Phytochemistry (v.68, #22-24)

Jeffrey B. Harborne (1928–2002) – An appreciation by Renée J. Grayer; Christine A. Williams (2708-2712).

Vegetable tannins are complex polyphenolic metabolites of plants based upon two principal structural themes – oligomeric flavan-3-ols (proanthocyanidins) and poly-3,4,5-trihydroxyaroyl esters (gallotannins and ellagitannins). The remarkable progress made in the last fifty years in the understanding of their chemistry and biochemistry is reviewed.After the early encouragement from the outstanding contribution in the early 1900s of Emil Fischer to an understanding of vegetable tannins the work of the following half-century had simply exemplified the complexity of the problems they presented. It was generally recognised [] that there was a broad division into condensed or non-hydrolysable and hydrolysable tannins but much else remained vague and untidy. In the 1950s Bate-Smith and Swain gave the lead into totally new ways of looking at these substances. They drew aside for the first time the curtains on the botanical aspects of these substances to reveal the rich vistas which lay beyond. It was to initiate remarkable progress in the next fifty years in the understanding of their chemistry and biochemistry; some of the principal developments of this work are reviewed herein.
Keywords: Vegetable tannins; Galloyl and hexahydroxydiphenoyl esters; Condensed proanthocyanidins; Structure and biosynthesis; Comparative biochemistry; Astringency;

Phenolic compounds are ubiquitous in plants which collectively synthesize several thousand different chemical structures characterized by hydroxylated aromatic ring(s).These compounds play several important functions in plants. They represent a striking example of metabolic plasticity enabling plants to adapt to changing biotic and abiotic environments and provide to plant products colour, taste, technological properties and putative health promoting benefits.Phenolic compounds represent the most studied phytochemicals and have been widely exploited as model systems in different areas of plant research. Initial studies in the field concerned the analytical characterization of a wide range of structures and of relevant enzymes with PAL being one of the most studied plant enzymes. This research is still active due to the complexity of the structures and the biosynthetic pathways As an example, the nature and functions of enzymes involved in lignin synthesis have been revisited several times, even in recent years.More recently, molecular biology and genomics have provided additional understanding of the mechanisms underlying the synthesis of these compounds with special emphasis on the regulation of gene expression by environmental factors. The extensive characterization of genes encoding the different enzymatic steps of flavonoid synthesis and cytochrome P450 genes have been among the most recent advances in this area.Metabolic engineering of lignins and flavonoids has been deeply investigated. Significant positive results have been obtained in both areas but the negative European opinion towards genetically modified organisms has considerably hampered potential applications. From a more basic point of view, global approaches (such as transcript and metabolite profiling) have investigated the repercussions of these engineered modulations of specific phenolics synthesis on other branches of plant metabolism. These studies have revealed a substantial and sometimes unexpected network of regulatory interactions.In the present time, the societal demand and an increasing interest for practical applications has stimulated a wide range of biological and epidemiological studies aiming at characterizing the health promoting properties of specific phenolic compounds with antioxidant activities towards cancer, cardiovascular and neurodegenerative diseases or for use in antiaging or cosmetic products.Increased emphasis on sustainable development should stimulate innovative investigations on phenolic synthesis for improving plant biomass and for a better control of plant and animal health.
Keywords: Phenolic compounds; Phenylpropanoids; Lignins; Flavonoids; Biosynthetic pathways; Multigene families; Transcription factors; Cytochrome P450 genes; Metabolic engineering; Antioxidant activities; Health promoting properties;

Justicidin B 7-hydroxylase, a cytochrome P450 monooxygenase from cell cultures of Linum perenne Himmelszelt involved in the biosynthesis of diphyllin by Shiva Hemmati; Bernd Schneider; Thomas J. Schmidt; Katja Federolf; A. Wilhelm Alfermann; Elisabeth Fuss (2736-2743).
A biosynthetic pathway for the formation of arylnaphthalene lignans like diphyllin is suggested. Justicidin B 7-hydroxylase (JusB7H) which catalyzes the last step in the biosynthesis of diphyllin was characterized as a cytochrome P450 monooxygenase from suspension cultures of Linum perenne Himmelszelt.Cell suspension cultures of Linum perenne L. Himmelszelt accumulate justicidin B as the main component together with glycosides of 7-hydroxyjusticidin B (diphyllin). A hypothetical biosynthetic pathway for these compounds is suggested. Justicidin B 7-hydroxylase (JusB7H) catalyzes the last step in the biosynthesis of diphyllin by introducing a hydroxyl group in position 7 of justicidin B. This enzyme was characterized from a microsomal fraction prepared from a Linum perenne Himmelszelt suspension culture for the first time. The hydroxylase activity was strongly inhibited by cytochrome c as well as other cytochrome P450 inhibitors like clotrimazole indicating the involvement of a cytochrome P450-dependent monooxygenase. JusB7H has a pH optimum of 7.4 and a temperature optimum of 26 °C. Justicidin B was the only substrate accepted by JusB7H with an apparent K m of 3.9 ± 1.3 μM. NADPH is predominantly accepted as the electron donor, but NADH was a weak co-substrate. A synergistic effect of NADPH and NADH was not observed. The apparent K m for NADPH is 102 ± 10 μM.
Keywords: Linum perenne; Linaceae; Lignan; Cytochrome P450; Justicidin B; Diphyllin; Justicidin B 7-hydroxylase;

Coniferin dimerisation in lignan biosynthesis in flax cells by Vickram Beejmohun; Ophélie Fliniaux; Christophe Hano; Serge Pilard; Eric Grand; David Lesur; Dominique Cailleu; Frédéric Lamblin; Eric Lainé; José Kovensky; Marc-André Fliniaux; François Mesnard (2744-2752).
The use of [8,9-13C2]-coniferin coupled with mass spectrometry and nuclear magnetic resonance showed the ability of a flax cell suspension to biosynthesise differently linked (neo)lignans: 8–8′, 8–5′ and 8-O-4′. The continuous synthesis and subsequent metabolisation of these coniferin-derived dimers were established all over the culture period.[13C2]-Coniferin was provided to a flax (Linum usitatissimum L.) cell suspension to monitor subsequent dimerisation by MS and NMR. The label was mainly incorporated into a 8–8′-linked lignan, lariciresinol diglucoside, a 8–5′-linked neolignan, dehydrodiconiferyl alcohol glucoside and a diastereoisomeric mixture of a 8-O-4′-linked neolignan, guaiacylglycerol-β-coniferyl alcohol ether glucoside. This latter compound is reported for the first time in flax. The strong and transient increase in these compounds in fed cells was concomitant with the observed peak in coniferin content. These results suggest (i) a rapid metabolisation of coniferin into lignans and neolignans and indicate the capacity of flax cells to operate different types of couplings, and (ii) a continuous synthesis and subsequent metabolisation of coniferin-derived dimers all over the culture period.
Keywords: Linum usitatissimum; Linaceae; Flax; LC-MS; MS/MS; NMR; Labelled coniferin; Lignan; Neolignan;

Homoisoflavanones from Pseudoprospero firmifolium of the monotypic tribe Pseudoprospereae (Hyacinthaceae: Hyacinthoideae) by Chantal Koorbanally; Sarisha Sewjee; Dulcie A. Mulholland; Neil R. Crouch; Anthony Dold (2753-2756).
Five homoisoflavanones: 3,5-dihydroxy-7,8-dimethoxy-3-(3′,4′-dimethoxybenzyl)-4-chromanone, 3,5-dihydroxy-7-methoxy-3-(3′,4′-dimethoxybenzyl)-4-chromanone, 3,5-dihydroxy-7,8-dimethoxy-3-(3′-hydroxy-4′-methoxybenzyl)-4-chromanone, 3,5,6-trihydroxy-7-methoxy-3-(3′-hydroxy-4′-methoxybenzyl)-4-chromanone and 3,5,7-trihydroxy-3-(3′-hydroxy-4′methoxybenzyl)-4-chromanone and the nortriterpenoid, 15-deoxoeucosterol, have been isolated from the bulbs of Pseudoprospero firmifolium, the sole representative of the tribe Pseudoprospereae (Hyacinthaceae: Hyacinthoideae).Five 3-hydroxy-type homoisoflavonoids, 3,5-dihydroxy-7,8-dimethoxy-3-(3′,4′-dimethoxybenzyl)-4-chromanone, 3,5-dihydroxy-7-methoxy-3-(3′,4′-dimethoxybenzyl)-4-chromanone, 3,5-dihydroxy-7,8-dimethoxy-3-(3′-hydroxy-4′-methoxybenzyl)-4-chromanone, 3,5,6-trihydroxy-7-methoxy-3-(3′-hydroxy-4′-methoxybenzyl)-4-chromanone and 3,5,7-trihydroxy-3-(3′-hydroxy-4′methoxybenzyl)-4-chromanone in addition to the nortriterpenoid, 15-deoxoeucosterol, have been isolated from the dichloromethane extract of the bulbs of Pseudoprospero firmifolium, the sole representative of the tribe Pseudoprospereae of the subfamily Hyacinthoideae of the Hyacinthaceae.
Keywords: Pseudoprospero; 3,5-Dihydroxy-7,8-dimethoxy-3-(3′,4′-dimethoxybenzyl)-4-chromanone; 3,5-Dihydroxy-7-methoxy-3-(3′,4′-dimethoxybenzyl)-4-chromanone; 3,5-Dihydroxy-7,8-dimethoxy-3-(3′-hydroxy-4′-methoxybenzyl)-4-chromanone; 3,5,6-Trihydroxy-7-methoxy-3-(3′-hydroxy-4′-methoxybenzyl)-4-chromanone; 3,5,7-Trihydroxy-3-(3′-hydroxy-4′methoxybenzyl)-4-chromanone; 15-Deoxoeucosterol;

Evolution and current status of the phytochemistry of nitrogenous compounds by Meinhart H. Zenk; Melanie Juenger (2757-2772).
With the discovery of nitrogen-containing plant products, 200 years ago, a success story began. Strongly bioactive pure chemicals from plants were isolated and used in medicine and, in parallel, organic and pharmaceutical chemistry developed. This success story continues today and multidisciplinary “phytochemistry” surely has a bright future.Nitrogen-containing and other secondary plant products have evolved as a consequence of the struggle between the plant and the animal kingdoms, the latter directly or indirectly thriving on plants. During evolution plants developed bioactive and exceedingly complicated chemical structures that serve the purpose of plant defense. It is this property of those plants that has been exploited by mankind as medicines, poisons and recreational drugs. Three classes of nitrogen-containing plant products are being reviewed in this article: the alkaloids, the cyanogenic glucosides/glucosinolates and the nonprotein amino acids. It is the interplay of different scientific disciplines such as chemistry, pharmacognosy, medicine, analytics, cell biology, molecular biology, botany and chemotaxonomy that form a new and exciting area called “phytochemistry”. It is foreseeable that this integration of disciplines across traditional borders will bring new achievements in phytochemistry, as history has taught us already.
Keywords: Alkaloids; Cyanogenic glucosides/glucosinolates; Nonprotein amino acids; History; Discovery; Medicinal use; Cell cultures; Raison d’être; Biosynthesis;

Functional characterisation of genes involved in pyridine alkaloid biosynthesis in tobacco by Suvi T. Häkkinen; Sofie Tilleman; Agnieszka Šwiątek; Valerie De Sutter; Heiko Rischer; Isabelle Vanhoutte; Harry Van Onckelen; Pierre Hilson; Dirk Inzé; Kirsi-Marja Oksman-Caldentey; Alain Goossens (2773-2785).
Potential catalysers of tobacco pyridine metabolism can be discovered by functional genomics-based analysis of a Nicotiana tabacum gene platform, established previously by means of integrated transcript and metabolite profiling.Although secondary metabolism in Nicotiana tabacum (L.) (tobacco) is rather well studied, many molecular aspects of the biosynthetic pathways and their regulation remain to be disclosed, even for prominent compounds such as nicotine and other pyridine alkaloids. To identify players in tobacco pyridine alkaloid biosynthesis a functional screen was performed, starting from a tobacco gene collection established previously by means of combined transcript profiling and metabolite analysis. First, full-length cDNA clones were isolated for 34 genes, corresponding to tobacco transcript tag sequences putatively associated with pyridine alkaloid metabolism. Full-length open reading frames were transferred to pCaMV35S-steered overexpression vectors. The effects of plant transformation with these expression cassettes on the accumulation of nicotine and other pyridine alkaloids were assessed in transgenic tobacco Bright-Yellow 2 (BY-2) cell suspensions and hairy root cultures. This screen identified potential catalysers of tobacco pyridine metabolism, amongst which a lysine decarboxylase-like gene and a GH3-like enzyme. Overexpression of the GH3-like enzyme, presumably involved in auxin homeostasis and designated NtNEG1 (Nicotiana tabacum Nicotine-Enhancing GH3 enzyme 1), increased nicotine levels in BY-2 hairy roots significantly. This study shows how functional genomics-based identification of genes potentially involved in biosynthetic pathways followed by systematic functional assays in plant cells can be used at large-scale to decipher plant metabolic networks at the molecular level.
Keywords: Nicotiana tabacum; Solanaceae; Tobacco; Functional genomics; Pyridine alkaloids; Nicotine; Lysine decarboxylase; GH3-like enzyme; Jasmonate; Auxin conjugates; cDNA-AFLP;

Chromatography is essential to phytochemistry and is the key to obtaining pure compounds for structure elucidation (by countercurrent chromatography, for example), for pharmacological testing or for development into therapeuticals. It also plays a fundamental role as an analytical technique for quality control and standardisation of phytotherapeuticals.Chromatography is the lynchpin of phytochemistry and is the key to obtaining pure compounds for structure elucidation, for pharmacological testing or for development into therapeuticals. It also plays a fundamental role as an analytical technique for quality control and standardisation of phytotherapeuticals. Although liquid chromatography is barely 100 years old, an extraordinary variety of instrumental and ancillary equipment is available, notably in the domain of high-performance liquid chromatography. It is impossible to touch all areas of chromatography in such a review but certain areas are worthy of mention: HPLC, HPTLC, UPLC and countercurrent chromatography.Another important addition has been the development of hyphenated techniques involving HPLC: LC/UV, LC/MS, LC/MSn and LC/NMR. These are indispensable nowadays for the early detection and identification of compounds in crude plant extracts.
Keywords: Chromatography; Liquid chromatography; Countercurrent chromatography; Thin-layer chromatography; High-performance liquid chromatography;

Recent advances in instrumentation have led to a remarkable set of techniques and applications of NMR spectroscopy. This review describes the role of high-resolution NMR spectroscopy in the structural analysis of plant constituents (e.g. proteins and small metabolites) and in the analysis of biosynthetic pathways and metabolite flux.Rapid progress in instrumentation and software made nuclear magnetic resonance spectroscopy (NMR) one of the most powerful analytical methods in biological sciences. Whereas the development of multidimensional NMR pulse sequences is an ongoing process, a small subset of two-dimensional NMR experiments is typically sufficient for the rapid structure determination of small metabolites. The use of sophisticated three- and four-dimensional NMR experiments enables the determination of the three-dimensional structures of proteins with a molecular weight up to 100 kDa, and solution structures of more than 100 plant proteins have been established by NMR spectroscopy. NMR has also been introduced to the emerging field of metabolomics where it can provide unbiased information about metabolite profiles of plant extracts. In recent times, high-resolution NMR has become a key technology for the elucidation of biosynthetic pathways and metabolite flux via quantitative assessment of multiple isotopologues. This review summarizes some of the recent advances of high-resolution NMR spectroscopy in the field of plant sciences.
Keywords: NMR spectroscopy; Metabolomics; Metabonomics; Biosynthesis;

Biosynthesis of the chromogen hermidin from Mercurialis annua L. by Elena Ostrozhenkova; Eva Eylert; Nicholas Schramek; Avi Golan-Goldhirsh; Adelbert Bacher; Wolfgang Eisenreich (2816-2824).
The isotopologue patterns in hermidin labelled with [U-13C6]glucose or 13CO2 indicate that the alkaloid is derived from the nicotinic acid pathway.Cut seedlings of Mercurialis annua L. were supplied with solutions containing 5.4 mM [U-13C6]glucose and 50 mM unlabelled glucose. The pyridinone type chromogen, hermidin, was isolated and analyzed by NMR spectroscopy. 13C NMR spectra revealed the presence of [4,5,6-13C3]hermidin in significant amount. NMR analysis of amino acids obtained by hydrolysis of labelled biomass showed the presence of [U-13C3]alanine, whereas aspartate was found to be virtually unlabelled. Photosynthetic pulse labelling of M. annua plants with 13CO2 followed by a chase period in normal air afforded [4,5,6-13C3]- and [2,3-13C2]hermidin with significant abundance. [U-13C3]Alanine and multiply 13C-labelled aspartate isotopologues were also found in significant abundance. The labelling patterns of hermidin obtained in the present study closely resemble those observed for the pyridine ring of nicotine under similar experimental conditions. This suggests that hermidin, like nicotine, is biosynthesized via the nicotinic acid pathway from dihydroxyacetone phosphate and aspartate. The data show that pulse/chase labelling of plants with 13CO2 generates isotopologue patterns that are similar to those obtained with totally labelled carbohydrate as tracer, but with the added advantage that experiments can be conducted under strictly physiological conditions. This experimental concept appears ripe for application to a wide variety of problems in plant physiology.
Keywords: Biosynthesis; NMR Spectroscopy; Hermidin; Alkaloid; Mercurialis;

Determination of saponins in Maesa lanceolata by LC-UV: Development and validation by Mart H.B.L. Theunis; Kenn Foubert; Jacob Pollier; Miguel Gonzalez-Guzman; Alain Goossens; Arnold J. Vlietinck; Luc A.C. Pieters; Sandra Apers (2825-2830).
A method is described to quantify the saponins in Maesa lanceolata extracts. The method is based on a H2O/MeOH extraction and a purification step on a C18 SPE cartridge. The identification is done by the combination of LC-UV/MS, while quantification is performed by LC-UV, using oleanolic acid as an external standard.Triterpene saponins are a class of plant natural products with a wide range of bioactivities, which makes them an interesting research subject. The small tree Maesa lanceolata, growing in African countries, is used in traditional medicine against various diseases. In previous work a triterpenoid saponin mixture was isolated from the leaves of M. lanceolata and the compounds were identified as closely related oleanane type triterpenes [Apers, S., Foriers, A., Sindambiwe, J.B., Vlietinck, A., Pieters, L., 1998. Separation of a triterpenoid saponin mixture from Maesa lanceolata: semi preparative reversed-phase wide pore high performance liquid chromatography with temperature control. J. Pharm. Biomed. Anal. 18, 737; Apers, S., De Bruyne, T.E., Claeys, M., Vlietinck, A.J., Pieters, L.A.C., 1999. New acylated triterpenoid saponins from Maesa lanceolata. Phytochemistry 52, 1121]. The compounds showed virucidal, haemolytic, molluscicidal and antiangiogenic activity [Apers, S., Baronikova, S., Sindambiwe, J.B., Witvrouw, M., De Clercq, E., Vanden Berghe, D., Van Marck, E., Vlietinck, A., Pieters, L., 2001. Antiviral, haemolytic and molluscicidal activities of triterpenoid saponins from Maesa lanceolata: establishment of structure–activity relationships. Planta Med. 67, 528; Apers, S., Bürgermeister, J., Baronikova, S., Vermeulen, P., Paper, D., Van Marck, E., Vlietinck, A.J., Pieters, L.A.C., 2002. Antiangiogenic activity of natural products: in vivo and in vitro test models. J. Pharm. Belg. 57 (Hors-série 1), 47]. Here we report the development of an extraction and quantification method to analyse saponin compounds in roots and leaves of M. lanceolata. After a purification step using C18 solid phase extraction (SPE) cartridges, the samples were analysed on a LC-UV/MS system. The identification of the peaks from the different saponins was confirmed based on the retention time and mass spectrum. The quantification was performed using the UV signals. The standard oleanolic acid curve was linear over a concentration range of 2.8–140.0 μg/mL. The recovery from the leaves was 94.5%. The precision of the method with respect to time and concentration was acceptable, with relative standard deviation (RSD%) values of 4.9 and 4.3, respectively.
Keywords: Maesa lanceolata ; Myrsinaceae; Method development; Method validation; HPLC; Mass spectrometry; Saponin;

The chemical diversity of plant secondary metabolism – formerly understood as part of metabolic excretion – is now recognized as an essential part of survival strategies of plants in a continuously changing environment.The isolation of morphine (‘principium somniferum’) by Friedrich Wilhelm Sertürner about 200 years ago is generally accepted as the beginning of scientific phytochemistry (plant secondary products research). For about 150 years this research addressed almost exclusively the isolation and structure elucidation of new plant products. It had great impact on the development of modern organic chemistry and pharmaceutical industry and provided the chemical basis for biological research on plant secondary metabolism, which began about 50 years ago. The historical development of this field to its present state of knowledge will be considered in this review from three angles of vision: mechanistic, functional and evolutionary perspectives. Mechanistic research started on the metabolite level and was initiated by the availability of radioactive nuclides in the early 1950s. By means of sophisticated tracer techniques, the biosynthetic routs of most secondary pathways were outlined and provided the basis for the enzymatic characterization of biosynthetic pathways in the 1970s and 1980s, followed by the identification of the corresponding genes beginning in the late 1980s. During this 50-year period of intensive research a change of paradigm occurred addressing the question: why do plants synthesize this immense rich diversity of secondary metabolites comprising more than 200,000 structures? For a long time regarded as waste products or assigned with various other attributes their indispensable role as components of the survival strategy of plants in a mostly hostile environment appears now generally accepted. Along with the great progress in the field of chemical ecology, the emerging field of molecular evolution provided crucial evidence that during evolution of secondary metabolism genes encoding enzymes of plant’s primary metabolism were duplicated, recruited and diversified for new functions under the everlasting and continuously changing selection pressure of the environment.
Keywords: Secondary metabolism; Review; History; Biosynthetic pathways; Turnover and degradation; Cell-type specific localization; Ecological functions; Molecular evolution;

Current status of metabolic phytochemistry by Maike Petersen (2847-2860).
Some current topics of research in metabolic phytochemistry will be shown:This review will give selected examples of topics of current research into plant secondary metabolism. Besides detection, isolation and characterisation of enzymes and genes involved in the formation of natural products, the structures of enzymes after crystallisation are now being investigated and this information gives us hints on the catalytic mechanisms as well as probable evolutionary origins of these enzymes. Manipulation of natural product formation is achieved by overexpression or down-regulation of genes encoding biosynthetic enzymes or regulators (transcription factors) as well as by transfer of those genes into foreign organisms (bacteria, yeast, plants). Techniques, strategies and methods are used to investigate plant secondary metabolism ensuring that this field remains challenging.
Keywords: Natural product biosynthesis; Biosynthetic enzymes; Metabolic engineering; Overexpression; RNAi; Transcription factors; Enzyme structure elucidation;

This article describes the state of the art in modern phytochemical analysis and affords one potential perspective of where it is heading in the future. Particular stress is given to unravelling the function of metabolites and the genes that control their accumulation or degradation.Like all biological disciplines, phytochemical research has seen profound changes in recent years. Whilst enzyme and metabolite purification and characterisation and pathway identification remain vastly important, the relative ease at which we can now obtain genome scale information has facilitated analysis at the level of the metabolic network. In addition, in recent years we have experienced an explosion in the number of plant proteins for which structural information is available. However, despite the presence of sequence information from a growing number of photosynthetic species, the function of many genes, let alone their in vivo roles, remains unclear. This review attempts to provide both an overview of the current state of the art and a perspective of the major challenges that remain.
Keywords: Metabolic profiling; Pathway analysis; Temporal resolution; Spatial resolution;

Transport of camptothecin in hairy roots of Ophiorrhiza pumila by Supaart Sirikantaramas; Hiroshi Sudo; Takashi Asano; Mami Yamazaki; Kazuki Saito (2881-2886).
Autofluorescence emitted from camptothecin, an anticancer monoterpene indole alkaloid, in the hairy root of Ophiorrhiza pumila suggests its localization in the vacuole. In this paper, we employed a pharmacological approach to elucidate camptothecin transport in the hairy root.We have investigated the subcellular accumulation and transport of camptothecin (CPT), a monoterpene indole alkaloid, in hairy roots of Ophiorrhiza pumila. This hairy root produces high amounts of CPT and excretes it into the culture medium. When the hairy roots were exposed to UV radiation, autofluorescence emitted from CPT showed subcellular localization of CPT in the vacuole. Treatment with several inhibitors suggested that CPT excretion is a transporter-independent passive transport controlled by the concentration gradient of the compound. Interestingly, the hairy roots treated with brefeldin A, a vesicle transport inhibitor, showed increased CPT excretion. This could be explained by an increased transport rate of CPT from the endoplasmic reticulum (ER) to the cytoplasm when transport of CPT to the vacuole is blocked. The much higher concentration of CPT in the cytoplasm resulted in the increased excretion rate. This result indicates that CPT is biosynthesized at the ER and transported to accumulate in the vacuole by the same machinery that is used for vacuolar protein sorting. How O. pumila is insensitive to CPT is discussed.
Keywords: Ophiorrhiza pumila; Hairy root; Excretion; Transport; Accumulation; Camptothecin;

The evolution of chemosystematics by Tom Reynolds (2887-2895).
Chemosystematics has been used by animals, including man, to distinguish plants and other beings useful for food and those best avoided. Originally unwritten, this knowledge has been formalized down the ages, until in modern times useful and harmful chemical constituents from relevant taxa have been identified and recorded. In return this knowledge has now been used to aid taxonomic distinctions of these plants, animals and micro-organisms. Advances in analytical instrumentation, in particular chromatography in all its forms, followed by electronic detection methods, has speeded these studies.Chemosystematics has been used to distinguish plants and other organisms that are useful for food and those best avoided. Originally unwritten, this knowledge has been progressively formalized with useful, harmful and inactive chemical constituents from relevant taxa now identified and recorded. This knowledge has led to insights into taxonomy of these plants, animals and micro-organisms. Advances in analytical instrumentation, in particular chromatography, followed by electronic detection methods, have speeded these studies, culminating in metabolic profiling, (“metabolomics”). The huge array of chemical constituents isolated from plants combined with morphological and cytological data take their place as part of the overall Natural History of the organism in its environment. The study of, DNA (genomics) and to a certain extent m-RNA (transcriptomics) and proteins (proteomics), has led to the immense subject of molecular biology which relates the phenotype of a taxon to its genome. This type of chemosystematics on its own does not of course describe the small molecules in plants, often called, perhaps misguidedly, “secondary compounds”, or how they relate to each other, to the plant containing them or to the environment. Economic uses flow from this knowledge, such as the topic of non-protein amino acids and amines, which from 1958 to the present has produced information from the chemotaxonomic to the severely practical. Literature on the subject from 1909 to the present charts developments in the discovery of new compounds and their use in systematics. Often a mere catalogue, a list of plant constituents is nevertheless part of the overall description of a plant.
Keywords: Chemical constituents; Phylogenetics; Symposia; Chromatography; Mass spectrometry; Non-protein amino acids;

The current status of chemical systematics by Peter G. Waterman (2896-2903).
Chemical systematics sets out to interpret the phylogenetic implications of the occurrence and distribution of secondary metabolites. In this review, a number of the major contributions from the 1960’s and 1970’s are identified and re-assessed in the light of recent evidence gained from DNA studies. It is shown that for the most part conclusions drawn on the basis of secondary metabolite distribution have been confirmed by the new techniques and it is concluded that chemical systematics can continue to provide useful insights into plant phylogeny.Chemical systematics sets out to interpret the phylogenetic implications of the occurrence and distribution of secondary metabolites. In this review, a number of the major contributions from the 1960’s and 1970’s are identified and re-assessed in the light of recent evidence gained from DNA studies. It is shown that for the most part conclusions drawn on the basis of secondary metabolite distribution have been confirmed by the new techniques and it is concluded that chemical systematics can continue to provide useful insights into plant phylogeny.
Keywords: Chemical systematics; Plant phylogeny; Rutales; Juniperus; Belalains; Iridoids; Alkaloids;

Recircumscription of plant systematics must be taken into account when drawing chemosystematic conclusions. They are highly dependent on up-to-date botanical classification.For almost a decade it has been acknowledged that the flowering plant dichotomy of monocotyledons and dicotyledons does not reflect the evolution of angiosperms. Despite this, conclusions in the field of chemosystematics are still drawn from, and rely on, non-phylogenetic botanical classifications such as those of Cronquist, Dahlgren and Takhtajan. In this paper the two alkaloids colchicine and camptothecin are used as examples of how phylogenetic systematics may be applied to alkaloid chemosystematics.
Keywords: Alkaloid; Camptothecin; Colchicine; Chemosystematics; Phylogeny;

The number of known isoflavonoid-producers increases slowly but continously. Isoflavonoids have been found in not less than sixty families falling into four classes of multicellular plants. Biosynthetically advanced structural types of isoflavonoids (rotenoids, pterocarpans, etc.) were recorded in both classes and all subclasses of flowering plants.Isoflavonoids are characteristic metabolites in legumes and an overwhelming number of reports concerning them come from the Leguminosae. Nevertheless, the spectrum of isoflavonoid producing taxa includes the representatives of four classes of multicellular plants, namely the Bryopsida, the Pinopsida, the Magnoliopsida and the Liliopsida. At least 59 non-leguminous families have been reported to produce isoflavones sensu lato; coumestans have been reported in 3 families, coumaronochromones in 3, pterocarpans in 9 and rotenoids in 8 families. Prenylated isoflavones have been found in 15 non-leguminous families and isoflavone dimers, heterodimers or oligomers in three families. More than two hundred different isoflavonoid aglycones have been reported in non-legumes altogether. The number of individual structures is even greater if the variety of glycosides are considered.Enzymology and genetics of isoflavonoid biosynthesis have been studied almost exclusively in legumes, with the exception of a few model plants (i.e. Beta vulgaris, Arabidopsis thaliana, Nicotiana tabacum and Zea mays). The key step at the very beginning of the isoflavonoid metabolic pathway is the oxidation of flavanone connected with the migration of aryl moiety from C2 to C3 mediated by a CYP450 enzyme isoflavone synthase (IFS), which has been identified and cloned in multiple legumes and in sugar beet (Beta vulgaris, Chenopodiaceae). No information is available about the enzyme(s) responsible for the biosynthesis of isoflavonoid core in other taxa. Experimental data demonstrates the capability of numerous enzymes of non-legume origin to metabolize isoflavones as alternative substrates to other phenolics.
Keywords: Isoflavonoid; Pterocarpan; Rotenoid; 3-Arylcoumarin; Coumestan; Coumaronochromone; Non-leguminous; Chemotaxonomy;

Evolution and current status of ecological phytochemistry by Francisco A. Macías; Jose L.G. Galindo; Juan C.G. Galindo (2917-2936).
Phytochemical studies have experienced a great change through the last century. This change has occurred mainly in two key points: the methodologies used in phytochemical studies and the point of view about why and for what reason secondary metabolites are present in plants and, by extension, in the rest of living organisms. The main question is to clarify the role that secondary metabolites play in the plant and if the resources invested in their production have or have not a reasonable reward in terms of advantages for survival. Some considerations in terms of evolution are made.Phytochemical studies have experienced a great deal of change during the last century, not only regarding the number of compounds described, but also in the concept of phytochemistry itself. This change has mainly been related to two key points: the methodologies used in phytochemical studies and the questions regarding ‘why secondary metabolites appeared in plants and in other living organisms?’ and ‘what is their role?’. This transformation in the field has led to new questions concerning such different subjects as evolution, paleobotany, biochemistry, plant physiology and ethnography. However, the main issue is to clarify the role that secondary metabolites play in the plant (and other organisms) and whether the resources invested in their production (C and N allocation, genes encoding their biogenetic pathways, specific enzymes, energy-rich molecules such as ATP and NADPH) have or lack a reasonable reward in terms of advantages for survival. Consequently, in this review the main emphasis will be placed on two subjects related to the evolution of phytochemical studies. The first aim is to describe briefly the influence that the development of the methodologies needed for compound isolation and structure elucidation have had on the field of phytochemistry. The second area to be covered concerns the new theories addressing the role of secondary metabolites from an ecological point of view: co-evolution of plants and their potential enemies (phytophagous insects, microbes, herbivores and other plants), chemical plant defence, adaptative strategies of phytophagues to plant toxins (among them sequestration will be briefly mentioned), and models and theories for carbon and nitrogen allocation. Some final remarks are made to summarize our opinion about the immediate future of phytochemical ecology and phytochemical studies.
Keywords: Phytochemical ecology; Co-evolution; Secondary metabolites; Semiochemicals; Phytoalexins; Juvenile hormones; Allelopathy; Ethnobotany; Sequestration;

Developments in aspects of ecological phytochemistry: The role of cis-jasmone in inducible defence systems in plants by John A. Pickett; Michael A. Birkett; Toby J.A. Bruce; Keith Chamberlain; Ruth Gordon-Weeks; Michaela C. Matthes; Johnathan A. Napier; Lesley E. Smart; Christine M. Woodcock (2937-2945).
Developments in ecological phytochemistry for food production and generation of bioenergy and biofuels will involve processes activated by natural products to “switch on” plant defence pathways. Plant breeding can exploit these systems and heterologous gene expression will eventually give rise to a new range of crops for food and energy.The challenges and opportunities for protecting agricultural production of food and other materials will be met through exploiting the induction of defence pathways in plants to control pests, diseases and weeds. These approaches will involve processes that can be activated by application of natural products, patented in terms of this use, to “switch on” defence pathways. Already, a number of secondary metabolite defence compounds are known for which the pathways are conveniently clustered genomically, e.g. the benzoxazinoids (hydroxamic acids) and the avenacins. For the former, it is shown that the small molecular weight lipophilic activator cis-jasmone can induce production of these compounds and certain genes within the pathway. Numerous groups around the world work on inducible defence systems. The science is rapidly expanding and involves studying the interacting components of defence pathways and the switching mechanisms activated by small molecular weight lipophilic compounds. Examples are described of how plant breeding can exploit these systems and how heterologous gene expression will eventually give rise to a new range of GM crops for food and energy, without the need for external application of synthetic pesticides.
Keywords: Secondary plant metabolites; Activators; Induction; Defence; Pest control; Benzoxazinoid; cis-Jasmone; Semiochemical;

The ability of plants to withstand herbivores relies on direct and indirect chemical defense. By using toxic phytochemicals, plants can deter and/or poison herbivores, while by releasing volatile organic compounds (VOCs) into the atmosphere plants can attract predators of the attacking insects. In this review, we will focus on rapid early events following insect feeding on plants that eventually lead to the production and release of phytochemicals.The ability of plants to withstand herbivores relies on direct and indirect chemical defense. By using toxic phytochemicals, plants can deter and/or poison herbivores, while by releasing volatile organic compounds (VOCs) into the atmosphere plants can attract predators of the herbivores. The activation of specific responses requires recognition and appropriate response towards the attacking enemy and most of the events which finally lead to gene activation (the signaling pathway) occur within a few minutes. Among the several signaling molecules involved, reactive oxygen species (ROS) and intracellular calcium signatures belong to early events, which are responsible for most of the ensuing cascades of chemical and molecular reactions. In this review, we will focus on rapid early events following insect feeding on plants that eventually lead to the production and release of phytochemicals. Furthermore, we compare and discuss the impact of mechanical and biotroph wounding.
Keywords: Plant–insect interaction; Direct and indirect defense; Calcium homeostasis; Reactive oxygen species; Volatile organic compounds;

Phytochemistry and pharmacognosy by J. David Phillipson (2960-2972).
The author met frequently with the first editors of phytochemistry, Tony Swain and Jeffrey Harborne, at scientific meetings of the Phytochemical Society. During the first 50 years of the Society there were tremendous advances in biological and chemical techniques that facilitated the isolation and structure determination of biologically active natural products. Today, we continue to rely on plants as sources of medicinal drugs and of herbal medicines. Pharmacognosy maintains strong links with phytochemistry and the Phytochemical Society of Europe.During the past 50 years there have been tremendous advances in chemical and biological techniques of analysis that have transformed research in pharmacognosy. The PSE has regularly held symposia of relevance to pharmacognosy and some of these are briefly reviewed in the area of natural products from higher plants. These symposia have charted the developments that link pharmacognosy with phytochemistry and illustrate the application of increasingly more sophisticated analytical techniques to the discovery of biologically active compounds. Plants have yielded clinical drugs, either as natural product molecules, or as synthetic modifications, particularly for chemotherapeutic treatment of cancer and malaria. Aspects of biotechnology, traditional medicines and herbal medicinal products are briefly discussed.
Keywords: Phytochemistry; Pharmacognosy; Natural products chemistry; Biological activities; Medicinal plants; Drug discovery;

Phytochemicals: The good, the bad and the ugly? by Russell J. Molyneux; Stephen T. Lee; Dale R. Gardner; Kip E. Panter; Lynn F. James (2973-2985).
The perception of phytochemicals can be good or bad, depending on viewpoint. This review describes phytochemicals isolated from poisonous plants of the genera Taxus, Veratrum, Lupinus, Swainsona, Astragalus, Oxytropis, Castanospermum and Ipomoea, some of which are currently in clinical use, others that have been used to produce animal models of human disease, and even more that have shown potential for development into drug candidates.Phytochemicals are constitutive metabolites that enable plants to overcome temporary or continuous threats integral to their environment, while also controlling essential functions of growth and reproduction. All of these roles are generally advantageous to the producing organisms but the inherent biological activity of such constituents often causes dramatic adverse consequences in other organisms that may be exposed to them. Nevertheless, such effects may be the essential indicator of desirable properties, such as therapeutic potential, especially when the mechanism of bioactivity can be delineated. Careful observation of cause and effect, followed by a coordinated approach to identify the responsible entities, has proved extremely fruitful in discovering roles for phytochemical constituents. The process is illustrated by selected examples of plants poisonous to animals and include the steroidal alkaloid toxin of Veratrum californicum (Western false hellebore), piperidine alkaloids of Lupinus species (lupines), and polyhydroxy indolizidine, pyrrolizidine and nortropane alkaloids of Astragalus and Oxytropis species (locoweeds), Castanospermum australe (Moreton Bay chestnut) and Ipomoea species (morning glories).
Keywords: Veratrum californicum; Lupinus spp.; Astragalus spp.; Oxytropis spp.; Castanospermum australe; Ipomoea carnea; Senecio spp.; Glycosidase inhibition; Anticancer; Antiviral;

Nutraceuticals: Facts and fiction by Juan Carlos Espín; María Teresa García-Conesa; Francisco A. Tomás-Barberán (2986-3008).
Nutraceuticals are pharmaceutical forms containing food phytochemicals as active principles. Scientific research supports the biological activity of many of these food phytochemicals, but the health claims attributed to the marketed nutraceuticals have often doubtful scientific foundation. Bioavailability and metabolism are key factors to understand the biological effects of these nutraceuticals.Epidemiological studies show a link between the consumption of plant-derived foods and a range of health benefits. These benefits have been associated, at least partially, to some of the phytochemical constituents, and, in particular, to polyphenols. In the last few years, nutraceuticals have appeared in the market. These are pharmaceutical forms (pills, powders, capsules, vials, etc.) containing food bioactive compounds as active principles. The bioactive phytochemicals have become a very significant source for nutraceutical ingredients. Scientific research supports the biological activity of many of these food phytochemicals, but the health claims attributed to the final marketed nutraceutical products have often little or doubtful scientific foundation. This is due to the fact that a lot of the scientific evidence is derived from animal testing and in vitro assays, whereas human clinical trials are scarce and inconclusive. Some key issues such as bioavailability, metabolism, dose/response and toxicity of these food bioactive compounds or the nutraceuticals themselves have not been well established yet. Amongst the phytochemicals, several groups of polyphenols (anthocyanins, proanthocyanidins, flavanones, isoflavones, resveratrol and ellagic acid) are currently used in the nutraceutical industry. In this report, we have reviewed the most recent scientific knowledge on the bioavailability and biological activity of these polyphenols (‘fact’), as well as the health claims (which are not always supported by scientific studies) ascribed to the polyphenols-containing nutraceuticals (‘fiction’). The in vitro antioxidant capacity, often used as a claim, can be irrelevant in terms of in vivo antioxidant effects. Bioavailability, metabolism, and tissue distribution of these polyphenols in humans are key factors that need to be clearly established in association to the biological effects of these polyphenols-containing nutraceuticals. The future trends of phytochemistry research regarding nutraceuticals are discussed.
Keywords: Phenolic compounds; Polyphenols; Anthocyanins; Flavanones; Isoflavones; Ellagitannins; Ellagic acid; Resveratrol; Proanthocyanidins; Procyanidins; Flavan-3-ols; Biological activity; Bioavailability; Nutraceuticals; Marketing; Health;