Resultados

Plants have been used for medicinal purposes as well as for the produc-tion of poisons, pigments and flavors since ancient times, even without knowledge of their chemical background. During the twentieth century, the progress in methodology resulted in the discovery of an enormous amount of natural compounds. Interest grew as to why these compounds appear in plants and other organisms and what their role may be.⁵ The question was, what evolutionary advantage does a plant gain by producing these secondary metabolites? In the year 1888, Stahl,⁶ who is frequently quoted but heavily criticized, described experiments on the subject of plants’ chemical protection against slugs and snails. He concluded that plant secondary compounds must have evolved under the selection pres-sures of herbivores. It was not until the 1980s that the functional aspects of secondary metabolism in plants were heralded as the “playground of biochemical evolution.”⁷However, at this time it was still thought that sec-ondary compounds were only ecologically important due to coincidence.⁸ Mueller and Börger^9,10 published the theory of phytoalexins in 1940,⁹ declaring phytoalexins to be “plant derived antibiotics that are synthe-sized de novo in living plant tissue as a response to pathogenic attack,”¹¹ thereby forming an immunologic system of defense. As a plant’s sensi-tivity to or resistance against disease is an evolutionarily advantage, and the secondary metabolic pathways that bring about such resistance are heritable, theories on the evolutionary role of secondary metabolites are supported. Around the same time, the theory of allelopathy (from Greek:

allelo= respective; pathos = damage) was developed. Its claims were and still are controversial, stating that plants can release compounds, be it volatile compounds through their leaves or liquid compounds through

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their roots, which are incorporated by other plants to cause harm or protection.

Phytochemical studies started at the end of the eighteenth century, when plants began to be analyzed for their major bioactive compounds. In the first half of the nineteenth century, crude plant extracts were purified by processes of crystallization and re-crystallization, liquid-liquid differential extraction, distillation and other separation techniques. The generation of secondary metabolites in plants was explained by analogous reactions in organic chemistry or by comparisons to known structures.⁵ Tracer tech-niques with radioactively labeled nuclides were developed in the 1950s.⁸ However, these techniques required large amounts of compounds and also great ability and imagination on the part of the chemists to solve the puzzle of chemical structures and biosynthesis routes.⁵Studies were much simpli-fied, when improved mass spectrometry (MS) and nuclear magnetic reso-nance (NMR) techniques allowed analysis without chemical degradation.

Later, MS combination systems with gas chromatography (GC-MS) or liq-uid chromatography (LC-MS) with advanced sensitivity were developed.⁸ Their widespread use in the 1970s led to great successes in understanding the chemistry of natural products.⁵Difficulties in isolating active enzymes were overcome in the early 1970s with the introduction of column chro-matography and the use of plant cell and organ cultures.⁸Box 1 gives more information about the development of analytic methods. In the 1960s and 1970s, phytochemical studies expanded to include microorganisms, insects, lichens, algae and marine organisms.⁵In the 1970s, a new discipline named (Phyto) Chemical Ecology came into being.⁸Over time, findings led to a new chemotaxonomic approach, using natural products as tools for the classification of species and taxa, which nowadays can be confirmed by DNA studies.⁵

The latter is regulated by continuous modification and new function-alization of duplicated genes. Because the duplicate copy is free from the demands on the parent gene, new traits can fuel the evolution of metabolic diversity.⁸

What accounts for the environmental pressure that necessitates the energetic expenses of secondary metabolism? A closer look on the his-tory of life on earth may bear the answer to this question: The earth was

Box 1: Development of analytic methods.⁵ X-Ray diffraction analysis

1895 Discovery of X-rays by W. C. Röntgen

1925 First use of X-rays with organic compounds (e.g. benzene) 1945 Elucidation of the structure of penicillin and Vitamin B12 1953 Revelation of the double helical structure of DNA

Today Routine technique

Drawback Only usable for crystalline compounds (not cells, which contain aqueous medium); individual stereoisomers only measurable with the presence of heavy atoms (e.g.

bromine)

NMR analysis

1957 First experiments; resonance of the N-atom was the most commonly used signal

1960 First H-atom resonance frequency for isobutyl and tert-butyl-groups

1960s Reliable results for chemical elucidation of natural products (e.g. In 1961 both alpha-thujaplicinol and pisatin — confirming the phytoalexin-theory of 1940) 1963 Elucidation of the stereochemistry of the methyl groups in betulin through long range spin-spin coupling and decoupling proton experiments

Since the 1960s

Increasing power of magnets has allowed higher

resolution spectra to be obtained with smaller amounts of the compound, resulting in the ability to examine minor metabolites.

Development of new techniques

e.g. TOCSY-1D, COSY, TOCSY, ROESY, NOESY, INADEQUATE, DOSY, HETCOR, HSQC and HMBC 1994–1996 Elucidation of the complete structure of marine

polyethers, the causative agents leading to poisonous effects of the red tide

(Continued)

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Box 1: (Continued) Chromatography methods

Important because NMR, as well as X-ray diffraction analysis, require high purity of the examined compound

Development from TLC on paper or silica gel to GC, HPLC and CCC Recently Combinations of chromatographic separation with

spectroscopic data collection and detection forms hyphenated tandem techniques, e.g. GC-MS, LC-UV-MS or LC-MS-UV-NMR

formed 4.6 billion years ago. Bacteria and primitive unicellular prokaryotic cells appeared 800 million years (MY) later. Autotrophic photosynthetic cyanobacteria emerged 3.1 billion years ago, which lead to the produc-tion of oxygen and its accumulaproduc-tion in the atmosphere, causing the first massive extinction of anaerobic organisms 2 billion years ago. Terrestrial bryophytes developed 550 MY ago, with fungi and lichen arising first, fol-lowed by the first terrestrial vascular plants 440 MY ago. These vascular plants formed the first forests. Only 40 MY later, i.e. about 400 MY ago, insects appeared on the earth. Since then, insects and plants have been living together, each influencing the course of the other’s evolution.⁵This persistent co-evolution between plants and their enemies (animal herbi-vores and parasites as well as other plant pathogens) is thought to be a major reason for the emergence of the great biodiversity found today on earth. Current theory states that plants synthesized and accumulated toxins as a response to the selection pressure of phytophagous insects. By adapting to and surviving the exposure to these toxins, a few insect species learn to tolerate these otherwise toxic plants. Thus, the plants evolved new or altered toxins to combat their assailants. This back-and-forth process resulted in highly specific relationships between insect and plant species that are closely linked ecologically, as was described in 1964 by Ehrlich and Raven.¹² Evi-dence of sequestration of toxic compounds by phytophagous organisms further supports the idea of co-evolution. Toxic compounds in the diets

of these insects serve in their own defense or as building blocks for insect venoms or pheromones. Certain organisms have formed a close, specialized plant-predator-relationship, such as the monarch butterfly (Danaus plex-ippus), which uses cardenolides from Asclepia scurassavica to make itself poisonous.⁵

Recently, the so-called “-omics” technologies (genomics, transcrip-tomics, proteomics, metabolomics) have found an entrance into chemi-cal ecology. In the past, medicinal plant compounds were discovered by bioassay-guided fractionation, examining the bioactivity of a first crude plant extract, which was then repeatedly fractionized and examined for bioactivity. Now, metabolomics allows for the identification and quan-tification of all primary and secondary metabolites in an organism.¹³ In ecogenetics and ecogenomics, the interactions of plants and herbivores are investigated, but not without several restraints. For one, there is necessarily reductionism in trying to understand the interactions of plants with their environments; combining potted plants in greenhouses with random her-bivores can only lead to very theoretical models.¹⁴Ideally, one should inves-tigate the interactions of plants and herbivores that share the same habitat under natural selection pressures of that habitat, that is to say, in their natu-ral environment. However, this is impractical, and techniques such as ecoge-netics can provide practical useful information. Plant micrometabolomics is a specialization of the metabolomics techniques to analyze the metabo-lites present in a certain plant cell or plant tissue.¹⁵

From time to time, compounds from the rich chemical pool of sec-ondary metabolites are adopted into primary metabolic pathways, gaining essential functions as phytohormones and signaling compounds. For instance, some flavonoids, known as floral color compounds, may affect auxin transport and developmental processes in Arabidopsis. Interestingly, these compounds predate the evolution of flowering plants. Some metabo-lites, such as canavanine, have acquired both primary and secondary func-tions. Canavanine is a toxic aginine-like antimetabolite that accumulates in the seeds of certain legumes. Its primary function is the remobilization of nitrogen during germination,⁸but it also serves as a protection against predators.

As new technologies are emerging, well established results are leading to new applications. Ethnobotanical and ecological approaches have given

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rise to the concept of ethnopharmacy, culminating in the discovery of new bioactive compounds to address diverse human afflictions. The issue of sustainability has been addressed as well. For example, sustainability concerns have arisen surrounding the Pacific yew tree (Taxus brefivolia), from which the anticancer-drug paclitaxel is obtained. Extensive harvesting endangered the survival of the species until a related analogue, bacattin III, was found in a more readily available source, Taxus baccata, allowing semisynthesis of the drug.⁵