**Secondary Metabolites**

Tânia da S. Agostini-Costa1, Roberto F. Vieira1, Humberto R. Bizzo2, Dâmaris Silveira3 and Marcos A. Gimenes1 *1Embrapa Genetic Resources and Biotechnology, Brasília 2Embrapa Food Technology, Rio de Janeiro, 3Health Sciences Quality, University of Brasilia, Brasília, Brazil* 

#### **1. Introduction**

130 Chromatography and Its Applications

Henrique, M. C.; Nunomura, S. M. & Pohlit, A. M. (2010). Alcaloides indólicos de cascas de

Jackson, A. U., Tata, A., Wu, C., Perry, R. H., Haas, G., West, L., & Cooks, R. G. (2009). Direct

Jácome, R. L. R. P.; Oliveira, A. B.; Raslan, D. S. & Wagner, H. (2004).Estudo químico e perfil

Jácome, R. L. R. P.; Souza, R. & Oliveira, A.B. (2003).Comparação cromatográfica entre o

Kobayashi, J; Sekiguchi, M.; Shimamoto, S.; Shigemori, H.; Ishiyama, H. & Ohsaki, A.,

Marques, M., Kato, L., Filho, H. & Reis, F., (1996). Indole alkaloid from Aspidosperma

Oliveira, A., (1999). Estudo de seis espécies do gênero Aspidosperma, utilizando CG,

Oliveira, V. B.; Freitas, M. S. M.; Mathias, L.; Braz-Filho, R. &Vieira, I. J. C.; (2009).Atividade

Phillipson, J. D.; Supavita, N. & Anderson, L. A. (1982). Separation of heteroyohimbine and

Stockigt, J.; Sheludko, Y.; Unger, M.; Gerasimenko, I.; Warzecha, H. & Stockigt, D., (2002).

Tanaka J.C., Silva C.C., Ferreira I.C.P., Machado G.M.C., Leon L.L. & Oliveira A.J.B., (2007).

Tanaka JCA, Silva C.C., Dias F., B.P., Nakamura C.V. & Oliveira A.J.B. (2006). Antibacterial

Verpoorte, R. & Baherheim, A. S. (1984).Chromatography of Alkaloids. Amsterdam,

Zocoler, M.A., Oliveira, A.J.B., Sarragiotto, M.H., Grzesiuk, V.L. & Vidotti, G.J., (2005).

alkaloid groups. *Journal of Chromatography* A, 967, 85-113.

analysis of *Stevia* leaves for diterpene glycosides by desorption electrospray

cromatográfico das cascas de *Aspidosperma parvifolium* A. DC. ("Pereira"). *Quim.* 

extrato de *Aspidosperma parvifolium* e o fitoterápico "Pau-Pereira". *Rev. Bras.* 

(2002). Subincanadines A-C, Novel Quaternary Indole Alkaloids from *Aspidosperma* 

CG/MS e HPLC: Análise Qualitativa e Quantitativa. Teste Bioautográfico; Cultura de Tecidos e Células Vegetais e rota de Preparação dos Compostos Diméricos Ramiflorina A e Ramiflorina B. Universidade de Campinas, Campinas. *Tese* 

biológica e alcalóides indólicos do gênero Aspidosperma (Apocynaceae): uma

oxindole alkaloids by reversed-phase high-performance liquid chromatography.

High-performance liquid chromatographic, capillary electrophoretic and capillary electrophoretic–electrospray ionisation mass spectrometric analysis of selected

Antileishmanial activity of indole alkaloids from *Aspidosperma ramiflorum*.

activity of indole alkaloids from *Aspidosperma ramiflorum*. *Braz J MedBiol Res 39*: 387-

Qualitative determination of indole alkaloids of *Tabernaemontana fuchsiaefolia*

*Aspidosperma vargasii* e *A. desmanthum*. *Química Nova*, 33, 284-287.

ionization mass spectrometry. *Analyst*, 134, 867-874.

*Farmacogn.*, v. 13, supl., p. 39-41, 2003. ISSN: 0102-695X

ramiflorum. *Phytochemistry*, v. 41, n. 3, pp.963-967.

*Nova,* Vol. 27, No. 6, 897-900.

*(doutorado em química orgânica)*.

revisão.*Rev. Bras. Pl. Med.*, *11*, 92.

*Phytomedicine* 14, 377-380.

391.

*Journal of Chromatography A*, 244, 91-98.

Netherlands, *Elselvier*, vol. 23B, p.31.

(Apocynaceae). *J. Braz. Chem. Soc.* 16, 1372—1377.

*subincanum*. *J. Org. Chem*., 67, 6449-6455.

Secondary metabolites are organic molecules that are not involved in the normal growth and development of an organism. While primary metabolites have a key role in survive of the species, playing an active function in the photosynthesis and respiration, absence of secondary metabolites does not result in immediate death, but rather in long-term impairment of the organism's survivability, often playing an important role in plant defense. These compounds are an extremely diverse group of natural products synthesized by plants, fungi, bacteria, algae, and animals. Most of secondary metabolites, such as terpenes, phenolic compounds and alkaloids are classified based on their biosynthetic origin. Different classes of these compounds are often associated to a narrow set of species within a phylogenetic group and constitute the bioactive compound in several medicinal, aromatic, colorant, and spice plants and/or functional foods.

Secondary metabolites are frequently produced at highest levels during a transition from active growth to stationary phase. The producer organism can grow in the absence of their synthesis, suggesting that secondary metabolism is not essential, at least for short term survival. A second view proposes that the genes involved in secondary metabolism provide a ''genetic playing field" that allows mutation and natural selection to fix new beneficial traits via evolution. A third view characterizes secondary metabolism as an integral part of cellular metabolism and biology; it relies on primary metabolism to supply the required enzymes, energy, substrates and cellular machinery and contributes to the long term survival of the producer (Roze et al, 2011).

A simple classification of secondary metabolites includes tree main groups: terpenes (such as plant volatiles, cardiac glycosides, carotenoids and sterols), phenolics (such as phenolic acids, coumarins, lignans, stilbenes, flavonoids, tannins and lignin) and nitrogen containing compounds (such as alkaloids and glucosinolates). A number of traditional separation techniques with various solvent systems and spray reagents, have been described as having the ability to separate and identify secondary metabolites. This chapter proposes to discuss major secondary metabolites classes (terpenoids, phenolic compounds and alkaloids) with different chemical structures and functions being screened, separated, fractionated, purified

Secondary Metabolites 133

The major constituents present in the *volatile oils* of different aromatic species may also be isolated or fractionated by silica gel on preparative TLC or CC. For example, the essential oil of *Tanacetum chiliophyllum* was subjected to silica gel CC, using a solvent mixture of nhexane and ethyl acetate, to isolate one of its major components, dihydro-α-cyclogeranyl

Djabou et al. (2010) have characterized the essential oils of *Teucrium massiliense* L. from Corsican and Sardinian islands, using a combination of capillary GC/retention indices, GC/MS and 13CNMR spectroscopy after fractionation on CC. A mixture of all Corsican oil samples was submitted to flash chromatography [FC; silica gel, elution with *n*-pentane, then with diethyl ether]. The polar fraction was separated on silica gel and 14 fractions were eluted with a mixture of *n*-pentane and diethyl ether of increasing polarity to give the major components: 6-methyl-3-heptyl acetate, 3-octyl acetate, isobutyl isovalerate, germacrene D and linalool. Successive CC revealed the unknown compound 6-methyl-3-heptyl acetate.

Chemical investigations on the essential oil of *Lippia integrifolia* performed by CC (silica gel using *n*-hexane with increasing amounts of diethyl ether), followed by HPLC, GC–MS, 1H and 13C-NMR spectroscopy led to the identification of 78 components. A new sesquiterpenic

Sutour et al. (2008) studied the essential oil of *Mentha suaveolens* ssp*. insularis* (Req.) Greuter, finding pulegone to be the major constituent. However, the second most abundant component of the essential oil remained unknown, being isolated by repeated chromatography on silica gel and submitted to a full set of NMR experiments, and identified as *cis-cis-p-*menthenolide. Fractions were eluted with a gradient of solvents of increasing

The essential oil from the leaves of *Piper hispidinervum*, a native species from the Amazon, is very rich in safrole (Maia et al., 1987). During an extensive agronomical study with this species, a population produced an essential oil rich in a different phenylpropanoid (up to 78%). The mass spectrum was identical to that of myristicin (Figure 1). However, a difference of near 30 units in retention index between myristicin and the phenylpropanoid was strong evidence that it was another compound, probably a myristicin isomer. No retention indices were available for the two possible isomers, croweacin and sarisan. For identification, the unknown compound was isolated by CC on silica gel and eluted with hexane-ethyl acetate mixtures. After 1H and 13CNMR studies, it was identified as sarisan

Fig. 1. Possible isomers of myristicin from the oil of a population of *Piper hispidinervum*;

alcohol, *trans*-africanan-1α-ol, was identified (Coronel et al. 2006).

polarity (pentane:diethyl ether 100:0–0:100).

sarisan was the actual compound present.

(Bizzo et al., 2001).

**2.1.1 Compound identification** 

hexanoate (Salamci et al, 2007).

or analyzed using various adsorbents and eluents through column chromatography (CC) and thin layer chromatography (TLC).
