teins, chlorophyll, nucleic acids, etc. are common to all plants and are .... bottom caftaric acid, chlorogenic acid, cichoric acid, cafeic acid, echina- coside and total ...
AFINIDAD REVISTA DE QUÍMICA TEÓRICA Y APLICADA EDITADA POR LA ASOCIACIÓN DE QUÍMICOS E INGENIEROS DEL INSTITUTO QUÍMICO DE SARRIÁ
Effects of atmospheric carbon dioxide fertilization on biomass and secondary metabolites of some plant species with pharmacological interest under greenhouse conditions (1, 3)
Savé, R.*
(1, 3)
, de Herralde, F.
(4)
(2, 3)
, Codina, C. Sánchez, X.
, Biel, C.
(1, 3)
(1)
IRTA, Centre de Cabrils, Ctra. de Cabrils, Km 2. E-08348 Cabrils (Barcelona). Spain MATGAS. Campus Universidad Autonoma de Barcelona. E-08193 Cerdanyola (Barcelona). Spain (3) agriGas. Joint Research Unit between IRTA and MATGAS. Ctra. de Cabrils, Km 2. E-08348 Cabrils (Barcelona). Spain (4) Department of Natural Products, Plant Biology and Edaphology. Pharmacy Faculty. University of Barcelona. Av. Joan XXIII, s/n. 08028 Barcelona (Spain) (2)
Efectos de la fertilización carbónica en la producción de biomasa y metabolitos secundarios en algunas plantas con interés farmacológico en condiciones de invernadero. Efectes de la fertilització carbònica en la producció de biomassa i metabòlits secundaris en algunes plantes amb interès farmacològic en condicions d’hivernacle. Recibido: 22 de enero de 2007; aceptado: 24 de abril de 2007
We want to say thanks to Dr. Joan Juli Bonet because he helped to promote the joint research unit “agriGas”, maintained the doubt about a lot of things as a source to maintain the spirit and increase the quality of this kind of job called research. Also we want say thanks for the possibility to meet him.
Afinidad (2007), 64 (528), 237-241
Effects of atmospheric carbon dioxide fertilization on biomass and secondary metabolites of some plant species with pharmacological interest under greenhouse conditions (1, 3)
Savé, R.*
(1, 3)
, de Herralde, F.
(4)
(2, 3)
, Codina, C. Sánchez, X.
, Biel, C.
(1, 3)
(1)
IRTA, Centre de Cabrils, Ctra. de Cabrils, Km 2. E-08348 Cabrils (Barcelona). Spain MATGAS. Campus Universidad Autonoma de Barcelona. E-08193 Cerdanyola (Barcelona). Spain (3) agriGas. Joint Research Unit between IRTA and MATGAS. Ctra. de Cabrils, Km 2. E-08348 Cabrils (Barcelona). Spain (4) Department of Natural Products, Plant Biology and Edaphology. Pharmacy Faculty. University of Barcelona. Av. Joan XXIII, s/n. 08028 Barcelona (Spain) (2)
Efectos de la fertilización carbónica en la producción de biomasa y metabolitos secundarios en algunas plantas con interés farmacológico en condiciones de invernadero. Efectes de la fertilització carbònica en la producció de biomassa i metabòlits secundaris en algunes plantes amb interès farmacològic en condicions d’hivernacle. Recibido: 22 de enero de 2007; aceptado: 24 de abril de 2007
We want to say thanks to Dr. Joan Juli Bonet because he helped to promote the joint research unit “agriGas”, maintained the doubt about a lot of things as a source to maintain the spirit and increase the quality of this kind of job called research. Also we want say thanks for the possibility to meet him.
RESUMEN
SUMMARY
El aumento del CO2 atmosférico debido al cambio global y/o a las prácticas hortícolas promueve efectos directos sobre crecimiento vegetal y el desarrollo. Estas respuestas pueden ocurrir en ecosistemas naturales, pero también se pueden utilizar para aumentar la producción de algunas plantas y de algunos compuestos secundarios. El actual trabajo intenta estudiar los efectos del enriquecimiento atmosférico del CO2 bajo condiciones de invernadero en el crecimiento y la concentración y la composición de metabolitos secundarios de Taxus bacatta, Hypericum perforatum y Echinacea purpurea en condiciones ambientales mediterráneas. La fertilización del CO2 muestra perspectivas interesantes para la mejorara y aplicabilidad de técnicas hortícolas para aumentar productividad de plantas medicinales, a pesar de diferencias claras entre la especie. En general esta técnica promueve aumentos importantes y significativos en producción primaria y, en algunos casos, también en compuestos secundarios. Esto tiene una gran importancia hortícola porque la productividad a nivel de cosecha total aumenta, directamente porque se aumenta la concentración e indirectamente porque se aumenta la biomasa.
The increase of atmospheric CO2 due to global change and/or horticultural practices promotes direct effects on plant growth and development. These responses may occur in natural ecosystems, but also can be used to increase the production of some plants and some secondary compounds. Present work tries to study the effects of atmospheric CO2 enrichment under greenhouse conditions on growth and in the concentration and composition of secondary metabolites of Taxus bacatta, Hypericum perforatum and Echinacea purpurea under Mediterranean environmental conditions. CO2 fertilization shows interesting perspectives to increase and improve horticultural techniques in order to increase plant medicinal productivity, in spite of clear differences among the species. In general this technique promotes important and significant increases in primary productivity and, in some cases, also in secondary compounds. This has a great horticultural relevance because the total productivity of this kind of products increase at crop level, directly because concentration is increased and /or indirectly because biomass is increased.
Palabras clave: Psicología de la planta. Taxus bacatta. Hypericum perforatum. Echinacea purpurea. Taxol. Flavonoides. Fenólicos totales.
Key words: Plant physiology. Taxus bacatta. Hypericum perforatum. Echinacea purpurea. Taxol. Flavonoids. Total phenolics.
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RESUM L'augment del CO2 atmosfèric a causa del canvi global i/o a les pràctiques hortícoles promou efectes directes sobre creixement vegetal i el desenvolupament. Aquestes respostes poden ocórrer en ecosistemes naturals, però també es poden utilitzar per a augmentar la producció d'algunes plantes i d'alguns compostos secundaris. L'actual treball intenta estudiar els efectes de l'enriquiment atmosfèric del CO2 sota condicions d'hivernacle en el creixement i la concentració i la composició de metabòlits secundaris de Taxus bacatta, Hypericum perforatum i Echinacea purpurea en condicions ambientals mediterrànies. La fertilització del CO2 mostra perspectives interessants per a la millora i aplicabilitat de tècniques hortícoles per a augmentar productivitat de plantes medicinals, a pesar de diferències clares entre l'espècie. En general aquesta tècnica promou augments importants i significatius en producció primària i, en alguns casos, també en compostos secundaris. Això té una gran importància hortícola perquè la productivitat a nivell de collita total augmenta, directament perquè s'augmenta la concentració i indirectament perquè s'augmenta la biomassa.
have a vital biochemical role in the process of building and maintaining plant cells. Recent research has shown a pivotal role of these chemicals in the ecophysiology of plants, in the relationships and interactions between plants and biotic and /or abiotic environment (Mooney et al. 1991).
OBJECTIVE Present work tries to study the effects of atmospheric CO2 enrichment under greenhouse conditions on growth and on the composition of secondary metabolites of Taxus bacatta, Hypericum perforatum and Echinacea purpurea under Mediterranean environmental conditions. This basic objective could be the first step in order to improve the horticultural technology for the production of this kind of metabolites, medical drugs.
MATERIAL AND METHODS Plant material. Assays were developed on seedlings of:
Mots clau: Psicologia de la planta. Taxus bacatta. Hypericum perforatum. Echinacea purpurea. Taxol. Flavonoids. Fenòlics totals.
Taxus bacatta, is a species that contains taxol that it is a chemotherapy drug that is given as a treatment for ovarian, breast and non-small cell lung cancer (Heinstein and Chang, 1994).
INTRODUCTION
Hypericum perforatum, of which leaves and flowers of are used for depression treatments and has its origins in the medical traditions of Europe well before the 1600’s (Upton, 1998).
The increase of atmospheric CO2 due to global change and/or horticultural practices promotes direct effects on plant growth and development (Bazzaz, 1990). The responses observed in different species show a wide range of patterns either in the biomass production or in the composition of secondary metabolites. These responses may occur in natural ecosystems, but also can be used to increase the production of some plants and some secondary compounds (Llusià et al. 1996; Peñuelas et al. 1996; Marks et al. 2000; Tisserat & Vaughn 2001). During last years there has been an increase in the use of medicinal plants products, which must be attributable to new way of life related to natural products: it is very easy to obtain this kind of products in a wide range of stores; an important number of these plants and their products are deeply introduced in the people’s culture; and there are only very soft rules about their commercial regulation (Brevoort 1998). Also there is an important interest in functional foods, which in some cases show a thin barrier with medicinal plants (Korver 1998). Consequently, along these years and even in these moments, it has been and is being developed a great research activity in phytochemeistry (bioactive compounds detailed and structural analysis), pharmacognosy (potential modes of action, target sites for active compounds) and horticulture (optimal cultivation). Horticultural research focused in this topic is very important because even in these moments an important amount of medicinal plant’s biomass is obtained by wild harvesting, which can promote loss of biodiversity, potential variability in plant’s and compounds quality, errors in plants identification and in some cases indiscriminate use of resources of native people (Briskin 2000). While primary products, such as carbohydrates, lipids, proteins, chlorophyll, nucleic acids, etc. are common to all plants and are involved in the primary metabolic processes of building and maintaining plant cells (Kaufmann et al., 1999; Wink, 1999), plant secondary products have historically been defined as chemicals that do not seem to
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Echinacea purpurea leaves and roots are used to treat colds, flu-like infection and upper respiratory infections (World Health Organization 1999). These three species were grown in a greenhouse located in IRTA (Cabrils, Barcelona, Spain; 2º 30’ E, 41º 45’ N). Each plant grew in 3 L plastic pots filled with peat and perlite (2:1: v:v). The plots were daily watered and fertilized by located irrigation system. The plastic greenhouse was divided in three compartments, each with a different CO2 gas concentration (Carburos Metalicos SA): high (750 ppm ECO2), intermediate (500 ppm ECO2) and ambient or control (350 ppm A CO2). In Taxus besides CO2 treatments were also imposed water different irrigation amounts of water (50 and 100% of ETP0). The minimum temperature was maintained above 12 ºC (Cortes et al. 2004; Biel et al. 2004). Biomass and secondary products composition measurement. Plants harvesting was carried out at the end of the experiment. 8 plants of each CO2 level were chosen randomly and their total biomass was separated into three compartments: leaves, stems and roots, and their dry weight was calculated after drying at 60 ºC (72 h). Leaf area was measured with a LI-COR area meter (LI-COR Model 3100, USA). Taxol, was measured by means of ELISA immunoassay technique (Heinstein and Chang, 1994). Flavonoids of H. perforatum were determined by HPLC at INIA’s lab (Madrid) and E. purpurea secondary metabolites were evaluated by HPLC in the laboratory of Pharmacy Faculty of University of Barcelona. Experimental design and data analysis. T. baccata experiment was designed as a double factorial with 2 irrigation treatment and 3 atmospheric CO2 treatments. H. perforatum was submitted to 3 atmospheric CO2 treatments E. purpurea was only submitted to 2 atmospheric CO2 treatments . The results were analyzed using the SAS system for the means analysis by GLM procedure.
RESULTS AND DISCUSSION Dry weight of leaves, stems and roots of Taxus plants did not change with CO2 or water treatment (Figure 1) while taxol concentration showed significant increase in leaves and bark, and considerably differences in bark concentration at high irrigation rate (Figure 2). This must be due to the plant productivity is focused to sink, as trunks and roots, no photosynthetic organs (Noarby et al. 1992) and consequently relative growth rate is delayed or stopped (Poorter et al. 1990). In Hypericum plants, leaf dry weight was 33% higher in ECO2 than in A CO2 (Figure 3). This difference also appeared in allocation of biomass, roots and stems (Figure 3). Flavonoids composition was affected by phenological stage of plants, so after blossom these were increased in ECO2 plants (Figure 4) (Croteau et al. 2000). Flavonoids are a class of plant secondary metabolites of phenolic nature which play a number of important roles in the interaction of plants with their environment. The concentration of flavonoids and other phenolic compounds in plants varies among organs, tissues and developmental stage, and is influenced by environmental factors. Among such factors are temperature, UV and visible radiation, nutrient and water availabilities, and atmospheric CO2 concentration (Estiarte et al. 1999). Plants of Echinacea purpurea subject to high CO2 conditions after 7 months showed significant differences between treatments, so plants grew in ECO2 had 79%, 339%, 546% and 57% greater dry weight productions of leaves, flowers, stems and roots with respect to the plants grown in ACO2 (Figure 5). ECO2 only promoted significant changes respect to ACO2 in amounts of caftaric acid and total phenols at root level, but there were significant differences in these compounds at leaf level and in cichoric, and echinacoside, where their concentration was approximately 4 times higher at ECO2 than ACO2 (Figure 6). It’s very difficult to give a general explanation about the effects of CO2 on plant productivity because there are involved a lot of environmental and physiological characteris-
Figure 1. Effects of CO2 enrichment and irrigation treatment on biomass allocation of Taxus baccata plants.
Figure 2. Effects of CO2 enrichment and irrigation treatment on taxol concentration in leaves (A) and bark (B) of Taxus baccata plants.
Figure 3. Effects of CO2 enrichment on biomass allocation of Hypericum perforatum plants.
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Figure 4. Effects of CO2 enrichment on flavonoids concentration before and after flowering of Hypericum perforatum plants.
tics that can promote important differences in the final response (Bazzaz, 1990, El-Gengaihi 1998, Briskin 2000). Thus, it seems clear that the increase of CO2 increases primary productivity, but it is more difficult to establish the response of secondary metabolism, above all when the interest is to obtain specific compounds with high pharmacological value (Biel et al. 2003).
Figure 5. Effects of CO2 enrichment on biomass allocation of Echinacea purpurea plants.
CONCLUSIONS CO2 fertilization shows interesting perspectives to improve horticultural techniques to increase medicinal plant productivity, in spite of clear differences among the species. In general, this technique promotes important and significant increases in primary productivity and in some cases also in secondary compounds. This has a great horticultural relevance because the total productivity of this kind of products increases at crop level, directly because concentration is increased and /or indirectly because biomass is increased.
ACKNOWLEDGEMENTS Figure 6. Effects of CO2 enrichment on secondary metabolites concentration of Echinacea purpurea plants. From left to right and from top to bottom caftaric acid, chlorogenic acid, cichoric acid, cafeic acid, echinacoside and total phenolics.
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Authors want thank to P. CABOT, Dra. M.A. CASES, Dra. C. OLIVELLA, S. JOLY, MC. BELLIDO and J. MONTERO for their assistance in field and laboratory tasks.
BIBLIOGRAPHY Bazzaz, F.A.: Annu. Rev. Ecol. Syst. 1990 (21):167-96. Biel, C.; Cristobal, R.; Cases, M.A.; Savé, R. 2003: «Effects of atmospheric carbon dioxide concentration on Thymus zygis and Thymus hyemalis». III World Congress on Medicinal and Aromatic Plants for Human Welfare. Chiang Mai (Tailandia). Biel, C.; R. Savé, A. Habrouk, J.M. Espelta, J. Retana. 2004: «Effects of restricted watering and CO2 enrichment in the morphology and performance after transplanting of nursery grown Pinus nigra seedlings». HortScience 39 (4): 1-6. Blumenthal M., Goldberg A., Brinckmann J., (2000): «Herbal Medicine: Expanded Commission E Monographs». Integrative Medicine Communications, Boston. Briskin, D.P. 2000: «Medicinal plants and phytomedicines. Linking plant biochemistry and physiology to human health». Plant. Physiol. 124:507-514. Cortes, P.; J.M. Espelta, R. Savé & C. Biel. 2004: «Effects of nursery CO2 enriched atmosphere in the germination and seedling morphology of two Mediterranean oaks with contrasting leaf habit». New Forest New Forest 28: 79-88. El-Gehgaihi S.E., Shalaby A.S., Agina E.A., Hendawy S.F. (1998): «Alhylamides of Echinacea sp. purpurea L. as influenced by plant ontogeny and fertilization». J. Herb. Spices Med. Plant. 5:35-41. Estiarte M., J. Peñuelas, J.; Kimball, B.A.; Hendrix, D.L.; Pinter, P.J.; Wall, G.W.; LaMorte, R.L.; Hunsaker, D.J. 1999: «Free-air CO2 enrichment of wheat: leaf flavonoid concentration throughout the growth cycle». Physiol. Plant. 105 (3), 423–433. Fornasiero R.B., Bianchi A., Pinetti A., (1998): «Anatomical and ultrastructural observations in Hypericum perforatum. L. leaves». J. Herb. Spices Med. Plant 5:21-33. Heinstein, P.F. and Chang, C.J. 1994: «Taxol». Annu. Rev. Plant Physiol. Plant Mol. Biol., 45: 663-674. Kaufman P.B., Cseke L.J., Warber S., Duke J.A., Brielmann H.L. (1999): «Natural products from plants». CRC Press Boca Raton, FL. Llusià, J.; Vila, R.; Cañigeral, S.; Biel, C.; Savé, R. and Peñuelas, J. 1996: «The effect of carbon dioxide supply and seasonally on allocation to terpenoids of Rosmarinus officinalis L». Investigación Agraria 11 (1): 33-43.
Mark S.J.: New Phytol. 2000 (146):437-451. Norby, R.J.; O’Neill, E.G.; Luxmoore, R.J.; Oechel, W.C.; Pitelka, L.F., 1991: «Effects of atmospheric CO2 enrichment on the growth and mineral nutrition of Quercus alba seedlings in nutrient-poor soil». Plant Physiol., 82: 83-89. Peñuelas et al.: Jour. Exper. Bot. 1996 Vol 47 (302): 14631467. Poorter, H.; Remkes, C.; Lambers, H., 1990: «Carbon and nitrogen economy of 24 wild species differing in relative growth rate». Plant Physiol. 94: 621-627. Savé, R.; Biel, C. and Sánchez, X. 2001: «Especie vegetal modificada del genero Taxus y procedimiento de obtención de la misma Patente A-149989». España & USA. Titular: Carburos Metálicos - IRTA. Savé, R.; Biel, C. and Sánchez, X. 2001: «Utilización del dióxido de carbono para incrementar la concentración de metabolitos secundarios de una especie vegetal Patente A-1519753». España & USA. Titular: Carburos Metálicos IRTA. Schulz V., Hänsel R., Tyler V. (1998): «Rational Phytotherapy: A Physician’s Guide to Herbal Medicine». Springer-Verlag, Berlin. Tisserat B; Vaughn S.F.: Plant Cell Reports 2001 (20):361368. Tyler V.E., (1999): «Phytomedicines: back to the future». J. Nat. Pro. 62.1589-1592. Upton R. (1998): «St. John’s Wort: Quality Control, Analytical and Therapeutic Monograph». American Herbal Phamacopoeia Compendium, Santa Cruz, CA. Wink M. (1999): «Introduction: biochemistry, role and biotechnology of secondary products». In M. Wink, ed, Biochemistry of Secondary Product Metabolism. CRC Press, Boca Raton, FL, pp 1-16. Wink M., Schimmer O., (1999): «Modes of action of defensive secondary metabolites». In M. Wink, ed, Functions of Plant Secondary metabolites and Their Exploitation in Biotechnology. CRC Press, Boca Raton, FL, pp. 17-112. «World Health Organization Monographs on Selected Medicinal Plants», Vol. 1 (1999). World Health Organization, Geneva.
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