ARTICLE IN PRESS Microbiological Research 163 (2008) 495—502
www.elsevier.de/micres
Phthalates production from Curvularia senegalensis (Speg.) Subram, a fungal species associated to crops of commercial value Esther M.F. Lucasa, Lucas M. Abreua, Ivanildo E. Marrielb, Ludwig H. Pfenningc, Jacqueline A. Takahashia, a
Departamento de Quı´mica, ICEx, Universidade Federal de Minas Gerais, Av. Antonio Carlos, 6627, CEP 31270-901, Belo Horizonte, MG, Brazil b Empresa Brasileira de Pesquisa Agropecua´ria, EMBRAPA Milho e Sorgo, Rodovia MG 424, km 45, Sete Lagoas, Caixa Postal 285, CEP 35701-970, Sete Lagoas, MG, Brazil c Departamento de Fitopatologia, Universidade Federal de Lavras, Campus universita´rio, Caixa Postal 3037, CEP 37200-000, Lavras, MG, Brazil Received 9 January 2007; received in revised form 31 January 2007; accepted 2 February 2007
KEYWORDS Curvularia senegalensis; Phthalates; Gas chromatography/mass spectrometry; GCMS
Summary The fungal species Curvularia senegalensis was isolated from a soil sample collected at a Brazilian region of cerrado transition. This microorganism was grown in vitro and the extract of the culture medium was fractionated by chromatographic methods yielding an oil rich in phthalates, from which seven derivatives were identified by infrared, 1H and 13C NMR and mass spectrometry as 1-hexyl-2-propylphthalate, 1ethyl-2-heptylphthalate, 1-hexyl-2-butylphthalate, 1-heptyl-2-proylphthalate, 1propyl-2-nonylphthalate and two positional isomers of 1-decyl-2-butane phthalate. This is the first report on the phthalates production by Curvularia senegalensis revealing a scientific basis for the use of this species on biodegradation experiments. Since C. senegalensis is a very common pathogen in some commercial crops, presence of highly toxic phthalates on the final feed products should be investigated. & 2007 Elsevier GmbH. All rights reserved.
Introduction Corresponding author. Tel.: +55 31 34995754;
fax: +55 31 34995700. E-mail address:
[email protected] (J.A. Takahashi).
Curvularia senegalensis (Speng.) Subram is a filamentous fungus regarded by some authors as a synonym of C. geniculata (Tracy & Earle) Boedijn (Hosokawa et al., 2003). This species develops
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ARTICLE IN PRESS 496 black and velvet colonies with an abundant septate mycelium (Santos et al., 2003). C. senegalensis exhibits importance in agriculture, medicine, veterinary and ecology. In agriculture its importance relies in the fact that C. senegalensis is able to cause diseases in crops of economical and agroecological interest, such as Bactris gasipaes (pupunha tree) and Archontophoenix sp. (real palm tree) (Santos et al., 2003). In medicine, it is already established that C. senegalensis is an etiologic agent of some allergic sinusitis (Vishnoi et al., 2005), keratitis (Wilheelmus and Jones, 2001) and endophtalmites in immunocompetent and immunosuppressed patients (Vishnoi et al., 2005). It was also proved that this fungal species possesses pathogenic potentiality to invade mammalians tissues, having lungs, liver and kidney as the target organs (Vishnoi et al., 2005). In veterinary this fungus is recognized to be the pathologic agent that causes nervous central system disturbance and wing paralysis in parrots (Clark et al., 1986) and mycetoma (maduromycosis) in horses and dogs (Boomker et al., 1977). This fungus was previously studied as an alternative agent to perform the biotransformation of polyurethane materials, since it is capable to degrade ester-based polyurethanes (Crabbe et al., 1994), pointing out to its use as biodegradation agents with ecological benefits. There is only one metabolite of this fungus described at literature (Biglimo et al., 1968), named radicinin (1). Therefore, there was not a scientific support for its use as a biodegradation agent. Phthalate esters result from condensation of 1,2benzenedicarboxylic acid with alcohols bearing chains comprising two to eight carbons. They are a group of substances that can be found in cosmetics, pesticides, adhesives, cellophane wrappings (Saillenfait and Laudet, 2005) and they are widely used as plasticizers to increase the flexibility and workability of high-molecular-weight polymers. In some plastics, this group of substances constitutes up to 50% of the total weight. Their low melting point and high boiling point make them very useful to transfer hot fluids and as carries. Although phthalates have low acute toxicity, they are classified as priority pollutant since they can cause endocrine disturbance (Chao et al., 2006). Some phthalates (di-n-buthyl, di-butylbenzyl and di-2-ethylhexyl phthalate) produce alterations of the developing of adult male reproductive system, and embryo/fetal toxicity. Phthalates can be easily released from the polymeric matrixes of plastic materials being able to contaminate not only the environment but also food wrapped by a phthalatecontaining plastic and also children products. In
E.M.F. Lucas et al. Europe there is a limit for adding this kind of plastic material in puericulture products (Saillenfait and Laudet, 2005). Phthalate contamination of children and adults occurs by mouth, skin or inhalatory system (for aerosols containing phthalates) and can be detected on blood and urine (Saillenfait and Laudet, 2005). Release of phthalate esters into environment may occur during production, distribution and the waste disposal of the products that use them as components. Due to their low solubility in water they readily get adsorbed to soil sediments and to suspended solids at the rivers (Chao et al., 2006). The potential risk of these substances to the ecosystem is concerned at di-butylphthalate toxicity upon aquatic organisms as algae, crustaceans and fishes from fresh and saltwater, in an order of milligrams per litter (Wezel et al., 2000). In the present work, the chemical study of an extract obtained from C. senegalensis was carried out, leading to the identification of some endogenous phthalate derivatives by gas chromatography coupled to mass spectrometry (GC/MS).
Experimental procedures Reagents and solvents All reagents or solvents were of analytical or chromatographic grade, purchased from Merck (Darmstadt, Germany) or Grupo Quı´mica (Sa ˜o Paulo, Brazil). For chromatographic columns were used silica-gel 60 (article Merck 7734) and, for thin layer chromatography, silica-gel 60G (article Merck 7731) was used.
Culture medium Culture medium components were purchased from Merck (Darmstadt, Germany) and Biobra ´s (Montes Claros, Brazil). The fungal strains were maintained on potato dextrose agar (PDA) and refrigerated at 7 1C. For metabolites production, C. senegalensis was grown on liquid medium of complex composition. Liquid medium composition was (g/l): Dextrose (20.0), Peptone (5.0), KH2PO4 (1.0), MgSO4.7H2O (0.5), NaCl (5.0).
Fungus cultivation and extract preparation C. senegalensis was isolated from a soil sample collected at Serra do Cipo ´ National Park, Minas Gerais, a typical Brazilian region of cerrado transition. The microorganism was inoculated into six conical flasks, containing each, one liter of
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liquid medium and the growth took place for 15 days, after which ethyl acetate was added and the mycelium was separated from the culture medium by filtration. The mycelium and the medium were extracted successively with ethyl acetate and butyl alcohol and both extracts were obtained separately.
using temperatures from 220 to 300 1C with a increase of 3 1C/min. MS analysis was carried out with a Hewlett-Packard model 5989A mass spectrometer after separation with a Hewlett-Packard model 5989A series II gas chromatograph (Palo Alto, CA). The carrier gas was helium. A 30 m 0.25 mm id SE-54 capillarity column was used at the same temperature program described above. Mass spectral scans (45–350 m.u.) were recorded at 70 ev, every 2 s, and the scans were started 2 min after injection.
Oil isolation 15 g of the butyl alcohol extract was chromatographed on a silica gel column (200 g) that was eluted with ethyl acetate and methyl alcohol. Fractions 20–22, eluted by a mixture of ethyl acetate and methyl alcohol (1:1) were joined; the solvent was removed yielding 240 mg of orange oil that was further chromatographed on a silica gel column (hexane, dichloromethane and methyl alcohol). Fractions 1–7, obtained by the elution with mixture of hexane and dichloromethane (9:1) yielded 63 mg of reddish oil that was submitted to analysis by GC and GC-MS. (Fig. 1)
Oil analysis by GC and GC-MS A Varian gas chromatograph model CP-3380 was used to achieve the oil composition. The carrier gas was hydrogen. A 30 m 0.25 mm SE30 column (Alltech, Deerfield, IL) was used. The injection volume of the sample was 1 mL; sample concentration was 1% in methanol. The injector and detector temperatures were both 300 1C. A split ratio of 1–100 was used and the spectra were obtained
Spectrometric analysis Infrared spectrum was obtained on a Spectrum One-FT-IR Spectrometer-Perkin-Elmer and the samples were loaded directly on the NaCl window. Four scans were used to cover the wave from 4000 to 600 cm 1.
Results and discussion The oil isolated from the extract of C. senegalensis was analyzed by nuclear magnetic resonance (NMR) spectroscopy. The 1H NMR spectrum showed signals centered at dH 7.70 and 7.49, typical of hydrogens bonded to aromatic carbons and a multiplete centered at dH 4.28, typical of hydrogens on oxygenated carbons. In the 13C NMR spectrum, a signal at dC 167.70 revealed the presence of an ester carbonyl. Signals at dC 132.46, 130.96 and 128.91 confirmed the presence
141.7 130 120 110 704.52
100 90
3441.41 3069.26
%T
80
1600.84 1580.42 2858.25
70
50
1379.55
2872.89
60
881.75
1842.10
1462.94
2957.65 2926.32
965.65 1039.74 741.12
40
1072.48
30
1121.32 1271.16
1726.05
20 10 -10 -14.8 4000.0
3000
2000
1500
1000
650.0
cm-1
Figure 1. Infrared spectrum of the reddish oil isolated from Curvularia senegalensis.
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Table 1. Values of retention times and relative abundance of components of the reddish oil isolated from C. senegalensis Peak
rT
Area
%Ar
Height
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
8.383 8.512 8.758 8.829 8.983 9.192 9.279 9.433 9.654 9.762 10.096 10.162 10.262 10.446 10.567 10.850 10.996 11.287 11.4 11.762
22.075 14.6 29.788 100.859 51.446 111.102 274.617 53.338 350.612 371.47 363.563 586.567 304.69 329.538 481.692 210.096 129.555 63.648 16.254 13.392
0.57 0.38 0.77 2.60 1.33 2.86 7.08 1.38 9.04 9.58 9.37 15.12 7.86 8.5 12.42 5.42 3.34 1.64 0.42 0.35
3.828 2.225 6.985 16.254 6.426 21.72 40.518 10.752 45.989 59.668 63.638 97.389 66.04 62.485 86.206 30.097 17.893 10.095 2.308 2.321
(0.6%), 8 min 829 s (2.6%); 9 min 279 s (7.1%), 9 min 654 s (9.0%), 10 min 162 s (15.12%, major compound), 10 min 567 s (12.4%), and 10 min 850 s (5.4%), present in the mixture. The mass spectra of such compounds are shown in Figs. 2–8. Not all phthalates were able to be identified by this technique and attempt to purify the remaining components by column chromatography was not successful as their behavior on column were identical. MS analysis of peaks with rT 8.383 and 8.829 showed a M++1 peak at m/z 293 indicating these compounds to have molecular formula C17H24O4. To phthalate with rT 8.383, it was also observed a peak at m/z 85, characteristic of six carbons aliphatic chains, suggesting this compound to be 1-hexyl-2-propylphthalate (2). Peak with rT 8.829, by its turn, showed a peak at m/z 99, characteristic of an aliphatic chain bearing seven carbons. This one seems to be the biggest carbon chain of this compound, the other chain possessing 2 carbons. By this data we suggest that this compound is 1-ethyl2-heptylphthalate (3). MS analysis of peaks with rT 9.279 and 10.850 suggested M++1 at m/z 307 indicating both these compounds having the molecular formula C18H26O4. To phthalate with rT 9.279, it was also observed a peak at m/z 85, indicating one aliphatic chain bearing six carbons, the other one containing 4 carbons (m/z 57). Then, this compound was identified as 1-hexyl-2-butylphthalate (4). Mass spectrum of phthalate with rT 10.850 presented a peak at m/z 99, characteristic of aliphatic chains of seven carbons, representing its biggest carbon chain, the other chain bearing three carbons. These data are in good agreement with the structure of 1-heptyl-2-proylphthalate (5). MS spectra of phthalates with rT 9.654, 10.162 and 10.567 showed in common, peaks at m/z 127,
57
60000 50000 Abundance
of an aromatic ring and at dC 77.88, 77.24, 76.60, 66.22, 65.91 and 64.41 indicated the presence of different lateral chains linked to the oxygen from the ester moiety. The assembled NMR data pointed out to the presence of a mixture of compounds possessing phthalate skeleton. The IR spectrum showed peaks at 3070, 1600, 1580 cm 1, characteristic of aromatic rings, at 2958, 2926, 2872, 2858, 1462 and 1379 cm 1, typical of long aliphatic chains, and at 1726 cm 1, characteristic of an ester carbonyl. The comparison of this spectrum with a spectra database Perkin-Elmer software, showed a strong resemblance with the spectrum of di-butyl phthalate. The oil was submitted to gas chromatography analysis and the retention times and relative abundances of compounds present in the oil are shown in Table 1. The mass spectra of representative peaks seen on the gas chromatogram were obtained, showing, in all cases, an intense peak at m/z 149, characteristic of phthalic esters with lateral chains bigger than two carbons (El-naggar, 1997). Another peak, at m/z 57, was also present in all mass spectra, corresponding to the fragment [CH3CH2CH2CH2]+, characteristic of molecules possessing an aliphatic chain bearing four carbon atoms, an indicative that butyl or dibutylphthalates were present in the oil. However, careful analysis of mass spectra made possible to establish the structure of seven phthalates with the following retention times: 8 min 383 s
149
40000 30000 20000 10000
69 50
104 85
127 121
167
0
293
40 60 80 100 120 140 160 180 200 220 240 260 280 300 m/z-->
Figure 2. Mass spectrum of the peak with retention time of 8 min and 383 s.
ARTICLE IN PRESS Phthalates production from Curvularia senegalensis
499
57
200000 180000
149
160000
Abundance
140000 71
120000 100000 80000
85
60000 40000
104 50
20000 0
12 7
16 7
128
40
60
80
17 6
207
275
293
100 120 140 160 180 200 220 240 260 280 300 m/z-->
Figure 3. Mass spectrum of the peak with retention time of 8 min and 829 s. 149
57
50000
Abundance
40000 71
30000 85
20000 10000
104
50
121
141
167
307
0 40
60
80
100 120 140 160 180 200 220 240 260 280 300 m/z-->
Figure 4. Mass spectrum of the peak with retention time of 9 min and 279 s.
149
800000
Abundance
700000
57
600000
71
500000 400000
85
300000 200000
127
100000 0
104
50
100
176
150
207217
200
275
250
293
300
332
350
400
m/z-->
Figure 5. Mass spectrum of the peak with retention time of 9 min and 654 s.
ARTICLE IN PRESS 500
E.M.F. Lucas et al.
Abundance
149
1300000 1200000 1100000 1000000 900000 800000 700000 600000 500000 400000 300000 200000 100000 0
57
71
85 127 10 4
50
100
150
275
207217
17 6
200
250
293
319 332
300
350
36 0
400
m/z-->
Figure 6. Mass spectrum of the peak with retention time of 10 min and 162 s. 149
1600000 1400000
Abundance
1200000 1000000 800000 57
600000
55
400000
85
200000 0
127 150
97
176 193
275 293307
217
360
40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 m/z-->
Figure 7. Mass spectrum of the peak with retention time of 10 min and 567 s. 149
100000
57
90000
Abundance
80000 70000 60000 71
50000 40000 30000 20000
10 4 50
10000
97
121
14 1
167
0 40
60
207
293 307
80 100 120 140 160 180 200 220 240 260 280 300 m /z-->
Figure 8. Mass spectrum of the peak with retention time of 10 min and 850 s.
characteristic of saturated aliphatic chains bearing nine carbons. This appeared to be the size of biggest carbon chain of these three compounds. For phthalate at RT 9.654, MS analysis showed a M+
peak at m/z 332, indicating this compound to have the molecular formula C20H28O4, suggesting it to be identified as 1-propenyl-2-nonylphthalate (6). MS analysis of phthalates with rT 10.162 and 10.567
ARTICLE IN PRESS Phthalates production from Curvularia senegalensis O O
O
OH
O 1 O OR1 OR2 O
Compound
R1
R2
2
C6H13
C3H7
3
C7H15
C2H5
4
C6H13
C4H9
5
C7H15
C3H7
6
C9H19
C3H6
7 and 8
C10H21
C4H9
Figure 9. Structure of radicin and identified compounds.
suggested a M+ peak at m/z 360, indicating for both a molecular formula C22H32O4. Another peak, present in both spectra, at m/z 307, corresponds to the fragment [Ph(COOdecyl) (COHOH)]+, suggesting those compounds to be two position isomers of 1-decyl-2-butane phthalate (7 and 8). (Fig. 9)
501 the enzymatic rote to catalyze internal degradation of endogenous phthalates and the relationship between the soil microflora and the level of phthalates can be established with interesting ecological impacts. It is possible to suggest that C. senegalensis plays an important role as a natural plastic biodegradation agent and can be further exploited for environmental purposes, since alternatives for phthalates degradation are sought worldwide. For medical sciences, the relationship between the pathogenicity of C. senegalensis and its capacity to produce phthalates deserves to be further investigated. Since C. senegalensis can be associated to crops used to produce fine gourmet products in many countries, the extension of phthalates contamination of agricultural products should also be evaluated.
Acknowledgments The authors thank International Foundation for Science (IFS, Grant F/3564-1), Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo ´gico (CNPq), Fundac-a ˜o de Amparo `a Pesquisa do Estado de Minas Gerais (FAPEMIG, Grant CEX 1088/05) and Empresa Brasileira de Pesquisa Agropecua ´ria (EMBRAPA) for financial support.
References Conclusion Many biological activities (El-naggar, 1997; Marchetti et al., 2002) have been described for phthalates but attention on their use is advised. Control on phthalate emission levels on the environment was already established since these substances can cause serious and irreversible toxic effects to mammalians (Saillenfait and Laudet, 2005). But up to now most of the efforts are related to the control of phthalate on the production of plastic material. Dibutyl phthalate was already isolated from the bacteria Streptomyces albidoflavus (Roy et al., 2006) but this is the first time that phthalates were obtained as metabolites from C. senegalensis. Considering that no plastic materials were used to handle this species since its isolation, the phthalates identified should be part of C. senegalensis secondary metabolism biosynthesis. This is an interesting achievement since this fungus is reported to be able to degrade esterbased polyurethanes (Yang-Hoon et al., 2005). Such biodegradation capacity is most probably related to
Biglimo G, Mazzoli G, Corsi E. In: Seminari di Chimica, Consiglio Nazionale delle Ricerche e Fondazone f. Giordani, vol. 11, 1968. p. 74–7. Boomker J, Coetzer JA, Scott DB. Black grain mycetoma (maduromycetoma) in horses. Onderstepoort J Vet Res 1977;44:249–51. Chao WL, Lim CM, Shiung JI, Kuo YL. Degradation of dibutyl-phthalate by soil bacteria. Chemosphere 2006; 63:1377–83. Clark FD, Jones lP, Panigrahy B. Mycetoma in a grand ecletus (Ecletus roratus roratus) parrot. Avian Dis 1986;30:441–3. Crabbe JR, Campbell JR, Thompson lW, Stephen l, Warren W. Biodegradetion of a colloidal ester-based polyurethane by soil fungi. Int Biodeterioration Biodegrad 1994;33:103–13. El-naggar MYM. Dibutyl phthalate and the antitumor agent f5a1, two metabolites produced by Streptomyces nasri submutant h35. Biomed. Lett. 1997;55: 125–31. Hosokawa M, Tanaka C, Tsuda M. Conidium morphology of Curvularia geniculata and allied species. Mycoscience 2003;44:227–37. Marchetti l, Sabbieti MG, Menghi M, Materazzi S, Hurley MM, Manghi G. Effect of phthatate esters on actin
ARTICLE IN PRESS 502 cytoskeleton of py1a rat osteoblasts. Histol Histopathol 2002;17:1061–6. Roy RN, Laskar S, Sen SK. Dibutyl phthalate, the bioactive compound produced by Streptomyces albidoflavus. Microbiol Res 2006;161:121–6. Saillenfait AM, Laudet H. Phthalates. Emc-toxicologie Patol 2005;2:1–13. Santos AF, Bezerra JL, Tessmann DJ, Poltronieri lS. Ocorre ˆncia de Curvularia senegalensis em pupunheira e palmeira real no Brasil. Fitopatologia brasileira 2003;28:204. Vishnoi S, Naidu J, Singh SM, Vishinoi R. Patogenicity of Curvularia geniculata (c. senegalensis) for albio rats:
E.M.F. Lucas et al. study of clinical isolate from blood of a cancer patient. J Micol Me´d 2005;15:97–102. Wezel AP, Vlaardigen P, Van Phostumus R, Crommentuijn GH, Sjim DTHM. Environmental risk limits for two phthalates, with special emphasis on endocrine disruptive properties. Ecotoxicol Environ Saf 2000;46:305–21. Wilheelmus K, Jones DB. Curvularia keratitis. Trans Am Ophthamol Soc 2001;99:111–32. Yang-Hoon K, Jiho M, Kyung-Dong B, Man Bock G, Jeewon L. Biodegradation of dipropyl phthalate and toxicity of its degradation products: a comparison of Fusarium oxysporum f. sp. pisi cutinase and Candida cylindracea esterase. Arch Microbiol 2005;184:25–31.
