Boletín de la Sociedad Chilena de Química
versión impresa ISSN 0366-1644
Bol. Soc. Chil. Quím. v.47 n.3 Concepción sep. 2002
Bol. Soc. Chil. Quím., 47, 273-278 (2002) ISSN 0366-1644
FLAVONOIDS AS CHEMOSYSTEMATIC MARKERS IN CHILEAN
SPECIES OF DRIMYS J.R. FORST. ET G. FORST. (WINTERACEAE).
1Departamento de Botánica, Facultad de Ciencias Naturales y Oceanográficas,
Universidad de Concepción. Casilla 160-C, Concepción, Chile.
2Departamento de Biología Molecular, Facultad de Ciencias Biológicas,
Universidad de Concepción.
(Received: May 10, 2002 - Accepted: June 23, 2002)
Thirteen flavonoids were isolated from leaves of three Chilean species of Drimys (Winteraceae), one endemic to the Juan Fernandez Islands (D. confertifolia) and two native from continental Chile (D. winteri and D. andina). Flavonoids were characterized by a combination of ultraviolet spectroscopy, colors reactions when fumed with NH4 and Rf values on paper chromatography. Comparisons of flavonoid were done by thin layer chromatography on polyamide, high performance liquid chromatography and descendent paper chromatography on Whatman Nº3 paper. With flavonoid patterns, a phenetic analyses using Baroni-Urbani and Buser index was done to evaluate chemical similarities among these species. The results suggest that the endemic species, D. confertifolia is closely related to D. winteri. The three species are very different in flavonoid contents as showed by the low similarity values. Chemical results are congruent with separation of this taxa at specific level.
Key words: Flavonoids, Drimys, Juan Fernandez Islands, chemotaxonomy.
Se aislaron trece flavonoides de las hojas de tres especies de Drimys (Winteraceae), una endémica de Juan Fernández (D. confertifolia) y dos nativas de Chile continental (D. winteri y D. andina). Los flavonoides fueron caracterizados por la combinación de sus características espectroscópicas al ultravioleta, valores de Rf y cambio de color al reaccionar con vapores de NH4 bajo luz ultravioleta. La comparación de flavonoides se hizo por cromatografia en placa fina sobre poliamida, cromatografía líquida de alta eficiencia y por cromatografía descendente en papel Whatman Nº3. Con los patrones de flavonoides, se realizó un análisis fenético usando el índice Baroni-Urbani & Buser para evaluar la similitud entre las especies. Los resultados sugieren que la especie endémica D. confertifolia está más relacionada con D. winteri. Las tres especies analizadas son muy distintas en sus contenidos de flavonoides. Los resultados químicos son congruentes con la separación de estos taxa a nivel específico.
Palabras clave: Flavonoides, Drimys, Archipiélago Juan Fernández, Quimiotaxonomía
The genus Drimys J.R. Forst. et G. Forst. (Winteraceae) consist of six species from Central and South America and five from Philippines and Tasmania1. The relationships within the New World taxa have been not clear and have result in different perspectives on specific and varietal limits. At one extreme, Hooker2 regarded all Central and South American taxa as belonging to a single species. At the other extreme, Miers3,4 who recognized nine species within the New World. An alternative intermediate view is Smith`s5, who treated four species, three of which possess infraspecific taxa (varieties). The taxonomic history of the Chilean (and adjacent Argentinean) species has been examined from different point of view. In Chile three species of Drimys are recognized. Drimys confertifolia Phil., (endemic to the Juan Fernandez Islands), Drimys winteri J.R. Forst. et G. Forst. and Drimys andina (Reiche) R.A. Rodr. et Quez. (both native from continental Chile). The endemic island species has been treated as a distinct species1,3,5,6,7 or as a variety of D. winteri 8,9,10. On the other hand, Drimys andina has been considered as a variety of D. winteri 5,7,8,11,12,13. Recently, D. andina was considered as different species14.
To give new insight about the relationships among Chilean species of Drimys, a chemotaxonomic study was carry out using the flavonoid distribution patterns of those species.
Flavonoids were isolated from leaves of two samples of D. andina, two of D. confertifolia and three of D. winteri (Table I). The Collected samples are deposited in the Herbarium of University of Concepción (CONC).
00000D. andina: 1. X Región, Osorno, up to Volcán Casablanca, at the vegetation limit, Nov, 1997, Ruiz 361. 2. IX Región, Angol, Nahuelbuta National Park, trail between cabins and Piedra del Aguila, Jan, 1998, Ruiz 561.
00000D. confertifolia: 3. V Región, Juan Fernández, Masafuera, up to Cordón Atravesado, en el Canelo Bajo, Nov, 1998, Ruiz 783. 4. V Región, Juan Fernández, Masatierra, Salsipuedes ridge, 470 m, Jan, 1997, Stuessy et al. 15239.
00000D. winteri: 5. IV Región, 5 km north from Los Vilos, small wet forest in the coastal side of the highway, Sept, 1997, Ruiz 301. 6. VIII Región, Between Concepción and Santa Juana, Patagual bridge, trail to Coronel, two km toward Coronel, Oct, 1997. Ruiz 330. 7. IX Región, Pto. Saavedra, from Pelaco, 2 km to the south, Nov, 1997, Ruiz 389.
Leaf material was extracted with methanol:water (80:20). The extract was partitioned with dichloromethane and ethyl acetate. Flavonoids were isolated from ethyl acetate fraction and purified by a combination of column and thin layer chromatography on polyamide and descending paper chromatography on Whatman Nº3 MM paper with the following solvents: 15%, 25% and 40% acetic acid and TBA (tertiary butyl alcohol: acetic acid: water, 3:1:1). The flavonoids were characterized by a combination of ultraviolet spectroscopy, colors reactions when fumed with NH4 and Rf values on paper chromatography15. Similar Rf values and time retention in High Performance Liquid Chromatography (HPLC, when necessary) were used to judge whether flavonoids from different collections represented the same compounds. Hydrolysis of glycosidic flavonoids was done with hydrochloric acid 5N at 50ºC for 2 h, using 10 mg of compound. The aglycone was separated from sugars by partitioning with water/ethyl acetate. Acetylation was done dissolving 5-10 mg of compound in 1 ml of pyridine and acetic anhydride for 24 h. The precipitate was filtered and washed with distilled water and recovered with ethyl acetate15.
Flavonoid patterns were phenetically analyzed using the Baroni-Urbani & Buser index which evaluate the similarities among taxa16. A dendrogram was built to reflect the similarities. Cluster analysis was done using UPGMA algorithm with MVSP 3.1. (Multi-Variate Statistical Package) program.
Thirteen flavonoids were isolated from the studied species of Drimys, representing three different classes: flavones, flavonols and dihydroflavonols (Table II). Some of them were partially identified, but they were useful for phenetic analysis.
*not completely identified. Numbers in parenthesis are different derivatives of taxifolin
There was not intraspecific variation in flavonoid contents, but patterns of distribution were very different among the species. Only two compounds were present in more than one species (compound 6 and 8) and only one was present in all species (compound 6) (Table III).
TABLE III. Distribution of flavonoids in analyzed species of Drimys
X = presence of flavonoid. Numbers correspond those Table II
Table IV shows the similarity values with Baroni-Urbani & Buser index. All values are very low (lower than 50% of similarity), however D. winteri and D. confertifolia are the most closely related species (Figure 1). The Cophenetic Correlation Index was very high (CCI = 0.87), indicating little distortion of the data in the phenogram.
Fig. 1. Phenogram of relationships of studied species of Drimys. CCI = 0.87
Secondary compounds, specially flavonoids, have been used in chemotaxonomic and evolutionary studies of other genera with species endemic to the Juan Fernandez Islands, including: Robinsonia 17, Dendroseris 18, Erigeron 19, Peperomia 20, Gunnera 21 Sophora 22,23 and Myrceugenia 24.
In this study, a maximum of six flavonoids were found in Chilean species of Drimys, confirming previous reports of low flavonoid diversity in several genera of Winteraceae25. This situation is different in other genera with species endemic to the Juan Fernandez Islands, possibly reflecting the primitive nature of the family Winteraceae. More rich and complex profiles of flavonoids are common in advanced Angiosperm families 26
In the present study, flavonoid distribution patterns were very variable and a unique compound was present in all studied species. Some flavonoids can function in the attraction of pollination vectors27. Probably the attract pollinator strategies in Chilean species of Drimys could be different in each species, evolving in different pathway after of colonization into the new habitat and radiation in different species, probably due to the three species occur at geographically distant and in different habitats14.
Flavonoid data are congruent with the habit of these species. Flavonoid patterns show D. confertifolia closely related to D. winteri both are big trees and D. andina is a very small shrub. The most probably alternative is that D. winteri was the ancestor species of D. confertifolia as is point out by Skottsberg 28.
Presence of many dihydroflavonols, specially in D. winteri, confirm the archaic position of these species into the Angiosperms 26.
Finally, the small similarity values agree with the separation of these taxa at specific level as actually is recognized 1.
The authors thank to FONDECYT, project 1960822, 7960015, 1990444 and Dirección de investigación for financial supports, CONAF V Región and CONAF Juan Fernández for permission and help to collect plant material.
1. R. Rogríguez and M. Quezada. Winteraceae. pp. 2-6 En C. Marticorena & R. Rodríguez (Eds.) Flora de Chile. vol. 2, Magnoliaceae - Ranunculaceae. (2001). [ Links ]
2. J.D. Hooker. Magniliaceae. pp. 229-230. In J.D. Hooker (ed.) The Botany of the Antarctic Voyage of H.M. Discovery Ships Erebus ans Terror in the years 1839-1843, Vol. 2. Reeve & Co., London (1845). [ Links ]
3. J. Miers. Ann. Mag. Nat. Hist., ser. 3, 2, 33-48. (1858) [ Links ]
4. J. Miers. Contrib. Bot. 1, 123-145 (1862) [ Links ]
5. A.C. Smith. J. Arnold. Arbor. 24, 1-33 (1943) [ Links ]
6. R.A. Philippi. Anales Univ. Chile 13, 157-169 (1856) [ Links ]
7. R. Rodríguez, O. Matthei, M. Quezada. Flora arbórea de Chile., Editorial de la Universidad de Concepción. Concepción, Chile (1983). [ Links ]
8. C. Reiche. Anales Univ. Chile 88, 80-81 (1895) [ Links ]
9. F. Johow. Estudios sobre la flora de las Islas de Juan Fernandez. Imprenta Cervantes, Santiago, Chile (1896). [ Links ]
10. C. Skottsberg. The phanerogams of the Juan Fernandez Islands. pp 127-128 In: C. Skottsberg ed. The natural history of Juan Fernandez and Easter Island. Uppsala, Almqvist & Wiksells Boktryckeri AB. Vol. 2, (1922) [ Links ]
11. O. Urban. Botánica de las plantas endémicas de Chile, Concepción (1934) [ Links ]
12. M. Muñoz. Flora del Parque Nacional Puyehue. Santiago (1980) [ Links ]
13. C. Marticorena and M. Quezada M. Gayana, Bot. 42, 1-157 (1985). [ Links ]
14. R. Rogríguez and M. Quezada. Gayana, Bot. 48, 111-114 (1991) [ Links ]
15. T.J. Mabry, K.R. Markham and M.B. Thomas. The systematic identification of flavonoids. Springer Verlag, New York (1970). [ Links ]
16. C. Baroni-Urbani and M.W. Buser. Syst. Zool. 25, 251-259 (1976) [ Links ]
17. P. Pacheco, D.J. Crawford, T.F. Stuessy and M. Silva. Amer. J. Bot. 72, 989-998 (1985). [ Links ]
18. P. Pacheco, D.J. Crawford, T.F. Stuessy and M. Silva. Amer. J. Bot. 78, 534-543 (1991). [ Links ]
19. H. Valdebenito, T.F. Stuessy, D.J. Crawford, and M. Silva. Syst. Bot. 17, 470-480. (1992a). [ Links ]
20. H. Valdebenito, T.F. Stuessy, D.J. Crawford, and M. Silva. Pl. Syst. Evol. 182, 107-119 (1992b) [ Links ]
21. P. Pacheco, D.J. Crawford, T.F Stuessy and M. Silva. Gayana, Bot. 50, 17-28 (1993). [ Links ]
22. M. Hoeneisen, M. Silva, M. Wink, T.F. Stuessy and D. Crawford. Bol. Soc. Chil. Quím. 38, 167-171 (1993). [ Links ]
23. E. Ruiz, C. Donoso, F. González, J. Becerra, C. Marticorena and M. Silva. Bol. Soc. Chil. Quím. 44, 351-356 (1999). [ Links ]
24. E. Ruiz, J. Becerra, M. Silva, D.J. Crawford, T.F. Stuessy. Brittonia 46, 187-193 (1994). [ Links ]
25. C.A. William and W.J. Harvey. Phytochemistry 21, 329-337 (1982). [ Links ]
26. T. Swain. Evolution of flavonoid compounds. pp. 1096-1129. In: J.B. Harborne and T.J. Mabry (eds.) The flavonoids. Academic Press, New York (1977) [ Links ]
27. J.B. Harborne. Introduction to ecological biochemistry. Academic. Press, New York. (1988) [ Links ]
28. C. Skottsberg. Derivation of the flora and fauna of Juan Fernandez and Easter Islands. pp. 193-439. In: Skottsberg, C. ed. The natural history of Juan Fernandez and Easter Island. Uppsala, Almqvist & Wiksells Boktryckeri AB. Vol. 1, (1956). [ Links ]