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Two phytoalexins from Allium cepa bulbs
Phytochemistry, Vol. 30, No. 3, pp. 799-800, 1991
0031-9422/91 $3.00+0.00 © 1991 Pergamon Press pie
Printed in Great Britain.
TWO PHYTOALEXINS FROM A L L I U M CEPA BULBS LEONID TVERSKOY, ALEXANDER DMITRIEV, ANATOLY KOZLOVSKY* and DMITRY GRODZINSKY Institute of Cell Biology and Genetic Engineering, Vasilkovskaya 31/17, 252627 Kiev, U.S.S.R.; *Institute of Biochemistry and Physiology of Microorganisms, 142292 Puschino, U.S.S.R. (Received in revised form 11 July 1990)
Key Word Index--Allium cepa; Liliaceae; bulbs; phytoalexins; 5-octyl-cyclopenta-l,3-dione; 5-hexyl-cyclopenta1,3-diane.
Abstract--Two new phytoalexins: 5-octyl-cyclopenta-1,3-dione and 5-hexyl-cyclopenta-1,3-dione were isolated from the bulbs of Allium cepa. Their structures were elucidated by H P L C and spectroscopic methods.
INTRODUCTION
Phytoalexins are low M, antimicrobial compounds produced by plants in response to infection by microorganism. These compounds are accumulated at the site of infection and are considered to be involved in the plant's defence response to potential pathogens [1-3]. Phytoalexins have not been isolated from members of the Liliaceae [4"1. Previous studies on AUium cepa have resulted in the isolation of a group of fungitoxic substances with closely related polarity [5"1. We gave them a working name tsibulins from the Ukrainian 'tsibulya', meaning onion. Besides sulphur-containing pre-formed and stress metabolites, we have found two phytoalexins (tsibulins 1 and 2) [6] which accumulated only in response to fungal infection. The present paper deals with the isolation and identification of these onion phytoalexins. RESULTS AND DISCUSSION
In response to inoculation with Botrytis cinerea Pers. ex Fr. mycelium, Allium cepa bulb scales showed limited or spread lesions confined to the tissue beneath mycellure-contained agar pieces. Growth of B. cinerea was restricted within limited lesions [7]. Examination of infected tissue extracts by TLC plate bioassays with Cladosporium spp. revealed the presence within the lesions of two antifungal compounds (phytoalexins) which were absent from fresh or wounded tissues. The phytoalexins were isolated by gel filtration, TLC and HPLC, and identified as 5-octyl-cyclopenta-l,3-
o•R 0
1 R m (CH2hMe 2 R " (CH2)sMe PHY 3 0 : 3 - F
dione (1) and 5-hexyl-cyclopenta-l,3-dione (2) as follows. Molecular weights and formulae given by high resolution mass spectrometry are presented in Table 1. The most intensive IR bands indicated the presence of aliphatic substitution. Absorption bands at I705 and I608 c m - i revealed the presence of a conjugated carbonyl group and double bond, respectively. The hydroxyl is indicated by 3554cm - t (bounded) and 3600cm - t (free enolic hydroxyl) bands. Mass and IR spectroscopy data showed the absence of sulphur-containing functional groups. There were two intense fragments (m/z 98 and 111) in the mass spectra of both I and 2 which indicates the existence of cyclic fragments and two types of fragmentation. Incomplete trimethylsilylation also confirmed indirectly that I and 2 exist as keto-enol tautomers. The presence of carbonyl group and conjugated double bond is also confirmed by the type and pH-dependence of the UV spectra, tH N M R spectrum in chloroform-d was in good agreement with previous data obtained by other methods and indicated keto-enolic tautomerism. Thus we conclude that the onion phytoalexins are 5-octyl-cyclopenta 1,3-dione (1) and 5-hexyl-cyclopenta 1,3-dione (2). This is the first time that compounds of such structure have been isolated either from onion or as phytoalexins
[8, 9]. EXPERIMENTAL IHNMR: 80 MHz in CDCI3, using CHCI 3 as int. standard. MS: direct inlet and by chromatomass-spectrometry at 70 eV. Plant material and infection. Fleshy scales dissected from onion bulbs (cv Skvirsky), were placed in plastic boxes on moist tissue paper, abaxial epidermis facing up. Small agar pieces (7 mm in diameter) containing 2-day-old Botrytis cinerea mycelium were mounted onto the epidermis. After incubation in closed boxes in the dark at 20° for 48 hr, the tissue was scraped from inoculation sites and analysed for the presence of antifungal compounds. TLC plate bioassays with Cladosporium spp. were carried out as previously described [lO'l. Extraction and isolation. Excised tissue was homogenised in redistilled 96% EtOH (at least 10 ml EtOH g- 1fr. wt tissue). The extract was evapd in vacua at 40°. The aq. phase was reextracted with benzol. The CeHe fr. was evapd up to the complete removal
799
800
L. TVERSKOY et al. Table 1. The analysis of mass-spectral main peaks of compounds 1 and 2
Compound, peak
M/z measured value
Tsibulin 1, [M] + Tsibulin 2 [M] + Fragment 1 Fragment 2
210,1607 182,1300 111,0386 98,0346
of H 2 0 and suspended in hexane-Et20 (3:1). The residue was removed by centrifugation (3 000 g) and the filtered supernatant was the tsibulin total preparation. The contents of I and 2 were 0.28 and 0.65 rag g- 1 of limited lesions. The phytoalexins were further purified by gel filtration on a Shott-filter funnel with gradient mixt. hexane-Et20. Frs used in further experiments consisted of 1 (20-25%) and 2 (80-75%). This fr. was sepd into pure 1 and 2 by prep. HPLC, (21.2 mm x 25 cm) with a mobile phase hexane-iso-PrOH (199:1). The yields of pure 1 and 2 were up to 50-60% of its content in necrotic tissue. Silylation.The trimethyl silylation of I and 2 was carried out under standard conditions Ell] using N,O-bis(trimethylsilyl)acetamide. Tsibulins 1 and 2. White to pale-yellow oils without odour. Odour of tsibulins total preparation is caused by the presence of trace amounts of sulphur-containing metabolites. IR v m,xr~rcm- 1: 1608, 1705 (CO-C--C), 3554 (OH), 3600 (OH); UV A~m t°n nm: 207, 220, 246; EIMS 70eV m/z 43, 57, 68, 85, 98 (C5H602) , 111 (C6H702) , 182 (C11HlsO2, tsibulin 1) and 43, 57, 68, 85, 98 (C5H602) , 111 (C6H702) , 210 (C13H2202, tsibulin 2).
Formula C13H2202 CllH1802
C6H702 C5H60 2
M/z calculated value 210,1620 182,1309 111,0446 98,0368
REFERENCES
1. Ku6, J. (1972) Ann. Rev. Phytopothol. 10, 207. 2. Cruickshank, K. A. M. (1977) Pontif. Acad. Sci. Set. Varia41, 509. 3. Keen, N. T. (1968) Jowa State J. Res. 60, 477. 4. Stewart, A. and Mansfield, J. W. (1984) Plant Pathol. 33, 401. 5. Dmitriev, A. P., T~erskoy, L. A. and Grodzinsky, D. M. (1988) Brighton Crop Protect. Conf. Pests Dis. 3, 1173. 6. Dmitriev, A. P., Tverskoy, L. A. and Grodzinsky, D. M. (1989) Prikladn. Biochim. Microbiol. (Russ., Engl. Summ.) 25, 232. 7. Dmitriev, A. P., Gushcha, N. I., Zakordonetz, O. A. and Grodzinsky, D. M. (1988) Mycol. Phytopathol. (Russ., Engl. Suture.) 1, 65. 8. Carson, J. F. (1987) Food Rev. lntern. 3, 71. 9. Coxon, D. T. (1982) in Phytoalexins (Bailey, J. A. and Mansfield, J. W., eds). Blackie, Glasgow. 10. Homans, A. L. and Fuchs, A. (1970) J. Chromatoor. 51, 327. 11. Zelenkova, N. P. (1988) ltogi Nauki Tech. Chromatographya VINITI (Russ., Engl. Summ.) 6, 134.
0031-9422/91 $3.00+0.00 © 1991 Pergamon Press pie
Printed in Great Britain.
TWO PHYTOALEXINS FROM A L L I U M CEPA BULBS LEONID TVERSKOY, ALEXANDER DMITRIEV, ANATOLY KOZLOVSKY* and DMITRY GRODZINSKY Institute of Cell Biology and Genetic Engineering, Vasilkovskaya 31/17, 252627 Kiev, U.S.S.R.; *Institute of Biochemistry and Physiology of Microorganisms, 142292 Puschino, U.S.S.R. (Received in revised form 11 July 1990)
Key Word Index--Allium cepa; Liliaceae; bulbs; phytoalexins; 5-octyl-cyclopenta-l,3-dione; 5-hexyl-cyclopenta1,3-diane.
Abstract--Two new phytoalexins: 5-octyl-cyclopenta-1,3-dione and 5-hexyl-cyclopenta-1,3-dione were isolated from the bulbs of Allium cepa. Their structures were elucidated by H P L C and spectroscopic methods.
INTRODUCTION
Phytoalexins are low M, antimicrobial compounds produced by plants in response to infection by microorganism. These compounds are accumulated at the site of infection and are considered to be involved in the plant's defence response to potential pathogens [1-3]. Phytoalexins have not been isolated from members of the Liliaceae [4"1. Previous studies on AUium cepa have resulted in the isolation of a group of fungitoxic substances with closely related polarity [5"1. We gave them a working name tsibulins from the Ukrainian 'tsibulya', meaning onion. Besides sulphur-containing pre-formed and stress metabolites, we have found two phytoalexins (tsibulins 1 and 2) [6] which accumulated only in response to fungal infection. The present paper deals with the isolation and identification of these onion phytoalexins. RESULTS AND DISCUSSION
In response to inoculation with Botrytis cinerea Pers. ex Fr. mycelium, Allium cepa bulb scales showed limited or spread lesions confined to the tissue beneath mycellure-contained agar pieces. Growth of B. cinerea was restricted within limited lesions [7]. Examination of infected tissue extracts by TLC plate bioassays with Cladosporium spp. revealed the presence within the lesions of two antifungal compounds (phytoalexins) which were absent from fresh or wounded tissues. The phytoalexins were isolated by gel filtration, TLC and HPLC, and identified as 5-octyl-cyclopenta-l,3-
o•R 0
1 R m (CH2hMe 2 R " (CH2)sMe PHY 3 0 : 3 - F
dione (1) and 5-hexyl-cyclopenta-l,3-dione (2) as follows. Molecular weights and formulae given by high resolution mass spectrometry are presented in Table 1. The most intensive IR bands indicated the presence of aliphatic substitution. Absorption bands at I705 and I608 c m - i revealed the presence of a conjugated carbonyl group and double bond, respectively. The hydroxyl is indicated by 3554cm - t (bounded) and 3600cm - t (free enolic hydroxyl) bands. Mass and IR spectroscopy data showed the absence of sulphur-containing functional groups. There were two intense fragments (m/z 98 and 111) in the mass spectra of both I and 2 which indicates the existence of cyclic fragments and two types of fragmentation. Incomplete trimethylsilylation also confirmed indirectly that I and 2 exist as keto-enol tautomers. The presence of carbonyl group and conjugated double bond is also confirmed by the type and pH-dependence of the UV spectra, tH N M R spectrum in chloroform-d was in good agreement with previous data obtained by other methods and indicated keto-enolic tautomerism. Thus we conclude that the onion phytoalexins are 5-octyl-cyclopenta 1,3-dione (1) and 5-hexyl-cyclopenta 1,3-dione (2). This is the first time that compounds of such structure have been isolated either from onion or as phytoalexins
[8, 9]. EXPERIMENTAL IHNMR: 80 MHz in CDCI3, using CHCI 3 as int. standard. MS: direct inlet and by chromatomass-spectrometry at 70 eV. Plant material and infection. Fleshy scales dissected from onion bulbs (cv Skvirsky), were placed in plastic boxes on moist tissue paper, abaxial epidermis facing up. Small agar pieces (7 mm in diameter) containing 2-day-old Botrytis cinerea mycelium were mounted onto the epidermis. After incubation in closed boxes in the dark at 20° for 48 hr, the tissue was scraped from inoculation sites and analysed for the presence of antifungal compounds. TLC plate bioassays with Cladosporium spp. were carried out as previously described [lO'l. Extraction and isolation. Excised tissue was homogenised in redistilled 96% EtOH (at least 10 ml EtOH g- 1fr. wt tissue). The extract was evapd in vacua at 40°. The aq. phase was reextracted with benzol. The CeHe fr. was evapd up to the complete removal
799
800
L. TVERSKOY et al. Table 1. The analysis of mass-spectral main peaks of compounds 1 and 2
Compound, peak
M/z measured value
Tsibulin 1, [M] + Tsibulin 2 [M] + Fragment 1 Fragment 2
210,1607 182,1300 111,0386 98,0346
of H 2 0 and suspended in hexane-Et20 (3:1). The residue was removed by centrifugation (3 000 g) and the filtered supernatant was the tsibulin total preparation. The contents of I and 2 were 0.28 and 0.65 rag g- 1 of limited lesions. The phytoalexins were further purified by gel filtration on a Shott-filter funnel with gradient mixt. hexane-Et20. Frs used in further experiments consisted of 1 (20-25%) and 2 (80-75%). This fr. was sepd into pure 1 and 2 by prep. HPLC, (21.2 mm x 25 cm) with a mobile phase hexane-iso-PrOH (199:1). The yields of pure 1 and 2 were up to 50-60% of its content in necrotic tissue. Silylation.The trimethyl silylation of I and 2 was carried out under standard conditions Ell] using N,O-bis(trimethylsilyl)acetamide. Tsibulins 1 and 2. White to pale-yellow oils without odour. Odour of tsibulins total preparation is caused by the presence of trace amounts of sulphur-containing metabolites. IR v m,xr~rcm- 1: 1608, 1705 (CO-C--C), 3554 (OH), 3600 (OH); UV A~m t°n nm: 207, 220, 246; EIMS 70eV m/z 43, 57, 68, 85, 98 (C5H602) , 111 (C6H702) , 182 (C11HlsO2, tsibulin 1) and 43, 57, 68, 85, 98 (C5H602) , 111 (C6H702) , 210 (C13H2202, tsibulin 2).
Formula C13H2202 CllH1802
C6H702 C5H60 2
M/z calculated value 210,1620 182,1309 111,0446 98,0368
REFERENCES
1. Ku6, J. (1972) Ann. Rev. Phytopothol. 10, 207. 2. Cruickshank, K. A. M. (1977) Pontif. Acad. Sci. Set. Varia41, 509. 3. Keen, N. T. (1968) Jowa State J. Res. 60, 477. 4. Stewart, A. and Mansfield, J. W. (1984) Plant Pathol. 33, 401. 5. Dmitriev, A. P., T~erskoy, L. A. and Grodzinsky, D. M. (1988) Brighton Crop Protect. Conf. Pests Dis. 3, 1173. 6. Dmitriev, A. P., Tverskoy, L. A. and Grodzinsky, D. M. (1989) Prikladn. Biochim. Microbiol. (Russ., Engl. Summ.) 25, 232. 7. Dmitriev, A. P., Gushcha, N. I., Zakordonetz, O. A. and Grodzinsky, D. M. (1988) Mycol. Phytopathol. (Russ., Engl. Suture.) 1, 65. 8. Carson, J. F. (1987) Food Rev. lntern. 3, 71. 9. Coxon, D. T. (1982) in Phytoalexins (Bailey, J. A. and Mansfield, J. W., eds). Blackie, Glasgow. 10. Homans, A. L. and Fuchs, A. (1970) J. Chromatoor. 51, 327. 11. Zelenkova, N. P. (1988) ltogi Nauki Tech. Chromatographya VINITI (Russ., Engl. Summ.) 6, 134.