation of Xanthomegnin Xanthomegnin exhibited no melting point but Two kilograms of powdered mycelium of cindered at 264°. It was insoluble in hydroxylic solvent, only slightly soluble in acetone, ethyl Trichophyton megnini were placed in a large acetate or benzene, and moderately soluble in Soxhlet extractor and defatted with petroleum
main constituents from a red contaminant. Further fractionization could be effected by
chloroform.
ether (b.p. 30—60°) for one week.
The residual mycelium was divided into three The mother liquors from the crystallization of xanthomegnin yielded another yellow pigment portions, each of which was extracted five times which possessed an aldehyde function, and hence with glacial acetic acid by adding the solvent, was named megninal. Megninal has not yet been stirring the slurry occasionally, and then allowing the mixture to stand overnight before filtering. purified. The Soxhlet extraction of the residual crude A total of 45 lbs. of acetic acid was used. The pigment was continued using benzene, and then bulk of the solvent was evaporated at 40° under chloroform. A silica gel chromatogram of these vacuum with a bubbler and a stream of nitrogen, extracts furnished the various pigment fractions and the remainder was removed in vacuo with a as shown in the separation scheme.* Since an rotating evaporator. The residue was freed of analysis of the dark brown flakes (0) showed traces of the solvent by azeotroping with benzene. The residue (ca. 227 gm.) was dissolved in 20 3.14% nitrogen, this fraction is suspected of lbs. of chloroform and the solution was filtered being chitinous material (10) chemically unrethrough celite. The residue was 101 gm. of a lated to the megnin pigments. The red pigment, dark, gummy gelatinous substance (A). The a very small amount of and purpuromegnin (Q), solvent was evaporated from the red-violet filtrate a yellow pigment (P) were eluted together and and the residue (126 gm.) was then defatted with were closely bound to less definable dark confive extractions with ether. Some red pigment taminants. Although purpuromegnin has not yet dissolved into the ether. The 71 gm. of etherbeen obtained completely free of inorganic im- insoluble material was a black vitreous solid (B) purities, the content has been reduced to such which was collected by filtration. trace amounts that we are assured the red color The pigment concentrate (B) was dissolved in is inherent to the organic chromophore and is 4.2 litres of acetic acid and then water was not due to metal chelation. added until the volume of solution was six litres (70% solution). The mixture was filtered through * letters on the following pages refer Capital glass wool to remove insoluble materials, and the to fractions listed in the separation scheme.
135
METABOLITES OF PATHOGENIC FUNGI FLOW-SHEET OF ISOL&TION PROCEDURE
Driedjmycelium
LExtd._with petroleum ether
Soluble fats
Residual mycelium Extd. with acetic acid
r
Solution Residual mycelium Evap. off solvent at 400 Residue Dissolved in chloroform
Red-violet solution Insoluble gummy residue (A) Evap. off solvent Residue Washed with ether Soluble fats (some red pigment)
Black glass (B) Dissolved in 70% acetic acid and passed through Amberlite IR-120
70% acetic acid eluate Glacial acetic Washed with pet. ether acid elution Evap. off solvent Brown sludge (C) Yellow-brown residue (D) (Soxhiet Extractor) I Ether extraction (one hour) Yellow fatty residue (E) Ether extraction (two days) Yellow solid (F) F Chromatographed on silica gel
[enzene Oily material (G)
r
Yellow band (J)
Chloroform Orange eluate (H) Mixed pigments (I) Rechromatographed benzene soln. through cellulose column I
Trailing material
Red band (E)
Mother liquor
Orange platelets
I
(Xanthomegnin) (M)
Megninal (L) Benzene extraction (one week)
r
Dark brown flakes (0)
Yellow-brown solid (N) Chromatographed on silica gel I
r1• Xanthomegnin
2. Megninal
Chloroform extraction (one week)
-
1. Yellow pigment (P) 2. Purpuromegnin (Q) Red-brown solid (R)
Insoluble residue (5)
filtrate was divided into four portions. Each bined 70% acetic acid eluates were extracted portion was deionized by passing the solution twice with petroleum ether to remove fatty conthrough a column (4 cm. x 60 cm.) of Amberlite stituents and the solvent was then removed IR-120 strong acid ion-exchange resin. The corn- under vacuum. The last traces of the solvent
136
THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
were removed by azeotropic drying with benzene megnin) represented 0.06% of the dried myand ethanol on a rotating evaporator in vacuo. celium. Chromatograms of xanthomegnin on The yield was 38.5 gm. of a yellow-brown solid Whatman No. 1 paper with the following solvent
(D). A brown sludge (C) was recovered from the systems showed a single spot: column by deionizing the ion-exchange resin with glacial acetic acid. A subsequent deionization and Sat'd aqueous benzene elution with glacial acetic acid containing a small Chloroform-hexane (7:3) amount of concentrated hydrochloric acid did Pyridine-fusel oil-water (6:3:1) not furnish any additional adsorbed material. Residue (D) was placed in a Soxhlet and after Anal. C H 0 extracting five times with ether the extraction
Rf
0.26 0.96
0.10 OCH,
furnished 5.93 gm. of yellow fatty material (E) which had been extracted. The Soxhlet extraction was then continued for two days with ether. The extraction yielded 12—13 gm. of yellow solid material (F). Extract (F) was dissolved in 400 ml of benzene and chromatographed on a column (3 cm. x 90 cm.) of dehydrated silica gel (B.D.H.). An oily substance (388 mg.) passed through the column unadsorbed. The eluting solvent was changed to chloroform and a vivid orange fraction which moved slowly with this solvent was collected. The total weight of the red-orange glass (H) obtained from this band was 6.30 gm. After elution of the orange band, the eluent was changed to chloroform-ethyl acetate (1:1 v/v) and additional pigments were eluted from the column. Ethyl acetate elution gave further pigmented material (trailing). The column was then washed with ethyl acetate-methanol (9:1 v/v) followed by ethyl acetate-acetic acid (9:1 v/v). All the fractions after the orange band (H) were combined (I). The orange glass (H) was dissolved in approximately 100 ml. of benzene and passed onto a dry-packed cellulose column (4.5 cm. x 43 cm.). Elution was carried out with benzene. An intense yellow band (J) which moved close to the solvent front was collected and furnished 5.41 gm. of material. The trailing substance in the benzene
I C.)
was interrupted. Evaporation of the solvent Found: Calc'd. for
62.46* 3.99t 32.52 31.57 62.50 4.20 33.30
10.87
3.01
11.45
5.22
3.86
10.80
5.23
12.40
C1 5H1 20 :
Calc'd. for
62.72
33.42
C301122012
* Average of 62.33, 62.58, 62.47, 62.48, 62.46.
f Average of 4.09, 3.76, 3.98, 4.01, 4.12.
The work-up of the mother liquors from the crystallization of xanthomegnin furnished another yellow pigment which contained an aidehyde function as indicated by the n.m.r. spectrum. This compound, megninal, has not yet been obtained in a pure state. The Soxhlet extraction of (D) was continued with benzene. Extraction for one week yielded 7.2 gm. of material (N). The extract was dissolved in 300 ml. of chloroform and chromatographed on 400 ml. of silica gel (B.D.H.). A dark brown material (729 mg.) (0) passed through the column unadsorbed. An analysis of a sample of this substance precipitated from benzene solution showed 3.14% nitrogen and 4.3% ash. It gave no melting point. An orange band followed slowly in the chloroform solvent which, upon combining the fractions, yielded 2.96 gm. or orange residue. Recrystallization from benzene gave 1890 mg. of crystalline material. Two recrystallizations from chloroform-benzene furnished 850 mg. of pure
eluates was kept separate, as was also a red xanthomegnin with the remainder apparently pigment (K) which was removed from the uppermost part of the column by changing to chloroform as the eluting solvent. Crystallization of (J) from benzene furnished
mixed with megninal. Further elution with the chloroform-ethyl acetate gave pigment mixtures similar to those obtained upon silica gel chroma-
chloroform-benzene yielded 1200 mg. of orange platelets, decomp. at 264° by cindering without
tected but could not be separated. The Soxhlet extraction of (D) was continued
melting. The pure orange platelets (xantho-
with chloroform for one week and 5.8 gm. of
tography of the ether extract (F). Two com2.86 gm. of orange crystals in two crops. Re- pounds, an extremely insoluble yellow pigment crystallization of this product three times from and a red pigment (purpuromegnin) were de-
METABOLITES OF PATHOGENIC FUNGI
137
extract (R) was obtained. A chromatograph of this extract on silica gel produced fractionation in the manner described above for the benzene extract. Some dark brown material (327 mg.) passed through unadsorbed, 1347 mg. was re-
2. RIvALIER, E: Mycologie médicale in Histoire
covered from the orange band, and the remainder consisted of red and brown material which could
development of the pigment of Trichophytort.
not be purified in the ethyl acetate elution
5. SILYA, M. AND BENHAM, R. W.: Nutritional
sequence.
Attempts to purify purpuromegnin by fractional crystallization or by rechromatographing on Davidson silica gel were unsuccessful. A sample gave the analysis: C 64.52; H 4.54; ash 2.4. After passing the sample through a strong acid ion-exchange with 70% acetic acid it gave the new analysis: C 63.51; H 4.19; ash. 1.2. SUMMARY
A method for the separation and isolation of several pigments from the mycelium of Trichophylom megnini was devised. The principal pig-
de Ia Botanique en France. 8e Congr. Intern. Botaniquc Paris-Nice 307—312, 1954.
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studies of the dermatophytes with special reference to Trichophyton megnini Blanchard
1896 and Trichophyton gallin.ce (Megnin
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A mutant with brilliant orange-red pigment
isolated in nine cases of ringworm of the skin and nails. J. Invest. Derm., 13: 339—350, 1949.
7. MIRE, P. D. : Pigments in Tnichophyton rubrum. Nature, 179: 1084—1085, 1957. 8. WIaTH, J. G., O'BRJEN, P. J., SCHMITT, F. L. AND SOHLER, A.: The isolation in crystalline form of some of the pigments of Trichophyton rubrum. J. Invest. Derm., 29: 47—53, 1957. 9. BLANK, F., SHORTLAND, F. S. AND JUsT, G.:
The free sterols of dermatophytes. J.
ment, xanthomegnin, was obtained in a pure Invest. Derm., 39: 91—94, 1962. 10. BLANK, F.: The chemical composition of the state. cell-walls of dcrmatophytes. Biochim. Bio-
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