39
Journal of Ethnapharmacology. 36 (1992) 39-41 Elsevier Scientific Publishers Ireland Ltd.
Antimicrobial
activity of benzylisoquinoline
alkaloids from
Annona s~~~~~n~~DC. Maqal de Q. Pauloa, JosC M. Barbosa-Filho”, Edeltrudes 0. Limaa, Ricardo F. Maia”, Rita de Cassia B.B.C. Barbosaa and Maria Auxiliadora C. Kaplanb a~~rat~ria
de Tecnoiogia Far~ac~utica, CCS, ~~iv~rsi~de Federal da Par&a. 58 Ooo. Jm-o Pessaa, PB and hhWeo de Pes~uisas de Produtos Naturais, CC’S, Universidade Federal do Rio de Janeiro, 21941, Rio de Janeiro. RJ (Brazil) (Received February 23, 1989; revision received February 27, 1991; accepted September 27, 1991)
Bark of Annona salzmarzii D.C. (Annonaceae), used in Brazilian folk medicine, was found to contain four benzylisoquinoline alkaloids, namely teticuline, anonaine, laureiliptine and isoboldine. Only anonaine possesses some antibacterial property while all four alkaloids show some antifungal activity. Key wora!s:Annana salzmanii; Annonaceae; benzylisoquinoline
alkaloids; antibacterial activity;
antifungal
activity
isolation of the const~tuenfs Amona salzmanii D.C. (Annonaceae) is a small leafy tree with a thick stalk which occurs in the southeast of Brazil from Bahia to Sao Paulo. Several of its organs are used in folk medicine as remedies for the treatment of a variety of human ailments such as verminosis, dysentery, ulcers and inflammatory conditions (Agra, 1977). The reasons for these observations are unknown, as is the chemical composition of the species. In the present report we wish to communicate the presence in the bark of the trunk, of four known benzylisoquinoline alkaloids, one benzyltetrahydroisoquinoline (reticuline) (Leboeuf, 1981) and three aporphines (anonaine (Urzua, 1977), laurelliptine (Hocquemiller, 1977) and isoboldine (Jossang, 1977)). Materials and Methods Plant material Bark of A. salzmani~was collected in Santa Rita, PB, by Maria de Fatima Agra and voucher specimens are on file at the Herbarium of Universidade Federal da Paraiba. Correspondence to: Maria Auxiliadora C. Kaplan, Nticteo de Pesquisas de Produtos Naturais, Centro de Ci&tcias da Saude Bloco H, Bha da Cidade Universitaria 21941, Rio de Janeiro, Brazil.
Dried stembark was reduced to powder (1.5 kg) and extracted with ethanol in a Soxhlet apparatus (48 h). The solution was filtered and evaporated to dryness under vacuum. The residue was washed successively with hexane and dichloromethane. The insoluble portions were suspended in aqueous NazCOs and the mixture was extracted with chloroform. The organic layer was evaporated to dryness under vacuum and the residue was fractionated by column chromatography on silica gel 60 (E. Merck), into four fractions which were purified by TLC on silica gel PFz% (E. Merck) giving reticuline (28 mg), anonaine (16 mg), laurelliptine (8 mg) and isoboldine (11 mg). Assay for antibacterial activity The microorganisms used included Staphylococcus aureus (ATCC-6538), S. epidermidis (ATCC-12.228), Bacillus cereus (ATCC-14.579), Escherichia coli (ATCC-11.105), Pseudomonas aeruginosa (ATCC-27.853) and ~rypt~~o~c~ neoforma~s (FCF-1 19), cultivated in Casoy agar. Solutions of the compounds were prepared containing 1 mg/ml, in aqueous 50% dimethyl sulfoxide (DMSO). To sterilized Petri dishes with a suspension of the microorganisms (1 ml) standardized by the McFarlane scale, liquefied culture medium (45”C, 20 ml) was added. After solidification the compound solutions (70 ~1) were introduc-
0378-8741192/%05.00 0 1992 Elsevier Scientific Publishers Ireland Ltd Printed and Published in Ireland
40
TABLE
1
ANTIMICROBIAL Compounds (1 wW
ACTIVITY
OF THE ALKALOIDS
Candida albicans
Candida tropicalis
FROM
Cryplococcus
A. SALZMANII
Bacillus cereus
IN SOLUTIONS
Escherichia coli
Pseudomonas aeruginosa
neoformans Reticuline Anonaine Laurelliptine Isoboldine Blank test for microorganism Blank test for solvent
20 mm
OF 1 mg/ml
8 mm
Staphylo-
Staphylo-
COCCUS
coccus
aureus
epidermidis
14 mm
12 mm
-
-
ed into preformed cavities. Controls were run for solvent and microorganisms. Each assay was incubated at 3.7”C for 24-48 h prior to the measurement of the inhibition halo (Moura, 1977) (Table 1). Assay for antifungal activity
The above procedure was also applied for the fungi Candida albicans (ICB-12) and C. tropicalis (ICB-19) (Table 1). Two additional fungi, Trichophyton rubrum (ICB-A04) and Microsporum gypseum (ICB-281) were tested by a technique involving progressive dilution of solutions in Sabouraud liquor. Each alkaloid was solubilized in DMSO and the solutions were diluted with the required volumes of Sabouraud liquor. Each assay was incubated at 37°C for 7-14 days prior to the measurement (McGuiness, 1980) (Table 2).
tropicalis, Cryptococcus neoformans and Pseudomonas aeruginosa, as well as for the action of the
other three alkaloids on all microorganisms mentioned so far and control experiments involving the same microorganisms and the same solvent. Only anonaine possesses some antibacterial activity (Table l), whereas all four alkaloids show some antifungal activity (Table 2). However it is again anonaine which possesses by far the greatest fungicidal power. These preliminary results suggest that the reported antidysenteric effect of the drug may be due to the aporphine alkaloid anonaine.
TABLE
2
ANITIMICROBIAL ACTIVITY OF ALKALOID SOLUTIONS; (+++) STRONG INHIBITION; (++) MEDIUM INHIBITION; INHIBITION
(+)
WEAK
Results and Discussion Compounds
The identification of the four alkaloids, anonaine, reticuline, isoboldine and laureliptine were based on the comparison of physical and spectral data ([a] D, UV, IR, ‘H and 13C NMR and MS) with data registered in the literature. All four compounds have been isolated previously from other species of the family Annonaceae. At the dilution employed (1 mg/ml in DMSO) only anonaine showed activity against bacteria, specifically against Bacillus cereus, Escherichia coli, Staphylococcus aureus and S. epidermidis. Inhibition potential as measured by the inhibition halo was respectively 20, 8, 14 and 12 mm. Zero values for inhibition halos were obtained for anonaine in the presence of Candida albicans, C.
Reticuline
&ml
INHIBITION;
Tricophyton rubrum
(0)
Microsporum gypseun
++
+
0
0
125 500 250 125 500 250 125 500 250 125
0
0
+++ +++ ++ ++
+++ ++ +
0
0
0
0
+++
+
0
0
0
0
Blank test for microorganisms
0
0
Blank test for solvent
0
0
Anonaine
Laurelliptine
Isoboldine
500 250
NO
0
41
References Agra, M. de F. (1977) Farmacop&a Popular da Paraiba. Universidade Federal da Paraiba (MEC), Jogo Pessoa. Hocquemiller, R., Cabalion, P., Bouquet, A. and Cavl, A. (1977) Chimie vigitale-L’isopiline, nouvel alcalorde aporphinique isoli de Paris, Ser. C. 285, Jossang, A., Leboeuf, Annonacees XVII
l’lsolona pilosa. C.R. Academy Science, 447-450. M. and Cavt, A. (1977) Alcaloi’des des : Alcaloi’des de I’hantia polycat-pa Engl.
et Diels. Pianta Medica. 32. 249-257.
Leboeuf, M., Leguet, C., Cave, A., Desconclois, J.F., Forgacs, P. and Jacquemin, H. (1981) Alcalo’ides des Annonacies XXIX: Alcaloi:des de I’Annona muricata L. Planta Medica. 42, 37-44. McGuiness, M.R. (1980) Laboratory Handbook of Medical Mycology. Academic Press, New York, pp. 41 l-446. Moura, R.A.A. (1977) Thicas de Laborat&io. Livraria Atheneu, Rio de Janeiro. Urztia, A. and Cassels, B.K. (1977) Revista Latinoamericana Quimica 8(3), 133-134.
de
Journal of Ethnapharmacology. 36 (1992) 39-41 Elsevier Scientific Publishers Ireland Ltd.
Antimicrobial
activity of benzylisoquinoline
alkaloids from
Annona s~~~~~n~~DC. Maqal de Q. Pauloa, JosC M. Barbosa-Filho”, Edeltrudes 0. Limaa, Ricardo F. Maia”, Rita de Cassia B.B.C. Barbosaa and Maria Auxiliadora C. Kaplanb a~~rat~ria
de Tecnoiogia Far~ac~utica, CCS, ~~iv~rsi~de Federal da Par&a. 58 Ooo. Jm-o Pessaa, PB and hhWeo de Pes~uisas de Produtos Naturais, CC’S, Universidade Federal do Rio de Janeiro, 21941, Rio de Janeiro. RJ (Brazil) (Received February 23, 1989; revision received February 27, 1991; accepted September 27, 1991)
Bark of Annona salzmarzii D.C. (Annonaceae), used in Brazilian folk medicine, was found to contain four benzylisoquinoline alkaloids, namely teticuline, anonaine, laureiliptine and isoboldine. Only anonaine possesses some antibacterial property while all four alkaloids show some antifungal activity. Key wora!s:Annana salzmanii; Annonaceae; benzylisoquinoline
alkaloids; antibacterial activity;
antifungal
activity
isolation of the const~tuenfs Amona salzmanii D.C. (Annonaceae) is a small leafy tree with a thick stalk which occurs in the southeast of Brazil from Bahia to Sao Paulo. Several of its organs are used in folk medicine as remedies for the treatment of a variety of human ailments such as verminosis, dysentery, ulcers and inflammatory conditions (Agra, 1977). The reasons for these observations are unknown, as is the chemical composition of the species. In the present report we wish to communicate the presence in the bark of the trunk, of four known benzylisoquinoline alkaloids, one benzyltetrahydroisoquinoline (reticuline) (Leboeuf, 1981) and three aporphines (anonaine (Urzua, 1977), laurelliptine (Hocquemiller, 1977) and isoboldine (Jossang, 1977)). Materials and Methods Plant material Bark of A. salzmani~was collected in Santa Rita, PB, by Maria de Fatima Agra and voucher specimens are on file at the Herbarium of Universidade Federal da Paraiba. Correspondence to: Maria Auxiliadora C. Kaplan, Nticteo de Pesquisas de Produtos Naturais, Centro de Ci&tcias da Saude Bloco H, Bha da Cidade Universitaria 21941, Rio de Janeiro, Brazil.
Dried stembark was reduced to powder (1.5 kg) and extracted with ethanol in a Soxhlet apparatus (48 h). The solution was filtered and evaporated to dryness under vacuum. The residue was washed successively with hexane and dichloromethane. The insoluble portions were suspended in aqueous NazCOs and the mixture was extracted with chloroform. The organic layer was evaporated to dryness under vacuum and the residue was fractionated by column chromatography on silica gel 60 (E. Merck), into four fractions which were purified by TLC on silica gel PFz% (E. Merck) giving reticuline (28 mg), anonaine (16 mg), laurelliptine (8 mg) and isoboldine (11 mg). Assay for antibacterial activity The microorganisms used included Staphylococcus aureus (ATCC-6538), S. epidermidis (ATCC-12.228), Bacillus cereus (ATCC-14.579), Escherichia coli (ATCC-11.105), Pseudomonas aeruginosa (ATCC-27.853) and ~rypt~~o~c~ neoforma~s (FCF-1 19), cultivated in Casoy agar. Solutions of the compounds were prepared containing 1 mg/ml, in aqueous 50% dimethyl sulfoxide (DMSO). To sterilized Petri dishes with a suspension of the microorganisms (1 ml) standardized by the McFarlane scale, liquefied culture medium (45”C, 20 ml) was added. After solidification the compound solutions (70 ~1) were introduc-
0378-8741192/%05.00 0 1992 Elsevier Scientific Publishers Ireland Ltd Printed and Published in Ireland
40
TABLE
1
ANTIMICROBIAL Compounds (1 wW
ACTIVITY
OF THE ALKALOIDS
Candida albicans
Candida tropicalis
FROM
Cryplococcus
A. SALZMANII
Bacillus cereus
IN SOLUTIONS
Escherichia coli
Pseudomonas aeruginosa
neoformans Reticuline Anonaine Laurelliptine Isoboldine Blank test for microorganism Blank test for solvent
20 mm
OF 1 mg/ml
8 mm
Staphylo-
Staphylo-
COCCUS
coccus
aureus
epidermidis
14 mm
12 mm
-
-
ed into preformed cavities. Controls were run for solvent and microorganisms. Each assay was incubated at 3.7”C for 24-48 h prior to the measurement of the inhibition halo (Moura, 1977) (Table 1). Assay for antifungal activity
The above procedure was also applied for the fungi Candida albicans (ICB-12) and C. tropicalis (ICB-19) (Table 1). Two additional fungi, Trichophyton rubrum (ICB-A04) and Microsporum gypseum (ICB-281) were tested by a technique involving progressive dilution of solutions in Sabouraud liquor. Each alkaloid was solubilized in DMSO and the solutions were diluted with the required volumes of Sabouraud liquor. Each assay was incubated at 37°C for 7-14 days prior to the measurement (McGuiness, 1980) (Table 2).
tropicalis, Cryptococcus neoformans and Pseudomonas aeruginosa, as well as for the action of the
other three alkaloids on all microorganisms mentioned so far and control experiments involving the same microorganisms and the same solvent. Only anonaine possesses some antibacterial activity (Table l), whereas all four alkaloids show some antifungal activity (Table 2). However it is again anonaine which possesses by far the greatest fungicidal power. These preliminary results suggest that the reported antidysenteric effect of the drug may be due to the aporphine alkaloid anonaine.
TABLE
2
ANITIMICROBIAL ACTIVITY OF ALKALOID SOLUTIONS; (+++) STRONG INHIBITION; (++) MEDIUM INHIBITION; INHIBITION
(+)
WEAK
Results and Discussion Compounds
The identification of the four alkaloids, anonaine, reticuline, isoboldine and laureliptine were based on the comparison of physical and spectral data ([a] D, UV, IR, ‘H and 13C NMR and MS) with data registered in the literature. All four compounds have been isolated previously from other species of the family Annonaceae. At the dilution employed (1 mg/ml in DMSO) only anonaine showed activity against bacteria, specifically against Bacillus cereus, Escherichia coli, Staphylococcus aureus and S. epidermidis. Inhibition potential as measured by the inhibition halo was respectively 20, 8, 14 and 12 mm. Zero values for inhibition halos were obtained for anonaine in the presence of Candida albicans, C.
Reticuline
&ml
INHIBITION;
Tricophyton rubrum
(0)
Microsporum gypseun
++
+
0
0
125 500 250 125 500 250 125 500 250 125
0
0
+++ +++ ++ ++
+++ ++ +
0
0
0
0
+++
+
0
0
0
0
Blank test for microorganisms
0
0
Blank test for solvent
0
0
Anonaine
Laurelliptine
Isoboldine
500 250
NO
0
41
References Agra, M. de F. (1977) Farmacop&a Popular da Paraiba. Universidade Federal da Paraiba (MEC), Jogo Pessoa. Hocquemiller, R., Cabalion, P., Bouquet, A. and Cavl, A. (1977) Chimie vigitale-L’isopiline, nouvel alcalorde aporphinique isoli de Paris, Ser. C. 285, Jossang, A., Leboeuf, Annonacees XVII
l’lsolona pilosa. C.R. Academy Science, 447-450. M. and Cavt, A. (1977) Alcaloi’des des : Alcaloi’des de I’hantia polycat-pa Engl.
et Diels. Pianta Medica. 32. 249-257.
Leboeuf, M., Leguet, C., Cave, A., Desconclois, J.F., Forgacs, P. and Jacquemin, H. (1981) Alcalo’ides des Annonacies XXIX: Alcaloi:des de I’Annona muricata L. Planta Medica. 42, 37-44. McGuiness, M.R. (1980) Laboratory Handbook of Medical Mycology. Academic Press, New York, pp. 41 l-446. Moura, R.A.A. (1977) Thicas de Laborat&io. Livraria Atheneu, Rio de Janeiro. Urztia, A. and Cassels, B.K. (1977) Revista Latinoamericana Quimica 8(3), 133-134.
de
