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Chemotaxonomic implications of the absence of alkaloids in Psychotria gitingensis
Biochemical Systematics and Ecology 45 (2012) 20–22
Contents lists available at SciVerse ScienceDirect
Biochemical Systematics and Ecology journal homepage: www.elsevier.com/locate/biochemsyseco
Chemotaxonomic implications of the absence of alkaloids in Psychotria gitingensis Mario A. Tan*, Jameson A. Eusebio, Grecebio Jonathan D. Alejandro The Graduate School and Research Center for the Natural and Applied Sciences, University of Santo Tomas, España 1015, Manila, Philippines
a r t i c l e i n f o Article history: Received 10 April 2012 Accepted 7 July 2012 Available online 4 August 2012 Keywords: Psychotria gitingensis Rubiaceae Alkaloid Vomifoliol Chemotaxonomy
1. Subject and source The pantropical Psychotria L. (Rubiaceae, Rubioideae, Psychotrieae) is the largest member of the coffee family and the third largest angiosperm genus with more than 1800 species worldwide (Davis et al., 2009). Most Psychotria species grow as shrubs or small trees in the understorey of moist tropical and subtropical forests with a variety of growth forms, including herbs and epiphytes (Nepokroeff and Sytsma, 1999). The taxonomy of the genus is complex and the relationships among the species are unclear, especially the endemic species found in Southeast Asia (Sohmer and Davis, 2007). Phytochemical studies have mostly been on the alkaloid-type metabolites (Faria et al., 2010; Porto et al., 2009; Lopes et al., 2004). There are 112 Psychotria species present in the Philippines and almost all of them are endemic to the country (Sohmer and Davis, 2007). These endemic species include Psychotria gitingensis Elmer, a species that is widely distributed throughout the country. Fresh leaves of P. gitingensis were collected from Mount Guiting–Guiting, Sibuyan Island, Romblon, Philippines in April 2011. The identity of the plant was confirmed by one of the authors (GJDA) as a Philippine Rubiaceae specialist and authenticated by Rosie S. Madulid, the Curator of the University of Santo Tomas Herbarium (USTH). A voucher specimen (USTH 5760) was deposited at the USTH, Research Center for the Natural and Applied Sciences, University of Santo Tomas. 2. Previous work There are no publications on the bioactive constituents of Psychotria from the Philippines.
* Corresponding author. Tel./fax: þ63 2 7314031. E-mail address: [email protected] (M.A. Tan). 0305-1978/$ – see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.bse.2012.07.016
M.A. Tan et al. / Biochemical Systematics and Ecology 45 (2012) 20–22
21
Fig. 1. Structure of vomifoliol.
3. Present study The air-dried, powdered leaves of P. gitingensis (864 g) were subjected to exhaustive extraction in a percolator for three days using MeOH (13.0 L) and filtered. The combined filtrates were concentrated under reduced pressure and this afforded the crude methanolic extract (104 g) that gave light orange spots when it was tested for the presence of alkaloids using TLC and Dragendorff’s spray reagent. The crude extract was dissolved in 1 M HCl (650 mL) and extracted thrice with EtOAc (3 x 100 mL). The aqueous layer was basified with sodium carbonate (pH 9–10) and exhaustively extracted with 5% MeOH/CHCl3 (1.5 L). The organic layer was dried with anhydrous Na2SO4, followed by evaporation under reduced pressure to afford the crude base extract (0.61 g). The crude base extract was subjected to initial gravity CC (CHCl3/MeOH gradient) to give nine major fractions (PgC-A to PgC-I). PgC-F (146.9 mg) and PgC-G (118.6 mg) gave light orange spots in TLC using Dragendorff’s reagent. PgC-F was purified using gravity CC (9:1 CHCl3/MeOH) to afford five fractions (PgC-F1 to PgC-F5). Further purification of PgC-F3 (25.1 mg) using gravity CC (99:1 EtOAc/MeOH) afforded vomifoliol (5.8 mg). PgC-G was purified using gravity CC (9:1 CHCl3/MeOH) to afford vomifoliol (5.0 mg). The structure of vomifoliol was elucidated by extensive spectroscopic analysis (1D and 2D NMR and MS) and comparison with the literature (Gonzalez et al., 1994). Antimicrobial assay of vomifoliol was also done using the agar diffusion paper-disc method (Quinto and Santos, 2004). At 0.5 mg/mL, vomifoliol exhibited moderate antibacterial activity against Klebsiella oxytoca with an average zone of inhibition of 12.5 mm. It showed negative results for Bacillus cereus, Staphylococcus aureus, and Escherichia coli. 4. Chemotaxonomic significance This study represents the first phytochemical work on Psychotria gitingensis from the Philippines. This also represents the first isolation of vomifoliol (Fig. 1) from the genus Psychotria. When the crude methanolic extract and major fractions from P. gitingensis were tested for the presence of alkaloids using Dragendorff’s reaction, light orange spots were observed on TLC plates that were interpreted as due to the alkaloids present in the plant. However, TLC-pure isolates (PgC-F3a and PgC-G1) were found to be vomifoliol based on the analysis of spectroscopic measurements. It is well established that certain compounds can give false-positive alkaloid reactions with Dragendorff’s spray reagent (Habib, 1980). Vomifoliol is a megastigmane sesquiterpene that has never been reported to exist in any species of Psychotria. This compound contains an a,bunsaturated ketone that might be responsible for the observed orange spots in Dragendorff’s test. Further collection and analysis of P. gitingensis from other locations are highly recommended to verify the absence of alkaloids in this species. This work adds to a number of reports regarding the absence of alkaloids in some species of Psychotria (Lopes et al., 2004; Leal and Elisabetsky, 1996; Raffauf, 1996). The results of this study present an implication on the chemotaxonomic relationship of P. gitingensis with other members of genus Psychotria. Although Psychotria species are easily recognized in the field, the assignment to species, however, is often quite difficult due to their frequent occurrence in forest habitats and the high within-genus diversity. In addition to alkaloids, other compounds can be used as chemotaxonomic markers for different Psychotria species. Achenbach et al. (1995) were able to isolate two megastigmanes, megastigm-5-ene-3,9-diol and S(þ)-dehydrovomifoliol, from the leaf extract of Psychotria correae. It is also interesting to note that Pimenta et al. (2011) isolated a megastigmane glycoside (i.e. byzantionoside B) from the leaves of Psychotria stachyoides. The presence or absence of vomifoliol or its structural analogs can be used to identify infrageneric relationships within the genus particularly in the assigning of their taxonomic sections. This is quite significant since molecular phylogeny of the whole Psychotria is currently not available. Therefore, chemotaxonomic and molecular studies should be done involving several, if not all, species of Psychotria aimed to establish the classification of the abundant species of the genus. Acknowledgments This work was made possible with the scholarship grant from the Department of Science and Technology – Science Education Institute and the Department of Biofunctional Molecular Chemistry, Graduate School of Pharmaceutical Sciences, Chiba University for the spectroscopic measurements. This study was also supported by the Research Center for the Natural and Applied Sciences of the University of Santo Tomas.
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M.A. Tan et al. / Biochemical Systematics and Ecology 45 (2012) 20–22
References Achenbach, H., Lottes, M., Waibel, R., Karikas, G.A., Correa, M., Gupta, M.P., 1995. Phytochemistry 38, 1537. Davis, A.P., Govaerts, R., Bridson, D.M., Ruhsam, M., Moat, J., Brummitt, N.A., 2009. Ann. Missouri Bot. Gard. 96, 68. Faria, E.O., Kato, L., de Oliveira, C.M.A., Carvalho, B.G., Silva, C.C., Sales, L.S., Schuquel, I.T.A., Silveira-Lacerda, E.P., Delprete, P., 2010. Phytochemistry Lett. 3, 113. Gonzalez, A.G., Guillermo, J.A., Ravelo, A.G., Jimenez, I.A., Gupta, M.P., 1994. J. Nat. Prod. 57, 400. Habib, A.A., 1980. J. Pharm. Sci. 69, 37. Leal, M.B., Elisabetsky, E., 1996. J. Ethnopharmacol. 54, 37. Lopes, S., von Poser, G.L., Kerber, V.A., Farias, F.M., Konrath, E.L., Moreno, P., Sobral, M.E., Zuanazzi, J.A.S., Henriques, A.T., 2004. Biochem. System. Ecol. 32, 1187. Nepokroeff, M., Sytsma, K.J., 1999. Speciation in cloud forest members of Psychotria section Notopleura (Rubiaceae). In: Nadkarni, N., Wheelwright, N. (Eds.), Monteverde: Ecology and Conservation of a Tropical Cloud Forest. Oxford University Press, Oxford. Pimenta, A.T.A., Uchoa, D.E.A., Braz-Filho, R., Silveira, E.R., Lima, M.A.S., 2011. J. Brazilian Chem. Soc. 22, 2216. Porto, D.D., Henriques, A.T., Fett-Neto, A.G., 2009. The Open Bioact. Comp. J. 2, 29. Quinto, E.A., Santos, M.A.G., 2004. Microbiology section. In: Guevara, B.Q. (Ed.), A Guidebook to Plant Screening: Phytochemical and Biological. Research Center for the Natural Sciences, University of Santo Tomas, Manila. Raffauf, R.F., 1996. Plant Alkaloids: A Guide to Their Discovery and Distribution. Food Products Press, New York. Sohmer, S.H., Davis, A.P., 2007. The Genus Psychotria (Rubiaceae) in the Philippine Archipelago. Botanical Research. Institute of Texas Press, Texas.
Contents lists available at SciVerse ScienceDirect
Biochemical Systematics and Ecology journal homepage: www.elsevier.com/locate/biochemsyseco
Chemotaxonomic implications of the absence of alkaloids in Psychotria gitingensis Mario A. Tan*, Jameson A. Eusebio, Grecebio Jonathan D. Alejandro The Graduate School and Research Center for the Natural and Applied Sciences, University of Santo Tomas, España 1015, Manila, Philippines
a r t i c l e i n f o Article history: Received 10 April 2012 Accepted 7 July 2012 Available online 4 August 2012 Keywords: Psychotria gitingensis Rubiaceae Alkaloid Vomifoliol Chemotaxonomy
1. Subject and source The pantropical Psychotria L. (Rubiaceae, Rubioideae, Psychotrieae) is the largest member of the coffee family and the third largest angiosperm genus with more than 1800 species worldwide (Davis et al., 2009). Most Psychotria species grow as shrubs or small trees in the understorey of moist tropical and subtropical forests with a variety of growth forms, including herbs and epiphytes (Nepokroeff and Sytsma, 1999). The taxonomy of the genus is complex and the relationships among the species are unclear, especially the endemic species found in Southeast Asia (Sohmer and Davis, 2007). Phytochemical studies have mostly been on the alkaloid-type metabolites (Faria et al., 2010; Porto et al., 2009; Lopes et al., 2004). There are 112 Psychotria species present in the Philippines and almost all of them are endemic to the country (Sohmer and Davis, 2007). These endemic species include Psychotria gitingensis Elmer, a species that is widely distributed throughout the country. Fresh leaves of P. gitingensis were collected from Mount Guiting–Guiting, Sibuyan Island, Romblon, Philippines in April 2011. The identity of the plant was confirmed by one of the authors (GJDA) as a Philippine Rubiaceae specialist and authenticated by Rosie S. Madulid, the Curator of the University of Santo Tomas Herbarium (USTH). A voucher specimen (USTH 5760) was deposited at the USTH, Research Center for the Natural and Applied Sciences, University of Santo Tomas. 2. Previous work There are no publications on the bioactive constituents of Psychotria from the Philippines.
* Corresponding author. Tel./fax: þ63 2 7314031. E-mail address: [email protected] (M.A. Tan). 0305-1978/$ – see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.bse.2012.07.016
M.A. Tan et al. / Biochemical Systematics and Ecology 45 (2012) 20–22
21
Fig. 1. Structure of vomifoliol.
3. Present study The air-dried, powdered leaves of P. gitingensis (864 g) were subjected to exhaustive extraction in a percolator for three days using MeOH (13.0 L) and filtered. The combined filtrates were concentrated under reduced pressure and this afforded the crude methanolic extract (104 g) that gave light orange spots when it was tested for the presence of alkaloids using TLC and Dragendorff’s spray reagent. The crude extract was dissolved in 1 M HCl (650 mL) and extracted thrice with EtOAc (3 x 100 mL). The aqueous layer was basified with sodium carbonate (pH 9–10) and exhaustively extracted with 5% MeOH/CHCl3 (1.5 L). The organic layer was dried with anhydrous Na2SO4, followed by evaporation under reduced pressure to afford the crude base extract (0.61 g). The crude base extract was subjected to initial gravity CC (CHCl3/MeOH gradient) to give nine major fractions (PgC-A to PgC-I). PgC-F (146.9 mg) and PgC-G (118.6 mg) gave light orange spots in TLC using Dragendorff’s reagent. PgC-F was purified using gravity CC (9:1 CHCl3/MeOH) to afford five fractions (PgC-F1 to PgC-F5). Further purification of PgC-F3 (25.1 mg) using gravity CC (99:1 EtOAc/MeOH) afforded vomifoliol (5.8 mg). PgC-G was purified using gravity CC (9:1 CHCl3/MeOH) to afford vomifoliol (5.0 mg). The structure of vomifoliol was elucidated by extensive spectroscopic analysis (1D and 2D NMR and MS) and comparison with the literature (Gonzalez et al., 1994). Antimicrobial assay of vomifoliol was also done using the agar diffusion paper-disc method (Quinto and Santos, 2004). At 0.5 mg/mL, vomifoliol exhibited moderate antibacterial activity against Klebsiella oxytoca with an average zone of inhibition of 12.5 mm. It showed negative results for Bacillus cereus, Staphylococcus aureus, and Escherichia coli. 4. Chemotaxonomic significance This study represents the first phytochemical work on Psychotria gitingensis from the Philippines. This also represents the first isolation of vomifoliol (Fig. 1) from the genus Psychotria. When the crude methanolic extract and major fractions from P. gitingensis were tested for the presence of alkaloids using Dragendorff’s reaction, light orange spots were observed on TLC plates that were interpreted as due to the alkaloids present in the plant. However, TLC-pure isolates (PgC-F3a and PgC-G1) were found to be vomifoliol based on the analysis of spectroscopic measurements. It is well established that certain compounds can give false-positive alkaloid reactions with Dragendorff’s spray reagent (Habib, 1980). Vomifoliol is a megastigmane sesquiterpene that has never been reported to exist in any species of Psychotria. This compound contains an a,bunsaturated ketone that might be responsible for the observed orange spots in Dragendorff’s test. Further collection and analysis of P. gitingensis from other locations are highly recommended to verify the absence of alkaloids in this species. This work adds to a number of reports regarding the absence of alkaloids in some species of Psychotria (Lopes et al., 2004; Leal and Elisabetsky, 1996; Raffauf, 1996). The results of this study present an implication on the chemotaxonomic relationship of P. gitingensis with other members of genus Psychotria. Although Psychotria species are easily recognized in the field, the assignment to species, however, is often quite difficult due to their frequent occurrence in forest habitats and the high within-genus diversity. In addition to alkaloids, other compounds can be used as chemotaxonomic markers for different Psychotria species. Achenbach et al. (1995) were able to isolate two megastigmanes, megastigm-5-ene-3,9-diol and S(þ)-dehydrovomifoliol, from the leaf extract of Psychotria correae. It is also interesting to note that Pimenta et al. (2011) isolated a megastigmane glycoside (i.e. byzantionoside B) from the leaves of Psychotria stachyoides. The presence or absence of vomifoliol or its structural analogs can be used to identify infrageneric relationships within the genus particularly in the assigning of their taxonomic sections. This is quite significant since molecular phylogeny of the whole Psychotria is currently not available. Therefore, chemotaxonomic and molecular studies should be done involving several, if not all, species of Psychotria aimed to establish the classification of the abundant species of the genus. Acknowledgments This work was made possible with the scholarship grant from the Department of Science and Technology – Science Education Institute and the Department of Biofunctional Molecular Chemistry, Graduate School of Pharmaceutical Sciences, Chiba University for the spectroscopic measurements. This study was also supported by the Research Center for the Natural and Applied Sciences of the University of Santo Tomas.
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M.A. Tan et al. / Biochemical Systematics and Ecology 45 (2012) 20–22
References Achenbach, H., Lottes, M., Waibel, R., Karikas, G.A., Correa, M., Gupta, M.P., 1995. Phytochemistry 38, 1537. Davis, A.P., Govaerts, R., Bridson, D.M., Ruhsam, M., Moat, J., Brummitt, N.A., 2009. Ann. Missouri Bot. Gard. 96, 68. Faria, E.O., Kato, L., de Oliveira, C.M.A., Carvalho, B.G., Silva, C.C., Sales, L.S., Schuquel, I.T.A., Silveira-Lacerda, E.P., Delprete, P., 2010. Phytochemistry Lett. 3, 113. Gonzalez, A.G., Guillermo, J.A., Ravelo, A.G., Jimenez, I.A., Gupta, M.P., 1994. J. Nat. Prod. 57, 400. Habib, A.A., 1980. J. Pharm. Sci. 69, 37. Leal, M.B., Elisabetsky, E., 1996. J. Ethnopharmacol. 54, 37. Lopes, S., von Poser, G.L., Kerber, V.A., Farias, F.M., Konrath, E.L., Moreno, P., Sobral, M.E., Zuanazzi, J.A.S., Henriques, A.T., 2004. Biochem. System. Ecol. 32, 1187. Nepokroeff, M., Sytsma, K.J., 1999. Speciation in cloud forest members of Psychotria section Notopleura (Rubiaceae). In: Nadkarni, N., Wheelwright, N. (Eds.), Monteverde: Ecology and Conservation of a Tropical Cloud Forest. Oxford University Press, Oxford. Pimenta, A.T.A., Uchoa, D.E.A., Braz-Filho, R., Silveira, E.R., Lima, M.A.S., 2011. J. Brazilian Chem. Soc. 22, 2216. Porto, D.D., Henriques, A.T., Fett-Neto, A.G., 2009. The Open Bioact. Comp. J. 2, 29. Quinto, E.A., Santos, M.A.G., 2004. Microbiology section. In: Guevara, B.Q. (Ed.), A Guidebook to Plant Screening: Phytochemical and Biological. Research Center for the Natural Sciences, University of Santo Tomas, Manila. Raffauf, R.F., 1996. Plant Alkaloids: A Guide to Their Discovery and Distribution. Food Products Press, New York. Sohmer, S.H., Davis, A.P., 2007. The Genus Psychotria (Rubiaceae) in the Philippine Archipelago. Botanical Research. Institute of Texas Press, Texas.