- Email: [email protected]
An antifungal chromene from Eupatorium riparium
Phytochemisny, Vol. 31, No. 6, pp. 1983-1985, 1992 hinted in Great Britain.
0
003 l-9422/92 S5.00 +O.OO 1992 Pergamon PressLtd
AN ANTIFUNGAL CHROMENE FROM EUPATORZUM RZPARZUM B. M. RATNAYAKE BANDARA,*CHANDRALAL M. HEWAGE,VERANJAKARUNARATNE,~ G. PERCYWANNIGAMAand N. K. B. ADIKARAM~ Department of Chemistry, University of Peradeniya, Peradeniya, Sri Lanka; $&partment of Botany, University of Peradeniya, Peradeniya, Sri Lanka (Received in reuisedjorm 4 October 1991)
Key Word Index-Eupatorium ripariwn;Compositae;antifungal activity;chromenes;methylripariochromeneA, avocado fruit;Colletotrichumgloeosporioides.
Abstract-Methylripariochromene
A (6-acetyl-7,8dimethoxy-2,2-dimethylchromene),
a
root
constituent
of
Eupatorium riparium, displayed antifungal activity against five ofthe seven fungal species tested. The chromene showed a toxicity to the fungus Colletotrichum gloeosporioides, a tropical pathogen, comparable to that of a commercial
fungicide, although the chromene appeared to be fungistatic.
INTRODUCTION
pR’
Eupatorium riparium Regel is a troublesome and invasive
weed growing in some tropical countries [l]. Several chromenes have been reported from the Australian [2], Jamaican [3] and Hawaiian [4] populations of this plant. Triterpenoids have been isolated from the Indian E. riparium [S, 61. In this paper, we describe the isolation of an antifungal chromene, four other known chromenes and three known triterpenoids from E. riparium growing in Sri Lanka.
0
*Present address: Department of Biochemistry, Tulane University School of Medicine, 1430 Tulane Ave, New Orleans, LA 70112, U.S.A. TPresent address: Department of Chemistry, University of British Columbia, 2036 Main Mall, Vancouver, British Columbia V6T lY6, Canada.
1
R’
1
2 3 4
RFSULTSANDDISCUSSION The root extracts of E. riparium exhibited antifungal activity against Cladosporium cladosporioides in the TLC bioassay. Chromatographic fractionation of the n-hexane extract, directed by the TLC bioassay, provided the active compound as a light brown oil. High resolution mass data suggested the molecular formula CiSH1s04. The ‘H (HOMOCOSY) and 13C (APT) NMR spectra corresponded to the presence of a 2,2-dimethylchromene having an acetyl and two methoxy groups and an unsubstituted aromatic carbon atom. The spectral data (IR and ‘H NMR) compared well with those of methylripariochromene A (1) previously isolated from the same species [2-4]. The antifungal activity of 1 and that of Benlate, a commercial fungicide, was evaluated against seven species of fungi using the TLC bioassay technique. The areas of fungal growth inhibition produced by varying amounts
m
ii0A3 9
R
5
R’ H H
R’ R3 OMe OMe OH Ohie OAc OH H OBui OH H H OH H
of 1 and Benlate are given in Table 1. The results show that 1 is inhibitory to five of the seven species tested, and that the sensitivity among the species varied considerably. Benlate showed a greater toxicity than 1 to most of the fungi. However, both 1 and Benlate displayed comparable toxicity to C. gloeosporioides. Colletrotrichum gloeosporioides is a common pathogen in the tropics having a wide host range, and is known to cause anthracnose disease in several fruit crops including avocado and mango [7]. Anthracnose is the most important disease of avocado in Sri Lanka and is responsible for over 30% fruit loss during post-harvest storage [8]. In view of a possible application of the weed E. riparium in the control of anthracnose in fruits, the antifungal properties of 1 were further examined against C. gloeosporioides, using the spore germination assay. The results shown in Table 2 confirmed that both 1 and Benlate had the same degree of antifungal activity against C. gloeosporioides. Next we determined whether the effect of 1 against C. gloeosporioides was fungistatic (temporary inhibition) or fungicidal (permanent damage). The conidia of the fungus that had been treated with a relatively large dose of 1 (2 mgml- ‘) for 6 hr were washed and separated by centrifugation followed by resuspension in water and
1983
B. M. RATNAYAKE BANDARAet al.
1984
Table 1. Area of fungal growth inhibition on the bioassay TLC plates corresponding to varying amounts of methylripariochromene A (1) and Benlate (B) Area of inhibItion (cm2) .-. _
--__ Fungus
VB
150
Cladosporium cladosporioides
1 B
ND*
Alternaria tenuis Nees.
1
0
0
Botrydiplodia theobromae
B 1 B
ND 0 ND
9.1 0 0
(Penz.) Penz. & Sacc.
(Pat.)
9.1
100
50
2s
1Opg
1.1 7.1
0.1 4.5
0
0
0
6.2 0 0
3.8 0 0
1.5 0 0 0
6.2
3.1
12.9
10.2
1
6.1
3.5
1.8
0.2
B
ND
19.7
12.6
8.0
3.7
Fusarium sp.
1 B
4.5
2.1
1.1
0
0
ND
7.1
4.5
3.4
1.1
Colletotrichum gloeosporioides
1
7.2
4.9
3.1
2.1
1.3
B
ND
4.9
2.3
2.0
1.6
1
5.3
3.1
2.1
0.8
0.3
B
ND
4.5
3.1
2.0
1.1
Aspergillus sp.
Cercospora nicotianae
Ell. & Ev. *ND, not determined.
Table 2. Percentage germination of conidia of Colletotrichum A (1) and Benlate (B)
gloeosporioides in methylripariochromene
% Germination of conidia Concentration of 1 or B (fig ml- I)*
with 1
with B
125 63 31 16 8 4 0t
0 5 11 16 20 28 31
0 5 10 16 19 29 31
*In 10% ethanol. tlO% ethanol.
allowed to germinate. The counts showed that the germination of the recovered conidia is similar to that of the controls, indicating that the inhibitory effect of 1 on the conidia is temporary and reversible and therefore fungistatic. We also isolated the following compounds from the root extracts of E. riparium by chromatographic fractionation: ripariochromene A (2) [2, 41, ripariochromene B (3) [2], ripariochromene C (4) [2], eupatoriochromene (5) [2], taraxasterol [5], taraxasteryl acetate [6] and stigmasterol [S]. These compounds did not exhibit antifungal activity against C. cladosporioides; however, their activities against other fungi (Table 1) were not examined. EXPEUIMENTAL Isolation of the antijiigal chromene (1) and other compounds. Dried and powdered roots of E. riparium (0.9 kg), collected at
Hakgala, Sri Lanka, were extracted with hot n-hexane and hot CH,Cl, successively. Removal of solvent gave brownish solids:
9 g and 5 g from the n-hexane and CH,Cl, extracts, respectively. Both extracts were tested for antifungal activity against Cladosporium cladosporioides using the TLC bioassay technique [9]
and found to be active. The hexane extract (8 g) was chromatographed on medium pressure silica gel CC using n-hexane and CH,Cl,. The fraction eluted with 40% CH,Cl, in n-hexane produced a distinct inhibition area at R, 0.7 (CHZC12). This fraction was chromatographed repeatedly on silica gel CC and prep. TLC (silica gel) to obtam the antifungal compound as a light brown oil (50 mg) wluch was identified as methylripariochromene A (1) 12-43; 13CNMR (CDCI,, 100 MHz): 6197.9 (MeC=O), 158.1, 154.1 and 150.9 (C-7, C-8 and C-9), 129.8 (C-5), 124.8 (C-6), 122.3 (C-4), 121.6 (C-3), 117.8 (C-lo), 77.7 (C-2), 61.5 and 60.9 (2 x OCH,). 31.1 (CH,C=O), 28.5 and 28.3 (2 x CH,); EIMS 70 eV, m/z (rel. int.): 262 CM] T (23), 248 (100). 233 (34), 218 (16), 203 (20), 189 (26); CM]” 262.1216. CI,H,,O, requires 262.1205. The CC fractions eluted with 2%. 4% and 10% CH,Cl, in nhexane, after further purification on short CC and by prep. TLC, gave taraxasteryl acetate (CM] + 468), mp 240-241’ (lit. [6] 243’), taraxasterol (CM]’ 426), mp 216217” (lit. [S] 218-219”), and eupatoriochromene (5) ([Ml’ 218), mp 76-77” (lit. [Z] mp 76”), respectively. Further separation by prep. TLC of the CC fraction eluted with 60% CH,Cl, in n-hexane, gave ripariochromene A (2) ([Ml’ 248), mp 89-90” (lit. [2] 88.S”),ripariochromene B (3) (CM]’ 276), mp 142-145’ (lit. [2] 145-146”), and ripariochromene C (4) ([Ml’ 276), mp 106-108” (lit. [2] 109-110”). The ‘H NMR spectra of the above compounds were similar to those reported previously [2, 5, 61. The CC fraction eluted with CH,Cl, gave stigmasterol identical with an authentic sample. Antifungal assay. The hexane and CH,Cl, extracts and the above CC fractions were spotted (2 mg each) on TLC plates and the plates were developed in CHCI,. The plates, after air-drying overnight, were sprayed with a suspension of conidia of Cladosporium cladosporioldes in Czapek-Dox nutrient soln and incubated in a moist chamber at 26+2” for 48 hr [9]. Compound 1 (4 mg) was dissolved in CH,Cl, (4 ml); Benlate [4 mg; 50% active ingredient, methyl I-(butylcarbonyl)-2benzimidazolecarbamate; Du Pont, U.S.A.] was dissolved in water (4 ml). Seven TLC plates were spotted with the above solns of 1 and Benlate. Each plate, after air-drying overnight, was
An antifungal
chromene
separately sprayed with each of the seven fungi (in Table 1) in a Czapek-Dox nutrient soln and kept in a moist chamber at 26 f 2”. Diameters of the zones of inhibition were measured after 48 hr except for the two slow-growing fungi, A. tenuis and C. nicotianae, which were kept for 96 hr. The conidia of Colletotrichum gloeosporioides isolated from an anthracnose lesion of a ripe avocado fruit [7], were used for the spore germination assay. Conidia in the final suspension, after removal of hyphae and washing with water, were adjusted to ca 5 x lo5 ml-‘. Aliquots (10 ~1) of test soln (1 or Benlate in 10% EtOH in water) or controls (10% EtOH in water) were placed on clean glass slides. Aliquots (10 ~1) of the conidia suspension were added on to the test soln drops and mixed well. The glass slides were then incubated in moist chambers at 26*2” for 6 hr and germination was stopped by adding a drop of lactophenol. Randomly selected conidia were counted for germination and the percentage was determined. AcknowledgementsWe thank NARESA (Sri Lanka), IFS (Sweden) and UNESCO for financial support, D. S. A. Wijesundara, Royal Botanic Gardens, Peradeniya, Sri Lanka for
PHYTO31:6-K
from Eupatorium
riparium
1985
identifying the plant material, and Anura Paranagama S. Fernando for technical assistance.
and I. H.
REFERENCES 1. Grierson, A. J. C. (1980) in A Revised Handbook to the Flora of Ceylon (Dassanayake, M. D. and Fosberg, F. R., eds), Vol. 1, pp. 145-146. Amerind, New Delhi. 2. Anthonsen, T. (1969) Acta Chem. Stand. 23, 3605. 3. Taylor, D. R. and Wright, J. A. (1971) Phytochemistry 10, 1665. 4. Banerjee, S., Jakupovic, J., Bohhnann, F., King, R. M. and Robinson, H. (1985) Phytochemistry 24, 2681. 5. Talapatra, B., Mukhopadhyay, R. and Talaptra, S. K. (1978) J. Indian Chem. Sot. 55, 296. 6. Patra, A., Mukhopadhyay, A. K. and Mitra, A. K. (1981) J. Indian Chem. Sot. 58, 1124. 7. Sivanathan, S. and Adikaram, N. K. B. (1989) J. PhytopathoL 125, 97. 8. Adikaram, N. K. B. (198687) Ceylon J. Sci. 19/20, 1. 9. Klarman, W. L. and Stanford, J. B. (1968) Life Sci. 7, 1095.
0
003 l-9422/92 S5.00 +O.OO 1992 Pergamon PressLtd
AN ANTIFUNGAL CHROMENE FROM EUPATORZUM RZPARZUM B. M. RATNAYAKE BANDARA,*CHANDRALAL M. HEWAGE,VERANJAKARUNARATNE,~ G. PERCYWANNIGAMAand N. K. B. ADIKARAM~ Department of Chemistry, University of Peradeniya, Peradeniya, Sri Lanka; $&partment of Botany, University of Peradeniya, Peradeniya, Sri Lanka (Received in reuisedjorm 4 October 1991)
Key Word Index-Eupatorium ripariwn;Compositae;antifungal activity;chromenes;methylripariochromeneA, avocado fruit;Colletotrichumgloeosporioides.
Abstract-Methylripariochromene
A (6-acetyl-7,8dimethoxy-2,2-dimethylchromene),
a
root
constituent
of
Eupatorium riparium, displayed antifungal activity against five ofthe seven fungal species tested. The chromene showed a toxicity to the fungus Colletotrichum gloeosporioides, a tropical pathogen, comparable to that of a commercial
fungicide, although the chromene appeared to be fungistatic.
INTRODUCTION
pR’
Eupatorium riparium Regel is a troublesome and invasive
weed growing in some tropical countries [l]. Several chromenes have been reported from the Australian [2], Jamaican [3] and Hawaiian [4] populations of this plant. Triterpenoids have been isolated from the Indian E. riparium [S, 61. In this paper, we describe the isolation of an antifungal chromene, four other known chromenes and three known triterpenoids from E. riparium growing in Sri Lanka.
0
*Present address: Department of Biochemistry, Tulane University School of Medicine, 1430 Tulane Ave, New Orleans, LA 70112, U.S.A. TPresent address: Department of Chemistry, University of British Columbia, 2036 Main Mall, Vancouver, British Columbia V6T lY6, Canada.
1
R’
1
2 3 4
RFSULTSANDDISCUSSION The root extracts of E. riparium exhibited antifungal activity against Cladosporium cladosporioides in the TLC bioassay. Chromatographic fractionation of the n-hexane extract, directed by the TLC bioassay, provided the active compound as a light brown oil. High resolution mass data suggested the molecular formula CiSH1s04. The ‘H (HOMOCOSY) and 13C (APT) NMR spectra corresponded to the presence of a 2,2-dimethylchromene having an acetyl and two methoxy groups and an unsubstituted aromatic carbon atom. The spectral data (IR and ‘H NMR) compared well with those of methylripariochromene A (1) previously isolated from the same species [2-4]. The antifungal activity of 1 and that of Benlate, a commercial fungicide, was evaluated against seven species of fungi using the TLC bioassay technique. The areas of fungal growth inhibition produced by varying amounts
m
ii0A3 9
R
5
R’ H H
R’ R3 OMe OMe OH Ohie OAc OH H OBui OH H H OH H
of 1 and Benlate are given in Table 1. The results show that 1 is inhibitory to five of the seven species tested, and that the sensitivity among the species varied considerably. Benlate showed a greater toxicity than 1 to most of the fungi. However, both 1 and Benlate displayed comparable toxicity to C. gloeosporioides. Colletrotrichum gloeosporioides is a common pathogen in the tropics having a wide host range, and is known to cause anthracnose disease in several fruit crops including avocado and mango [7]. Anthracnose is the most important disease of avocado in Sri Lanka and is responsible for over 30% fruit loss during post-harvest storage [8]. In view of a possible application of the weed E. riparium in the control of anthracnose in fruits, the antifungal properties of 1 were further examined against C. gloeosporioides, using the spore germination assay. The results shown in Table 2 confirmed that both 1 and Benlate had the same degree of antifungal activity against C. gloeosporioides. Next we determined whether the effect of 1 against C. gloeosporioides was fungistatic (temporary inhibition) or fungicidal (permanent damage). The conidia of the fungus that had been treated with a relatively large dose of 1 (2 mgml- ‘) for 6 hr were washed and separated by centrifugation followed by resuspension in water and
1983
B. M. RATNAYAKE BANDARAet al.
1984
Table 1. Area of fungal growth inhibition on the bioassay TLC plates corresponding to varying amounts of methylripariochromene A (1) and Benlate (B) Area of inhibItion (cm2) .-. _
--__ Fungus
VB
150
Cladosporium cladosporioides
1 B
ND*
Alternaria tenuis Nees.
1
0
0
Botrydiplodia theobromae
B 1 B
ND 0 ND
9.1 0 0
(Penz.) Penz. & Sacc.
(Pat.)
9.1
100
50
2s
1Opg
1.1 7.1
0.1 4.5
0
0
0
6.2 0 0
3.8 0 0
1.5 0 0 0
6.2
3.1
12.9
10.2
1
6.1
3.5
1.8
0.2
B
ND
19.7
12.6
8.0
3.7
Fusarium sp.
1 B
4.5
2.1
1.1
0
0
ND
7.1
4.5
3.4
1.1
Colletotrichum gloeosporioides
1
7.2
4.9
3.1
2.1
1.3
B
ND
4.9
2.3
2.0
1.6
1
5.3
3.1
2.1
0.8
0.3
B
ND
4.5
3.1
2.0
1.1
Aspergillus sp.
Cercospora nicotianae
Ell. & Ev. *ND, not determined.
Table 2. Percentage germination of conidia of Colletotrichum A (1) and Benlate (B)
gloeosporioides in methylripariochromene
% Germination of conidia Concentration of 1 or B (fig ml- I)*
with 1
with B
125 63 31 16 8 4 0t
0 5 11 16 20 28 31
0 5 10 16 19 29 31
*In 10% ethanol. tlO% ethanol.
allowed to germinate. The counts showed that the germination of the recovered conidia is similar to that of the controls, indicating that the inhibitory effect of 1 on the conidia is temporary and reversible and therefore fungistatic. We also isolated the following compounds from the root extracts of E. riparium by chromatographic fractionation: ripariochromene A (2) [2, 41, ripariochromene B (3) [2], ripariochromene C (4) [2], eupatoriochromene (5) [2], taraxasterol [5], taraxasteryl acetate [6] and stigmasterol [S]. These compounds did not exhibit antifungal activity against C. cladosporioides; however, their activities against other fungi (Table 1) were not examined. EXPEUIMENTAL Isolation of the antijiigal chromene (1) and other compounds. Dried and powdered roots of E. riparium (0.9 kg), collected at
Hakgala, Sri Lanka, were extracted with hot n-hexane and hot CH,Cl, successively. Removal of solvent gave brownish solids:
9 g and 5 g from the n-hexane and CH,Cl, extracts, respectively. Both extracts were tested for antifungal activity against Cladosporium cladosporioides using the TLC bioassay technique [9]
and found to be active. The hexane extract (8 g) was chromatographed on medium pressure silica gel CC using n-hexane and CH,Cl,. The fraction eluted with 40% CH,Cl, in n-hexane produced a distinct inhibition area at R, 0.7 (CHZC12). This fraction was chromatographed repeatedly on silica gel CC and prep. TLC (silica gel) to obtam the antifungal compound as a light brown oil (50 mg) wluch was identified as methylripariochromene A (1) 12-43; 13CNMR (CDCI,, 100 MHz): 6197.9 (MeC=O), 158.1, 154.1 and 150.9 (C-7, C-8 and C-9), 129.8 (C-5), 124.8 (C-6), 122.3 (C-4), 121.6 (C-3), 117.8 (C-lo), 77.7 (C-2), 61.5 and 60.9 (2 x OCH,). 31.1 (CH,C=O), 28.5 and 28.3 (2 x CH,); EIMS 70 eV, m/z (rel. int.): 262 CM] T (23), 248 (100). 233 (34), 218 (16), 203 (20), 189 (26); CM]” 262.1216. CI,H,,O, requires 262.1205. The CC fractions eluted with 2%. 4% and 10% CH,Cl, in nhexane, after further purification on short CC and by prep. TLC, gave taraxasteryl acetate (CM] + 468), mp 240-241’ (lit. [6] 243’), taraxasterol (CM]’ 426), mp 216217” (lit. [S] 218-219”), and eupatoriochromene (5) ([Ml’ 218), mp 76-77” (lit. [Z] mp 76”), respectively. Further separation by prep. TLC of the CC fraction eluted with 60% CH,Cl, in n-hexane, gave ripariochromene A (2) ([Ml’ 248), mp 89-90” (lit. [2] 88.S”),ripariochromene B (3) (CM]’ 276), mp 142-145’ (lit. [2] 145-146”), and ripariochromene C (4) ([Ml’ 276), mp 106-108” (lit. [2] 109-110”). The ‘H NMR spectra of the above compounds were similar to those reported previously [2, 5, 61. The CC fraction eluted with CH,Cl, gave stigmasterol identical with an authentic sample. Antifungal assay. The hexane and CH,Cl, extracts and the above CC fractions were spotted (2 mg each) on TLC plates and the plates were developed in CHCI,. The plates, after air-drying overnight, were sprayed with a suspension of conidia of Cladosporium cladosporioldes in Czapek-Dox nutrient soln and incubated in a moist chamber at 26+2” for 48 hr [9]. Compound 1 (4 mg) was dissolved in CH,Cl, (4 ml); Benlate [4 mg; 50% active ingredient, methyl I-(butylcarbonyl)-2benzimidazolecarbamate; Du Pont, U.S.A.] was dissolved in water (4 ml). Seven TLC plates were spotted with the above solns of 1 and Benlate. Each plate, after air-drying overnight, was
An antifungal
chromene
separately sprayed with each of the seven fungi (in Table 1) in a Czapek-Dox nutrient soln and kept in a moist chamber at 26 f 2”. Diameters of the zones of inhibition were measured after 48 hr except for the two slow-growing fungi, A. tenuis and C. nicotianae, which were kept for 96 hr. The conidia of Colletotrichum gloeosporioides isolated from an anthracnose lesion of a ripe avocado fruit [7], were used for the spore germination assay. Conidia in the final suspension, after removal of hyphae and washing with water, were adjusted to ca 5 x lo5 ml-‘. Aliquots (10 ~1) of test soln (1 or Benlate in 10% EtOH in water) or controls (10% EtOH in water) were placed on clean glass slides. Aliquots (10 ~1) of the conidia suspension were added on to the test soln drops and mixed well. The glass slides were then incubated in moist chambers at 26*2” for 6 hr and germination was stopped by adding a drop of lactophenol. Randomly selected conidia were counted for germination and the percentage was determined. AcknowledgementsWe thank NARESA (Sri Lanka), IFS (Sweden) and UNESCO for financial support, D. S. A. Wijesundara, Royal Botanic Gardens, Peradeniya, Sri Lanka for
PHYTO31:6-K
from Eupatorium
riparium
1985
identifying the plant material, and Anura Paranagama S. Fernando for technical assistance.
and I. H.
REFERENCES 1. Grierson, A. J. C. (1980) in A Revised Handbook to the Flora of Ceylon (Dassanayake, M. D. and Fosberg, F. R., eds), Vol. 1, pp. 145-146. Amerind, New Delhi. 2. Anthonsen, T. (1969) Acta Chem. Stand. 23, 3605. 3. Taylor, D. R. and Wright, J. A. (1971) Phytochemistry 10, 1665. 4. Banerjee, S., Jakupovic, J., Bohhnann, F., King, R. M. and Robinson, H. (1985) Phytochemistry 24, 2681. 5. Talapatra, B., Mukhopadhyay, R. and Talaptra, S. K. (1978) J. Indian Chem. Sot. 55, 296. 6. Patra, A., Mukhopadhyay, A. K. and Mitra, A. K. (1981) J. Indian Chem. Sot. 58, 1124. 7. Sivanathan, S. and Adikaram, N. K. B. (1989) J. PhytopathoL 125, 97. 8. Adikaram, N. K. B. (198687) Ceylon J. Sci. 19/20, 1. 9. Klarman, W. L. and Stanford, J. B. (1968) Life Sci. 7, 1095.