Proceedings of the First Workshop of the Italian Working Group on Ecology of Plant Terpenoids

Rome (Montelibretti) Research Area
National Research Council
July16, 1998
Rome Italy

Ecological roles of terpenoid mixtures in conifers

Marco Michelozzi
Istituto Miglioramento Genetico delle Piante Forestali, CNR, Firenze, Italy

Terpenoids are the largest family of plant metabolites with the skeletons consisting of different numbers of five-carbon isoprene units joined together. All plants produce terpenoids, some of which are primary metabolites including those found in chlorophyll, steroids, carotenoids, and growth regulators such as abscissic acid and gibberelline. Isoprenoids classified as secondary compounds include monoterpenoids (C10), sesquiterpenoids (C15) and diterpenoids (C20) that are constituents of essential oils and resin. Recently, these secondary metabolites have received great attention regarding their multiple contributing effects on community and ecosystem properties. Terpenoids are involved in mediation of vital interactions viz., plant-plant, plant-pathogens, plant-mammal herbivores, insect-insect and plant-insects. Terpenoids may also be recycled into primary compounds after serving in the primary role of defence substances. Terpenoids released in the soil can affect the soil microbial community and they can produce phytotoxic effects regulating patterns of plant populations within communities. Volatile terpenes can influence the tropospheric chemistry and isoprene and monoterpenes can contribute to ozone formation.
Conifers are prolific producers of oleoresin that can be constitutive and induced or newly synthesised in response to biotic stresses. The relative proportions (percentages) of constitutive monoterpenes in mature tissues (which make up the constitutive monoterpene profile or the constitutive mixture) are strongly inherited and analyses of constitutive monoterpenoid mixtures have found many applications in chemotaxonomy. For example, A. alba Mill. and A. nebrodensis (Lojac.) Mattei were clearly identified into distinct taxonomic groupings based on monoterpene composition in agreement with allozyme, DNA markers, and sequence data. A strong variation was evident among P. halepensis Mill., Pinus brutia Ten. and their artificial hybrid P. brutia x P. halepensis. Variations in the constitutive mixture of monoterpenoids have important consequences in the chemical defence of plants since monoterpenoids show antimicrobial, fungistatic and insecticidal properties and act as antifeedant deterrents to mammalians herbivores. Certain constitutive monoterpene profiles in slash pine (Pinus elliottii Engelm. var. elliottii), loblolly pine (Pinus taeda L.) clones and jack pine families (Pinus banksiana Lamb.) tended to be less susceptible to fusiform rust than other chemotypes. In addition, induced defence chemistry plays an important role in tree survival; in general, the largest proportionate accumulation in the most toxic monoterpenes can occur in response to attacks by insects and fungi. Significance of constitutive and induced terpenoid defences in conifer resistance is briefly presented. A better knowledge of the mechanism of action of individual terpenes and about the combined (additive and synergistic) effects of compounds, will have important implications for forest and agricultural management of pests and pathogens.
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Possibilities to utilise species from the Italian forest biomass for the production of essential oils

Veriano Vidrich
Dipartimento di Scienza del Suolo e Nutrizione della Pianta, Università degli Studi di Firenze, Italy

Resins and their components, essential oils, have been known to man since the beginning of time. Their use has always ranged from uses as glues and medicines, to the production of perfumes and liqueurs, to the fabrication of paints and liqueurs, and for wartime uses.
At the present time, many components of essential oils are produced by synthesis but we are currently witnessing an increasing requests for natural compounds extracted from various plants since the organolettic characteristics of natural oils are better and different from those of stoichiometric mixtures of the same synthesised components. This fact is particularly true for the production of liqueurs and perfumes and also for herbalist applications (aromatherapy).
In Italy there is considerable presence of indigenous and/or allogenic species which are or can be used for the production of essential oils. This type of industry is not very developed except for small “workshop” as handicraft. Our country is the largest exporter of Juniperus communis L. berries, the most precious berries for the production of liqueur. In Alto Adige and Trentino small handicraft workshops exist near Masi where essential oils are produced from the branches of logged or thinned Pinus cembra L., Pinus sylvestris L., J. Communis etc. In the wide variety of indigenous and allogenic plant species, an industrial use of Mirtus communis L., Eucalyptus sp., and Pinus genus can be foreseen.
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The use of terpenes in the pharmaceutical and cosmetic fields

Franco F. Vincieri President, Italian Society of Phytochemistry
The use of terpenes, or better their natural blends which are essential oils, has been a part of man’’ existence since the beginning of history: Indeed, their use has developed along with man and the documentation of this development coincides with the earliest expressions in ancient texts. Their use, as described in these texts, was prevalently tied to sacred cults in that the priests of the time played the role of physicians and wizards, as well.
In more recent times, two dates have determined historical changes in the use of essential oils: 1930 when Rideal and Walker defined The Phenolic Index, comparing the antimicrobal action of essential oils with that of phenol and 1977 when Paul Belalche established of the Aromatic Index.
Thus, it was possible to begin a rational study of the use of essential oils in therapies with the objective of obtaining phytotherapics which, like all drugs, have characteristics of both safety and efficacy. Another cornerstone in the validity of a medicament is quality: also in the case of essential oils it must be accurately established and controlled. This involves, in addition to a simple quali-quantitative analysis of the essential oil components, an exact determination of the plant and of the drug provenance as well as the balsamic time, system of extraction and the absence of particular impurities and/or contaminants.
With quality controlled essential oils it is possible to intervene in various pathologies such as cutaneous infections, respiratory tract diseases, digestive and urinary tract infections and vaginal infections. Some essential oils are used also as revulsants and other as flavouring and currently the use of some oils as modifiers for drug transdermal penetration is being carefully evaluated.
The evolution of pharmaceutical technology has led to the use of new and sophisticated systems of essential oil extraction such as supercritical carbon dioxide, and also to new formulations such as microencapsulation and microinclusion in cyclodextrins. Thus, it is possible to have stable and bioavailable products of high quality.
In the cosmetic field, essential oils are used in ointments, gel emulsions and lotions, as well as in powders as flavouring, perfums, lenitive stimulators, dermotonifiers and deodorants.
Essential oils, therefore, have great potential applications in both the pharmaceutical and cosmetic fields, and the ever more sophisticated systems for obtainment, formulation and quality control will allow them in the future to be used even more rationally.
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International regulations for natural products used as food additives

Enrico Casadei Food and Nutrition Division FAO, Roma, Italy
Foods moving in international trade are subject to a variety of constraints, including basic food quality and safety laws and regulations. These are officially applied by importing countries to protect consumers, to ensure fair food trading practices and to prevent commercial fraud.
Food control agencies of importing countries generally apply regulations which give them authority over such things as food safety, hygiene, quality, packaging, labelling, handling and storage. In general, these regulations include precise requirements, which must be met if food products are to be admitted into the importing country. For example, regulations often indicate levels of contaminants (microbiological, agricultural and veterinary, environmental and radioactive) and levels of additives that must not be exceeded. These are often referred to as sanitary requirements. Other regulations, which are commonly referred to as quality requirements, include parameters concerning the essential composition, labelling and description of foods. Food products, which do not comply with these requirements, will often result in their rejection or detention.
While the need to protect consumers from health hazards and deception is beyond question, the potential for applying national regulations in an inequitable or discriminatory manner is ever present. The application of such inequitable or discriminatory practices amounts to non-tariff technical barriers, which impede, rather than facilitate, international trade in foods.
The importance of non-tariff technical barriers to trade in impeding international trade in foods is recognised by the major food exporting and importing nations. The Codex Alimentarius Commission, which is administered by the Joint FAO/WHO Food Standards Programme, was established in part in response to the potential for the application of such non-tariff barriers to trade. The work of the Commission has been specifically recognised under the World Trade Organisation (WTO) Agreements on Sanitary and Phytosanitary Measures and on Technical Barriers to Trade.
In undertaking its work on the establishment of maximum levels for food additives in foods, the Commission relies on the use of independent scientific advice provided by FAO and WHO through the Joint FAO/WHO Expert Committee on Food Additives (JECFA). For almost 40 years, the recommendations of JECFA have formed the essential basis for countries to judge the acceptability and safety of these compounds and have set the parameters for fruitful intergovernmental Codex discussions on additives. Similar advice has been provided on an ad hoc basis by FAO and WHO in the areas of Contaminants (including radionuclides), Food Hygiene, Nutrition, Analytical Methods, Protein Quality Evaluation and Labelling.
The use of food additive is regulated at international level by Codex standards. Only food additives, which have been evaluated by JECFA and found acceptable for use in foods, are included in the Codex General Standard and are permitted for use in foods. Food additives are only included in Codex standard when the substance does not present any risk to the health of consumer at the levels of use proposed. Acceptable Daily Intake, or equivalent assessment, established for the additives and its probable daily intake from all sources are taken into account before the inclusion of food additives in Codex standards.
Food additives used in accordance with the Codex General standard, should be of appropriate food grade quality and should at all times conform with the applicable Specifications of Identity and Purity recommended by the Codex Alimentarius Commission or, in the absence of such specifications, with appropriate specifications developed by responsible national or international bodies.
Food additives are classified according their functional class but can be distinguished between natural and synthetic products. The division between natural and synthetic food additives can not be considered separated by a net mark, because many natural products are produced synthetically and many synthetic products are produced modifying natural products or using for their production biological systems such as fermentation.
One of the main difference between natural and synthetic food additives consists in the fact that for synthetic products it is quit easy to establish Specifications of Identity and Purity while for natural products it is more elaborate to establish such specifications due to the complex nature of the products and to some differences depending by areas of production, climatic and soil conditions and sources which can influence notably the composition of the product.
More than 500 substances have been evaluated and provided with specifications for purity and identity by JECFA. Specifications of food additives are intended to serve as a guide for manufacturers and users of the additives, as well as the basis for new or revised national legislation or regulations of member countries of FAO and WHO.
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Isoprenoids as protective compounds against biotic and abiotic stresses in Mediterranean and boreal forests

Francesco Loreto (1) and Sebastiano Delfine (2) (1) Istituto di Biochimica ed ecofisiologia vegetali, CNR, Roma, ITALY –(2) Dipartimento di Scienze Animali, Vegetali e dell’Ambiente, Università del Molise, Campobasso, ITALY
Isoprenoid emission is widespread in forests and may lead to high concentrations of isoprenoid in air. This, in turn, may contribute to chemical processes leading to the accumulation of stratospheric ozone and of tropospheric trace-gases. Plants that store high amount of isoprenoids in specialised organs (ducts, glands) may use them to protect themselves against pathogenes. Plants that have no reservoirs, mainly emit isoprenoids. The reason of the emission, an apparent loss of carbon, is unknown. Water stress stimulates isoprene emission by leaves, and isoprene or monoterpenes fumigation protects leaves against high temperature damages. These results suggest that isoprenoids are involved in the protection of photosynthetic membranes. Alternatively, isoprenoids may serve to remove radicals which are formed when oxidative agents are dissolved in the cellular membranes. Irrespective of the rightness of these mechanicistic hypotheses, isoprenoids seem to constitute an important defence from oxidative damages. As such, they may have a relevant role in protecting against ozone stress. To study the protective functions of isoprenoid against biotic and abiotic stresses, the biochemistry and the physiology underlying their formation has to be unravelled. This is now possible because new methods have been developed to accurately measure the emission and the internal content of isoprenoids in leaves and other plant organs. A study is proposed to investigate the synthesis and emission of isoprenoids by Mediterranean and boreal forests in relation to stress conditions.
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Role of biogenic VOC in photochemical smog formation

Paolo Ciccioli Istituto sull’Inquinamento Atmosferico, CNR, Roma, ITALY
Isoprene, monoterpenes and sesquiterpenes are emitted by plants in substantial amounts. It has been estimated that emission of biogenic VOC exceeds at a global scale that of anthropogenic components released by man-made activities. The reason why plants do emit these components is not clear yet although it has been demonstrated that they play a role in the protection of membranes from heat stress and in the defence from animals and parasites. In any case, the reactivity of isoprenoids in air is quite high as they react with ozone, OH radicals and NO3 radicals at rates comparable to those of the most reactive anthropogenic compounds. If enough NOx are present in rural and remote sites, biogenic VOC can produce ozone and OH radicals in the atmosphere by keeping alive photochemical smog processes in areas where direct pollution is low. The limited knowledge on the emission rates, transport, reactivity and deposition processes of biogenic VOC has prevented a precise assessment of ozone production at meso- and global scale levels. Without a detailed knowledge of these processes it is not possible to define proper strategies to control ozone through the reduction of VOC, NOx emissions. In this presentation, the main processes leading to photochemical ozone formation will be reviewed by emphasising how ozone production depends upon the content of VOC/NOx ratios and the nature of the emissions. It will be also discussed what the present knowledge of biogenic VOC emission is and what is the direction where research is concentrating efforts to evaluate emission rates and fluxes. Examples of the activities carried out in Europe in this field will be also presented.
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Terpenoid emission of Mediterranean oaks and their relation to taxonomy

Olav Csiky and Guenther Seufert European Commission, Joint Research Centre, Environment Institute, Ispra (Varese), Italy
We presented results of a laboratory screening study of biogenic emissions from Mediterranean oak species. The experiment aimed at improving our understanding of oak contributions to overall emissions of volatile organic compounds and to the atmospheric chemistry in the Mediterranean area. We measured type and amount of terpenoid emissions (isoprene, mono- and sesquiterpenes) from 14 different Quercus species of Mediterranean origin under standard conditions of light and temperature. Tree saplings were exposed in a controlled environment chamber, leaf level trace gas exchange was analysed with the help of a minicuvette system and of gas chromatography. We also studied the seasonal fluctuations of the emission factors over the year from one species. At the same time we compared the relation between the emission types and emission spectra found and the Quercus taxonomy. The holarctic group Lepidobalanus and the North American groups Erythrobalanus and Protobalanus were found to be strong isoprene emitters. The Eurasian Sclerophyllodrys oaks emit monoterpenes, Cerris oaks include mostly non-emitters, but also an isoprene and a monoterpene emitter has been found in this group. Results are discussed with respect to their implications for presently used emission scenarios.
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The impact of an elevated atmospheric CO2 on biogenic trace gas emissions

Carlo Calfapietra and Paolo De Angelis Dipartimento Scienze dell’Ambiente Forestale e delle sue Risorse, Università della Tuscia, Viterbo, Italy
The growing pressure of human activities on ecosystems, is modifying the structure and functioning of biosphere. These human driven “global changes” are progressively assessed by the scientific community, in the context of International Panel of experts (Houghton et al., 1995).
The increase of atmospheric concentration of carbon dioxide affects both the energy balance of planet, because CO2 is a greenhouse gas, and the primary production of ecosystems, because is also a source of carbon for photosynthesis.
The complex interaction between the increasing carbon dioxide and the air quality of the troposphere is related, also, with the secondary metabolism of plants. In fact, the emission of reactive organic compound as isoprene may be affected by an increased atmospheric concentration of CO2.
To study the impact of a doubled atmospheric [CO2] on a natural forest community, clump of vegetation were enclosed in large Open Top Chamber for several years (De Angelis, 1998). Preliminary measurements on VOC emission were conducted on Q. ilex trees at ambient and doubled [CO2].
Under limited water availability, as typical in summer for this ecosystems, the VOC emission was lower than normally reported for this species and without any evident effect of elevated CO2. Photosynthetic rate and stomatal conductance were in both treatments close to zero, during all the day.
Seasonal variation of physiological traits and of VOC emission will be further investigated in this site, in the context of an European research project: “ECOVOC”.
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Use of semiochemicals in agriculture

Giuseppe Rotundo, Antonio De Cristofaro and Salvatore Germinara Dipartimento di Scienze Animali, Vegetali e dell’Ambiente, Università del Molise, Campobasso, ITALY
Our research group is mainly involved in the study of intraspecific (sexes) and interspecific (plant-insect; plant-insect-parasitoid) communication to set up new control methods of insect pests.
Our group acquired experience in insect rearing, isolation and extraction (SPME, solvents, adsorbents, from air and pheromones glands) of sex pheromones, purification (on column and thin layer GC), electrophisiology (EAD, EAG, SCR), behaviour (olfactometry, wind-tunnel), laboratory analysis (GC, GC-MS, GC-EAG) and microanalysis.
The identified and synthesised compounds, in the proper dispensers and traps, are tested in laboratory conditions and open field for their practical use (monitoring and mating disruption).
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Volatile organic compounds emission from flowers of fruit trees

Rita Baraldi (1), Francesca Rapparini (1), Federica Rossi (1) and Paolo Ciccioli (2) (1) Istituto di Ecofisiologia delle Piante Arboree da Frutto, CNR, Bologna, ITALY – (2) Istituto di Inquinamento Atmosferico, CNR, Roma, ITALY
The emission of volatile organic compounds (VOCs) from different species of fruit trees has been investigated at blooming stage. Measurements were made on three years old potted plants incubated in a purpose-built growth chamber. After a total incubation time of 16 h, the headspace air of the Teflon chamber was sampled on absorbent traps and analysed by thermaldesorption gaschromatography mass spectrometry. The tested species showed their own distinct monoterpene emission fingerprint. Differences in the emission occurred mainly between pome and stone fruit plants. These preliminary results provide evidence of a species-dependent emission pattern of VOCs.
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Trends in pharmacological and phytotherapeutical researches for new active terpenoids

Andrea Pieroni
Dipartimento di Scienza del Suolo e Nutrizione della Pianta, Università degli Studi di Firenze, ITALY
If antiseptic, spasmolytic and sedative properties of terpenoids mixtures (mostly monoterpenes and sesquiterpenes), which are contained in essential oils, are since long well-known, pharmacological investigations were especially directed in the last three years to study the rule in human biological systems of more complex terpenoids, such as iridoids and diterpenes. In particular analgesic and anti-arthritic properties of procumbide and harpagoside from devil’s claw (Herpagophytum procumbens), endemic species of south-west Afrika, where clearly demonstrate and the first pharmaceutical preparations are entering in the European market. Recently also oleuropein from olive trees (Olea europaea) and its metabolites demonstrated inhibit angiotensin converting enzyme (ACE), what definitively confirmed the hypotensive properties of the plants, which were known since centuries in the Mediterranean popular medicine. Diterpenoid quinones from the Chinese Salvia miltiorrhiza showed to play a central role in the positive effects of the plant in threating myocardial ischemia (also in this case confirming an old prescription of the traditional Chinese medicine), while a certain success accompanied the first effective data after that two pharmaceutical products based on taxolderivates are commonly available in anti-cancer therapy in USA and Europe. On the other hand, further pharmacological and clinical studies are in progress about other diterpenes as phorbolesters from Euphorbia tirucalli, which is well know in Brazil as anti-cancer in some popular preparations (“Aveloz”).
Moreover, aucubin and agnuside form chaste tree (Vitex agnus castus) showed to be able to inhibit prolactine secretion and plant extracts gave arise positive results in patients with various premenstrual problems (the traditional use of the aerial parts and seeds of the plants as men anaphrodisiac in Middle Age, especially in cloisters, seems to find exact pharmacological confirms). At the same time the industrial interest (especially in Japanese and American companies) for the sweetener stevioside (200 times more potent than glucose) extracted from Stevia rabaudiana grows remarkably.
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