Table of Contents Author Guidelines Submit a Manuscript
International Journal of Analytical Chemistry
Volume 2012, Article ID 721494, 5 pages
Research Article

Uptake of Seeds Secondary Metabolites by Virola surinamensis Seedlings

1Instituto de Química, Universidade de São Paulo, CP 26077, 05599-970 São Paulo, SP, Brazil
2Núcleo de Pesquisa em Produtos Naturais e Sintéticos (NPPNS), Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, 14040-903 Ribeirão Preto, SP, Brazil
3Lychnoflora Pesquisa e Desenvolvimento em Produtos Naturais LTDA, Incubadora Supera, Campus da USP, 14040-900 Ribeirão Preto, SP, Brazil
4Núcleo de Bioensaio, Biossíntese e Ecofisiologia de Produtos Naturais (NuBBE), Instituto de Química, Universidade Estadual Paulista, CP 355, 14800-900 Araraquara, SP, Brazil

Received 2 September 2011; Accepted 19 December 2011

Academic Editor: Ernani Pinto

Copyright © 2012 Massuo Jorge Kato et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


The major secondary metabolites and fatty acids occurring in the seeds of Virola surinamensis were monitored by GC-MS during germination and seedling development. The role as carbon source for seedling development was indicated considering that both classes of compounds were similarly consumed in the seeds and that no selective consumption of compounds could be detected.

1. Introduction

Several neotropical trees produce fruits with large and heavy seeds [1]. Virola surinamensis is a myristicaceous tree growing in the Amazonian flooded plains and produces seeds during the rainy season [2, 3]. Seeds are viable shortly after ripening and are adapted to be dispersed by water or by large birds such as toucans and araçaris. The seedling formation can be divided in two distinct phases: seed germination and seedling development [4]. The cotyledons are hidden in the seed coat (cryptocotylar) and are storage organs of fatty material and polysaccharides that are recruited for the maintenance of seedling during its growth and development [5]. A study carried out on V. venosa revealed that the major lignans cubebin and dihydrokusunokinin accumulated in the seeds were not detected in its seedlings which accumulated a polyketide instead [6]. The major constituent identified in the seedling roots was shown to be the lignan sesamin, a minor constituent in the seeds. A different result were observed with V. sebifera in which a possible translocation of hydroxytetralone lignans and a preferential accumulation of a lignan hydroxy-otobain was observed in the whole seedlings [7].

In view of the lack of systematic investigation regarding this important event in the reproduction of tropical trees, the translocation of secondary metabolites occurring in large seeds to be used as a defensive compounds in the seedlings remains as a hypothesis [8, 9].

Virola surinamensis seeds contain 15.4% of soluble tannins as a dry mass and the highest concentration of compounds with a probable defensive function yet recorded [10]. Their cotyledons are rich in triacylglycerols and free fatty acids. Phytochemical analysis of V. surinamensis seeds collected at Combu Island demonstrated the occurrence of lignoids, propiophenone, and γ-lactones in these organs [11]. Analysis of seedling leaves of V. surinamensis growing in the field, in greenhouse conditions and in micropropagated plantlets revealed the absence of lignans and the exclusive occurrence of juruenolide C (8a) (Figure 1) [12]. Herein, we wish to report the analyses of fatty acids and major secondary compounds in seeds of V. surinamensis in order to evaluate a selective consumption during the germination process.

Figure 1: Chemical structures of the isolated substances: 4-hydroxy-3-methoxypropiophenone (1), galbulin (2), guaiacin (3), galbacin (4a), galbelgin (4b), calopeptin (5a), veraguensin (5b), 7,2′-dihydroxy-4′-methoxy-isoflavone (6), α,2′-dihydroxy-4,4′-dimethoxydihydrochalcone (7), juruenolide C (8a), and juruenolide D (8b).

2. Experimental Section

2.1. General Procedures

Preparative thin-layer chromatography (prep. TLC) was carried out on silica gel GF-254 (Merck) and column chromatography (CC) on silica gel 60H (0.005–0.045 mm) (Merck). The 1H NMR (200 MHz) and 13C NMR (50 MHz) spectra of samples were recorded on a Bruker-AC 200 in CDCl3 with tetramethylsilane (TMS) as an internal standard. EIMS was obtained at 70 eV on HP 5988-A.

2.2. Plant Material

Seeds of Virola surinamensis (Rol.) Warb. were collected in February 1995 at Combu Island (01°30′10′′S; 048°27′42′′W), near Belém, Pará State, Brazil. A dry voucher sample (LOPES-037) has been deposited in the SPF-Herbário do Instituto de Biociências da Universidade de São Paulo. Mature seeds were frozen for analysis or germinated as previously reported [13] and maintained at greenhouse facilities of Instituto de Química-USP.

2.3. Standards Isolation

One dried seed (320 mg), after the germination process, was extracted with CH3OH (3x 50 mL). The concentrated extract (70 mg) was suspended in CH3OH/H2O (6 : 4) and filtered through a Millipore membrane (0.45 μm). The filtered extract was submitted to preparative on HPLC (RP-8, 10 μm, 250 × 22 mm column; CH3OH/H2O 60 : 40 → CH3OH 100% (50 min), 8 mL·min−1, optimized conditions), followed by prep. TLC (silica gel; Hexane/EtOAc/i-PrOH or CH2Cl2/Me2CO) to yield 4-hydroxy-3-methoxypropiophenone (1, 1.6 mg) [14], galbulin (2, 5.5 mg) [15], guaiacin 3 (1.4 mg) [15], galbacin (4a, 2.0 mg) [16], galbelgin (4b, 1.0 mg) [17], calopeptin (5a, 1.6 mg) [18], veraguensin (5b, 5.0 mg) [19], 7,2′-dihydroxy-4′-methoxy-isoflavone (6, 1.5 mg) [20], α,2′-dihydroxy-4,4′-dimethoxydihydrochalcone (7, 1.8 mg) [21], juruenolide C (8a, 1.2 mg) [12], and juruenolide D (8b, 1.3 mg) [11]. All these compounds were identified by comparison of spectroscopic data with that reported in the literature.

2.4. Fatty Acids Analyses

Individual seeds before and after germination process were extracted (3x) with 200 mL of n-hexane. The transesterification of oils was carried out according to a procedure described by Maia and Rodrigues-Amaya, 1993 [22]. The methyl esters were dissolved with n-hexane (2 mg·mL−1), and 1 μL was injected in a Hewlett-Packard 5890 gas chromatograph coupled to a Hewlett-Packard 5988 mass spectrometer in the condition previously described [12, 23].

2.5. Analyses of Secondary Compounds

Individual seeds, before and after the germination process, were extracted (3x) with 20 mL of CH3OH. The extract was concentrated to dryness and the residue dissolved with CH2Cl2 to obtain 2 mg·mL−1 as the final concentration, and 1 μL was injected. All the analyses were performed with seven replicates in a Hewlett-Packard 5890 gas chromatograph coupled to a Hewlett-Packard 5988 mass spectrometer. The sample was injected (250°C) on a DB-5 column (30 m × 0.25 mm ID × 0.25 μm of film tickness). The column temperature was initially 120°C (2 min), then programmed to 230°C at 7°C·min−1, kept at 230°C for 10 min, and then increased to 290°C in 15 min. The mass spectra were recorded at 70 eV. The identification of individual constituents was carried based on injection of isolated substances and comparison of their mass spectra.

2.6. Statistical Analysis

Statistical analyses were performed with the graphPad InStat software. All values were reported as means ± SEM, and were analyzed for statistical significance by two way analysis of variance followed by Student test. The minimum level of significance considered was .

3. Results and Discussion

Two groups of seeds of V. surinamensis, before germination (BG) and 6-7 months after germination (AG), were analyzed for fatty acids and major secondary metabolites. The second group (AG) showed a decrease of 30% in dry weight, but without significant changes in the extraction yield (Table 1). These results are in agreement with Durian’s hypothesis, in which seeds are a nutrient storage organ to supply the seedling during the growth process [8]. The analyses of fatty acids content carried out in seeds of V. surinamensis before and after germination showed similar relative content of lauric acid (16%), myristic acid (70%), palmitic acid (6%), and stearic acid (8%). This result is similar to that previously reported [23], and since no preferential uptake of fatty acids could be detected, the major role of fatty material as carbon source is clearly supported (Table 2).

Table 1: Arithmetic mean of dry weight extracts and yields of V. surinamensis seeds.
Table 2: Relative contents of secondary metabolites and fatty acids in V. surinamensis seeds.

The secondary metabolites in both groups of seeds of V. surinamensis were analyzed by GC-MS. The chromatographic profile observed for both groups exhibited the predominance of galbulin (2), galbacin (4a), and veraguensin (5b) as the major compounds (Figure 2). After statistical analyses, no significant variation was observed in the relative content of monitored compounds, except to compound 1 ( ) (Table 2).

Figure 2: GC profile of secondary metabolites before (a) and after (b) germination of V. surinamensis seeds.

From V. surinamensis, new substances were isolated [24] and some neolignans showed allelopathic properties [25]. Recently, other neolignans showed antiinflammatory and antileishmanial activities [26, 27]. In addition, the increase of phenolic compounds was observed after elevated CO2 submission in V. surinamensis [28] and a strong inhibition of CO2 assimilation by sun exposure [29]. However, the analyses of the composition occurring in the seeds of this species during germination and seedling processes had not been studied yet.

In summary, the germination of V. surinamensis seeds and the seedling development are processes in which both fatty acids and secondary metabolites (lignans, isoflavonoids, and juruenolides) are equally consumed in the seeds indicating their physiological role as energy and carbon source, or by other physiological function. In spite of the large concentration of lignans in the seeds (8.5% as dry weight basis), no specific translocation to the seedlings and no consumption of a specific compound from the seeds could be detected. The lignans could have biological importance to the seeds, but after the lignans uptake to the seedling, our results, in addition to the previous phytochemical investigations [12], reinforce the use of these compounds as energy and carbon source by the seedlings.


This work was supported by financial aid provided by FAPESP and PADCT/CNPq. This work is dedicated to Professor Otto Richard Gottlieb.


  1. K. S. Bawa, P. S. Ashton, and S. M. Nor, “Reproductive ecology of tropical forest Plants: management issues,” in Reproductive Ecology of Tropical Forest Plants, K. S. Bawa and M. Hadley, Eds., p. 3, UNESCO & Parthenon Publishing Group, Paris, France, 1990. View at Google Scholar
  2. J. M. Ayres, As Matas de Várzea do Mamirauá, MST-CNPq, Brasília, Brasil, 1993.
  3. W. A. Rodrigues, “Revisão taxonomica das espécies de Virola Aublet (Myristicaceae) do Brasil,” Acta Amazonica, vol. 10, supplement, pp. 1–127, 1980. View at Google Scholar
  4. H. F. Howe, “Seed dispersal by birds and mammals implications for seedling demography,” in Reproductive Ecology of Tropical Forest Plants, K. S. Bawa and M. Hadley, Eds., pp. 191–218, UNESCO & The Parthenon Publising Group, Paris, France, 1990. View at Google Scholar
  5. A. Hladik and S. Miquel, “Seedling types and plant establishment in an African rain forest,” in Reproductive Ecology of Tropical Forest Plants, K. S. Bawa and M. Hadley, Eds., pp. 261–282, UNESCO & The Parthenon Publising Group, Paris, France, 1990. View at Google Scholar
  6. M. J. Kato, M. Yoshida, and O. R. Gottlieb, “Flavones and lignans in flowers, fruits and seedlings of Virola venosa,” Phytochemistry, vol. 31, no. 1, pp. 283–287, 1991. View at Google Scholar · View at Scopus
  7. A. P. Danelutte, A. J. Cavalheiro, and M. J. Kato, “Lignoids in seedlings of Virola sebifera,” Phytochemical Analysis, vol. 11, no. 6, pp. 383–386, 2000. View at Publisher · View at Google Scholar · View at Scopus
  8. D. H. Janzen, “The ecology and evolutionary biology of seed chemistry as relates to seed predation,” in Biochemical Aspects of Plant and Animal Coevolution, J. B. Harborne, Ed., pp. 163–206, Academic Press, London, UK, 1978. View at Google Scholar
  9. S. A. Foster, “On the adaptive value of large seeds for tropical moist forest trees: a review and synthesis,” The Botanical Review, vol. 52, no. 3, pp. 260–299, 1986. View at Publisher · View at Google Scholar · View at Scopus
  10. H. F. Howe and G. A. V. Kerckhove, “Removal of wild nutmeg (Virola surinamensis) crops by birds,” Ecology, vol. 62, no. 4, pp. 1093–1106, 1981. View at Google Scholar
  11. N. P. Lopes, E. E. De Almeida Blumenthal, A. J. Cavalheiro, M. J. Kato, and M. Yoshida, “Lignans, γ-Lactones and propiophenones of Virola surinamensis,” Phytochemistry, vol. 43, no. 5, pp. 1089–1092, 1996. View at Publisher · View at Google Scholar · View at Scopus
  12. N. P. Lopes, S. C. Franca de, A. M. S. Pereira et al., “A butanolide from seedlings and micropropagated leaves of Virola surinamensis,” Phytochemistry, vol. 35, no. 6, pp. 1469–1470, 1994. View at Publisher · View at Google Scholar · View at Scopus
  13. M. A. Cardoso, R. Cunha, and T. S. Pereira, “Germinação de sementes de Virola surinamensis (Rol.) Warb. (Myristicaceae) e Guarea guidonea (L.) Sleumer (Meliaceae),” Revista Brasileira de Sementes, vol. 16, no. 1, pp. 1–5, 1994. View at Google Scholar
  14. J. M. Bárbosa-Filho, M. S. D. Silva, M. Yoshida, and O. R. Gottlieb, “Neolignans from Licaria aurea,” Phytochemistry, vol. 28, no. 8, pp. 2209–2211, 1989. View at Google Scholar · View at Scopus
  15. M. M. M. Pinto, A. Kijjoa, I. O. Mondranondra, A. B. Gutiérrez, and W. Herz, “Lignans and other constituents of knema furfuracea,” Phytochemistry, vol. 29, no. 6, pp. 1985–1988, 1990. View at Google Scholar · View at Scopus
  16. L. E. S. Barata, P. M. Baker, O. R. Gottlieb, and E. A. Rùveda, “Neolignans of Virola surinamensis,” Phytochemistry, vol. 17, no. 4, pp. 783–786, 1978. View at Google Scholar · View at Scopus
  17. M. A. Sumathykutty and J. M. Rao, “8-Hentriacontanol and other constituents from Piper attenuatum,” Phytochemistry, vol. 30, no. 6, pp. 2075–2076, 1991. View at Google Scholar · View at Scopus
  18. R. W. Doskotch and M. S. Flom, “Acuminatin, a new bis-phenylpropide from Magnolia acuminata,” Tetrahedron, vol. 28, no. 18, pp. 4711–4717, 1972. View at Google Scholar · View at Scopus
  19. B. Talapatra, P. K. Chaudhuri, and S. K. Talapatra, “(-)-Maglifloenone, a novel spirocyclohexadienone neolignan and other constituents from Magnolia liliflora,” Phytochemistry, vol. 21, no. 3, pp. 747–750, 1982. View at Google Scholar · View at Scopus
  20. R. Braz Filho, O. R. Gottlieb, A. A. De Moraes et al., “The chemistry of Brazilian myristicaceae. IX. Isoflavonoids from amazonian species,” Lloydia, vol. 40, no. 3, pp. 236–238, 1977. View at Google Scholar · View at Scopus
  21. J. C. Martinez and L. E. Cuca, “Flavonoids from Virola calophylloidea,” Journal of Natural Products, vol. 50, no. 6, pp. 1045–1047, 1987. View at Google Scholar · View at Scopus
  22. E. L. Maia and D. B. Rodrigues-Amaya, “Avaliação de um método simples e econômico para a metilação de ácidos graxos com lipídios de diversas espécies de peixes,” Revista do Instituto Adolfo Lutz, vol. 53, no. 1/2, pp. 27–35, 1993. View at Google Scholar
  23. D. H. S. Silva, N. P. Lopes, M. J. Kato, and M. Yoshida, “Fatty acids from Myristicaceous seeds of myristic acid-rich species,” Anais da Associação Brasileira de Química, vol. 46, pp. 232–235, 1997. View at Google Scholar
  24. N. P. Lopes, P. A. Dos Santos, M. J. Kato, and M. Yoshida, “New butenolides in plantlets of Virola surinamensis (Myristicaceae),” Chemical and Pharmaceutical Bulletin, vol. 52, no. 10, pp. 1255–1257, 2004. View at Publisher · View at Google Scholar · View at Scopus
  25. F. C. Borges, L. S. Santos, M. J. C. Corrêa, M. N. Oliveira, and A. P. S. Souza Filho, “Allelopathy potential of two neolignans isolated from Virola surinamensis (Myristicaceae) leaves,” Planta Daninha, vol. 25, no. 1, pp. 1045–1047, 2007. View at Google Scholar
  26. L. E. S. Barata, L. S. Santos, P. H. Ferri, J. D. Phillipson, A. Paine, and S. L. Croft, “Anti-leishmanial activity of neolignans from Virola species and synthetic analogues,” Phytochemistry, vol. 55, no. 6, pp. 589–595, 2000. View at Publisher · View at Google Scholar · View at Scopus
  27. A. A. V. Carvalho, P. M. Galdino, M. V. M. Nascimento et al., “Antinociceptive and antiinflammatory activities of grandisin extracted from Virola surinamensis,” Phytotherapy Research, vol. 24, no. 1, pp. 113–118, 2010. View at Publisher · View at Google Scholar · View at Scopus
  28. P. D. Coley, M. Massa, C. E. Lovelock, and K. Winter, “Effects of elevated CO2 on foliar chemistry of saplings of nine species of tropical tree,” Oecologia, vol. 133, no. 1, pp. 62–69, 2002. View at Publisher · View at Google Scholar · View at Scopus
  29. G. H. Krause, E. Grube, A. Virgo, and K. Winter, “Sudden exposure to solar UV-B radiation reduces net CO2 uptake and photosystem I efficiency in shade-acclimated tropical tree seedlings,” Plant Physiology, vol. 131, no. 2, pp. 745–752, 2003. View at Publisher · View at Google Scholar · View at Scopus