Componentes de instalación de red

Basil (Ocimum basilicum L.) is one of the most important species belonging to the genus Ocimum, in the Lamiaceae family. The genus Ocimum encompasses a huge number of medicinal species and varieties, characterized by a large variability in morphology and habitats, flavours, scents, and uses (Putievsky and Galambosi, 1999). A lot of them are mainly cultivated to be used for culinary preparations. This species includes a large number of varieties and cultivars with distinct morphological traits and chemotypes (Simon et al., 1999), which range from typical green-leaf varieties (Genovese, Lettuce leaf, Gigante) to purple-colored genotypes (Dark Opal, Red Rubin) or lemon-flavoured cultivars (Citriodorum).

Basil is cultivated worldwide, and is also grown hydroponically (Miceli et al., 2003).

Whereas some varieties are used as ornamental plants, basil is mainly used for food preparations (Makri and Kintzios, 2007). The fresh green leaves of some cultivars (sweet basil; for example Genovese) are commonly used for the preparation of the well-known Italian ‘pesto’ sauce, now largely diffused all over the world (Miele et al., 2001).

Basil is also an important source of essential oils and of rosmarinic acid (Kiferle et al., 2011). The essential oils are extensively used in food and pharmaceutical industry, perfumery, cosmetics and herbal medicine (Makri and Kintzios, 2007; Hussain et al., 2008).

The composition and concentration of the essential oils is largely variable in dependence of cultivars and growing conditions. However, linalool, chavicol and methyl-chavicol, eugenol and methyl-eugenol, estragole, methyl-cinnamate, have all been reported as the dominant volatile constituents (Lee et al., 2005; Makri and Kintzios, 2007; Klimánková et al., 2008).

The relative content of each constituent can often enable to differentiate among distinct cultivars (Klimánková et al., 2008).

Rosmarinic acid is one of the most abundant antioxidant phenolic compounds accumulated by basil (Jayasinghe et al., 2003; Li et al., 2007; Makri and Kintzios, 2007;

Juliani et al., 2008; Lee and Scagel 2009). Rosmarinic acid is widely distributed in the plant kingdom, but represents a characteristic secondary metabolite of several medicinal plants (e.g.

Salvia officinalis, Mentha x piperita, Thymus vulgaris, Melissa officinalis) in the Boraginaceae and Lamiaceae families (Petersen and Simmonds, 2003; Petersen et al., 2009).

As a caffeic acid derivative, rosmarinic acid belongs to the class of phenylpropanoids (Kurkin, 2013). The molecule is formally obtained by esterification of the carboxylic group of

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caffeic acid with the alpha hydroxyl group of 3,4-dihydroxyphenyllactic acid. The pure compound was isolated for the first time in Rosmarinus officinalis by Scarpati and Oriente (1958), while the complete biosynthetic pathway from the precursors tyrosine and phenylalanine was fully elucidated 45 years later by Petersen and Simmonds (2003).

Rosmarinic acid is a strong free radical scavenging agent. The antioxidant properties of this secondary metabolite are due to the presence of two couples of hydroxyl groups, each couple being located in the ortho positions of a benzene ring. A large number of additional biological activities have been described for rosmarinic acid: adstringent, anti-inflammatory, anti-mutagen, anti-bacterial and anti-viral properties have been attributed to this compound (Petersen and Simmonds, 2003; Juliani et al., 2008).

Likewise the vast majority of plant secondary metabolites, rosmarinic acid accumulation for a given genotype is strongly affected by many factors, including growing and environmental conditions, phenological stage, plant organ (Del Baño et al., 2003; Juliani et al., 2008; Shiga et al., 2009).

Experiments at the University of Pisa

This section reports a synthesis of the main results obtained in a series of experiments carried out at the University of Pisa (Italy) with the green-leaf basil cultivar Genovese grown in floating system (Kiferle et al., 2011, 2012, 2013). These experiments were aimed at studying the applicability of greenhouse hydroponics to the agro-industrial production of rosmarinic acid (hereafter indicated as RA).

When grown hydroponically, basil plants showed a fast growth and leaf concentration of RA ranged from 4 to 29 mg/g DW (Kiferle et al., 2011). Roots also contained significant concentrations of RA. Although the shoot accounted for more than 90% total dry mass, in principle the whole plant could be processed for RA extraction, as the floating system facilitates also the harvesting of clean root tissues (Kiferle et al., 2011). All the determinations were conducted on non-dehydrated fresh or frozen (-80°C) samples, as desiccation at 70°C was found to reduce the content of RA in basil tissues up to 40% (Kiferle et al., 2011).

Leaf RA concentrations reported in the literature for sweet basil varied from less than 0.1 mg/g DW (Sgherri et al., 2010) to nearly 100 mg/g DW (Javanmardi et al., 2002). This wide range is probably the consequence of differences in plant genotype and growing conditions, or in the method used for the determination of rosmarinic acid.

In some experiments attempts were made to increase the production of RA in sweet basil while maintaining the same biomass production. In particular, the plants were exposed to a moderate NaCl salinity stress, to a moderate hypoxia condition or to a change in N nutrition.

In order to study the effect of salinity, a control treatment and two different saline treatments were compared. In the latter treatments, proper amounts of NaCl were added to the control nutrient solution (Kiferle et al., 2012). Although the response to saline stress may change in different cultivars (Attia et al., 2011; Omer et al., 2008), the cultivar Genovese resulted moderately tolerant to a NaCl-induced salinity stress (Kiferle et al., 2012). In agreement with these results, other authors found that NaCl concentrations up to 50 mM did not affect the growth of sweet basil grown in water culture (Attia et al., 2009; Tarchoune et al., 2009, 2010). In contrast, Bernstein et al. (2010) reported that NaCl salinity reduced

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Hydroponic Production of Medicinal Plants 105

significantly root and shoot growth in hydroponically-grown sweet basil, especially at concentrations higher than 50 mM. The content of RA in leaf tissues resulted unaffected by NaCl salinity (Kiferle et al., 2012). This result was in disagreement with those observed under slightly different growing conditions by other authors (Tarchoune et al., 2009), which found that 50 mM NaCl markedly reduced the leaf concentration of RA (as well as those of caffeic and vanillic acids) in basil cultivar Genovese. In contrast, the root content of RA was found to increase significantly in both saline treatments (Kiferle et al., 2012).

In another experiment, hypoxia conditions were easily induced in the culture by simply disconnecting the aeration of the nutrient solution. The cultivar Genovese appeared moderately tolerant to a moderate hypoxia stress. Hypoxia did not affect significantly shoot growth, while a marked reduction was observed in root dry weight (Kiferle et al., 2012). This result was in agreement with those of other studies (Drew, 1983; Incrocci et al., 2000; Shi et al., 2007). The root tissues were affected by hypoxia also for the accumulation of RA, whereas the leaf content of this metabolite was not modified by the oxygen level in the nutrient solution (Kiferle et al., 2012). In contrast to these findings, some authors observed an increase in the level of phenolic compounds in plants grown under root hypoxia, for instance in both shoots and roots of Hypericum brasiliense (Nacif de Abreu and Mazzafera, 2005) and in the stems of Eucalyptus marginata (Burgess et al., 1999). In the latter work, the increase in the concentration of phenolic compounds was also linked to the increased activity of some enzymes involved in their biosynthesis, such as phenylalanine ammonia lyase, 4-coumarate coenzyme A ligase and cinnamyl alcohol dehydrogenase.

Two distinct types of experiments were carried out concerning the influence of N nutrition. In the first one, N was entirely supplied as NO₃^-, at the concentrations of 10, 5 or 0.5 mM (Kiferle et al., 2013). The former concentration is the standard level of NO₃^- that is considered as optimal, and similar concentrations are generally employed in hydroponic cultivation (Pardossi et al., 2006; Sonneveld and Voogt, 2009). In the second experiment, the total N content was kept constant at the optimal concentration value, and the NO₃^-/NH₄⁺ ratio was modified (Kiferle et al., 2013).

Overall, the growth parameters were higher at the optimal concentration of 10 mM, except the root dry matter, which increased at the lowest NO₃^- concentration. This was an expected result, as it is known that N deficiency inhibits shoot growth while stimulating root growth (Clarkson, 1985), because this adaptive mechanism enhances the plant’s ability to absorb nutritive ions from the growing medium. In a similar way, several growth parameters indicated that the supply of NH4+, alone or in mixture with NO₃^-, had a marked detrimental effect on plant growth (Kiferle et al., 2013). In contrast with these findings, in pot-grown sweet basil the biomass production resulted unaffected by the use of salt fertilizers containing NO₃^- or NH₄⁺ (Tesi, 1995; Adler et al., 1989).

The decrease in NO₃^- concentration increased the content of RA in the tissues (Kiferle et al., 2013). In agreement with this outcome, an increase in leaf RA content of sweet basil grown under limited N availability was reported by Nguyen and Niemeyer (2008). The concentration of RA was affected also by the N form. In leaf tissues, the presence of NH₄⁺ ion had the undesired effect of decreasing the level of rosmarinic acid, even in the presence of NO₃^- (Kiferle et al., 2013).

The following table (Table 1) summarizes the main effects that were determined in the leaf tissues of sweet basil cultivar Genovese by a change in the composition of the nutrient solution (Kiferle et al., 2012, 2013).

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Table 1. Effect of modifications of the nutrient solution on biomass production and leaf content of rosmarinic acid in sweet basil (Ocimum basilicum L.), cultivar Genovese.

The symbols + and ¯ indicate higher or lower values than those obtained under standard growing conditions while the letters ‘ns’ indicate no significant change. DW: dry weight.

See text for details

Salinity^a

(NaCl addition)

Low N level^b (N as NO3-)

NH4+ addition^b (constant total N)

Hypoxia^a

Dry biomass (g/plant) ns ns _ ns

Rosmarinic acid (mg /g DW) ns + _ ns

a Kiferle et al., 2012.

b Kiferle et al., 2013.

Overall, salinity or hypoxia did not have a significant effect either on the leaf biomass production or on the content of RA, which remained unchanged in leaf tissues. On the other hand, a clearly detrimental effect was observed for both growth and RA production in the presence of NH₄⁺. Thus, the use of this ion in the nutrient solution should be avoided for hydroponic cultivation of sweet basil. The best results were provided by a decrease in the level of NO₃^- supplied to the plants, compared to the standard concentrations generally used in hydroponic culture (Pardossi et al., 2006; Sonneveld and Voogt, 2009). When plants were grown with a NO₃^- concentration of 5 mM, leaf and total RA content was significantly greater than at 10 mM, the typical concentration of hydroponic nutrient solutions.

All these findings suggested the potential of greenhouse hydroponic culture of sweet basil for the agro-industrial production of RA, as a large amount of biomass with a high concentration of this antioxidant compound could be produced in a few weeks. The concentration of RA could be further increased by a proper change in the composition of the nutrient solution, specifically by a decrease in the NO3- level compared to the typical concentration of hydroponic nutrient solutions (10 mM or higher).

The above reported results also have some important operative and environmental implications, as they suggest that poor quality (i.e. moderately saline) irrigation water can be used in water culture of sweet basil and that the aeration of nutrient solution is not a crucial factor for optimal plant growth and RA production of this species. Furthermore, the reduction of NO₃^- concentration in the culture solution results in lower environmental impact, as less N fertilisers are applied and the leaching of NO₃^- with nutrient solution discharge is limited.

A further outcome of the experiments described in this section was that different basil genotypes accumulated different amounts of RA (Kiferle et al., 2011) and that, as a consequence, cultivar selection is recommended for production improvement.

Conclusion

Greenhouse hydroponic technology is currently applied to the commercial-scale production of fresh or minimally-processed herbs (including basil) for the vegetables market.

This well-known and commonly employed technology could be easily applied also to the production of biomass for the extraction of bioactive molecules.

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Hydroponic Production of Medicinal Plants 107

The production efficiency of this growing system could be further improved by accurate variety selection, as the content of bioactive compounds in medicinal plants is strongly dependent on the genotype.

It is generally acknowledged that greenhouse hydroponic cultivation is a profitable system for medicinal plants production in terms of biomass yield and quality of the raw material, which is clean and easy to be harvested and processed. A low-cost greenhouse hydroponic system such as the floating raft system, which has found actual commercial application for the production of high density, short cycle leafy vegetables, may also result economically profitable, especially if the species to be grown are selected both for their economic value and bio-active properties.

With greenhouse hydroponics, the cultural cycle can be sensibly shortened. While this is an evident advantage for biomass production, it may be a limiting factor for the synthesis and accumulation of sufficient amounts of bioactive substances in the tissues, as we found in Echinacea angustifolia (Maggini et al., 2012).

On the other hand, we found that the floating raft system provided a suitable growing method for the agro-industrial production of RA from basil (Kiferle et al., 2011, 2012, 2013).

The greenhouse hydroponic growing of this species may be considered as a model system for the production of plant material for the extraction of bioactive compounds, as a large amount of biomass with high concentration of bioactive compound could be produced in few weeks.

In addition, proper manipulation of the characteristics of the nutrient solution (e.g. N concentration) may increase the production of the metabolite(s) of interest.

In our experiments on basil, the determinations were conducted on fresh or frozen (-80°C) samples, which contained much more RA than oven dried tissues (Kiferle et al., 2011).

Medicinal plant material generally undergoes desiccation, as dried tissues are easier to handle and process. This common post-harvest practice prevents undesired microbial degradation and facilitates storage and transportation to the processing unit. In contrast, a special production scheme is required for the processing of fresh material. In particular, the greenhouses for the cultivation of medicinal plants should be located close to the processing units, a short-term storage should be planned before extraction and suitable cold rooms should be available. In order to evaluate the profitability of this scheme, which resembles the one for the industrial production of fresh-cut vegetables, the overall costs to obtain secondary metabolites from fresh or dry plant material should be compared.

The literature data evidence that there is still a lack of information on the suitable growing practices for medicinal plants production in hydroponics, and suggest that specific cultural protocols should be developed for each species individually.

References

Abeles, FB; Morgan, PW; Saltveit, ME, Jr. Ethylene in plant biology. San Diego, CA, USA:

Academic Press; 1992.

Adler, P. 1989. Nitrogen form alters sweet basil growth and essential oil content and composition. Horticultural Science, 1989, 24, 789-790.

Ahloowalia, BS; Prakash, J. Physical components of tissue culture technology. In: Low cost options for tissue culture technology in developing countries. Proceedings of a Technical

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Meeting organized by the Joint FAO/IAEA Division of Nuclear Techniques in Food and Agriculture and held in Vienna, 26–30 August 2002; 17-29.

Ahloowalia, BS; Savangikar, VA. Low cost options for energy and labour. In: Low cost options for tissue culture technology in developing countries. Proceedings of a Technical Meeting organized by the Joint FAO/IAEA Division of Nuclear Techniques in Food and Agriculture and held in Vienna, 26–30 August 2002; 41-47.

Almeida-Cortez, JS; Shipley, B; Arnason, JT. Do plant species with high relative growth rates have poorer chemical defences? Functional Ecology, 1999, 13, 819–827.

Atanassova, M; Georgieva, S; Ivancheva, K. Total phenolic and total flavonoid contents, antioxidant capacity and biological contaminant in medicinal herbs. Journal of the University of Chemical Technology and Metallurgy, 2011, 46, 1, 81-88.

Attia, H; Karray, N; Ellili, A; Msilini, N; Lachaâl, M. Sodium transport in basil. Acta Physiologia Plantarum, 2009, 31, 1045-1051.

Attia, H; Ouhibi, C; Ellili, A; Mslini, N; Bouzaïen, G; Karray, N; Lachaâl, M. Analysis of salinityeffects on basil leaf surface area, photosynthetic activity, and growth. Acta Physiologia Plantarum, 2011, 33, 823-833.

Azarmi, F; Nazemieh, H; Dadpour, MR. Growth and essential oil production of Aloisya citriodora L. grown in greenhouse using soil and floating system. Research Journal of Biological Sciences, 2012, 7(4), 148-151.

Belitz, AR; Sams, CE. The effect of water stress on the growth, yield, and flavonolignan content in milk thistle (Silybum marianum). Acta Horticulturae, 2007, 756, 259-265.

Bernstein, N; Kravchik, M; Dudai, N. Salinity-induced changes in essential oil, pigments and salts accumulation in sweet basil (Ocimum basilicum) in relation to alterations of morphological development. Annals of Applied Biology, 2010, 156, 167-177.

Berti, M; Wilckens, R; Fischer, S; Hevia, F. Effect of harvest season, nitrogen, phosphorus and potassium on root yield, echinacoside and alkylamides in Echinacea angustifolia L.

in Chile. Acta Horticulturae, 2002, 576, 303-310.

Brechner, ML; Albright, LD; Weston, LA. Impact of a variable light intenity at a constant light integral: effects on biomass and production of secondary metabolites by Hypericum perforaturm. Acta Horticulturae, 2007, 756, 221-228.

Briskin DP. Medicinal plants and phytomedicines. Linking plant biochemistry and physiology to human health. Plant Physiology, 2000, 124, 507-514.

Britto, DT; Kronzucker, HJ. NH4+ toxicity in higher plants. Journal of Plant Physiology, 2002, 159, 567-584.

Bryant, JP; Chapin, FS; Klein, DR. Carbon nutrient balance of boreal plants in relation to vertebrate herbivory. Oikos, 1983, 40, 357-368.

Burgess, T; McComb, JA; Colquhoun, I; Hardy, GE. Increased susceptibility of Eucalyptus marginata to stem infection by Phytophthora cinnamomi resulting from root hypoxia.

Plant Pathology, 1999, 48, 797-806.

Canter, PH; Thomas, H; Ernst, E. Bringing medicinal plants into cultivation: opportunities and challenges for biotechnology. Trends in Biotechnology, 2005, 23, 180-185.

Cao, H; Ge, Y; Liu, D; Cao, Q; Chang, S.X; Chang, J; Song, X; Lin, X. Nitrate/ammonium ratios affect ryegrass growth and nitrogen accumulation in a hydroponic system. Journal of Plant Nutrition, 2011, 34, 206–216.

Complimentary Contributor Copy

Hydroponic Production of Medicinal Plants 109

Cárdenas-Navarro, R; López-Pérez, L; Lobit, P; Ruiz-Corro, R; Castellanos-Morales, VC.

Effects of nitrogen source on growth and development of strawberry plants. Journal of Plant Nutrition, 2006, 29, 1699-1707.

Clarkson, DT. Factors affecting mineral nutrient acquisition by plants. Annual Review of Plant Physiology, 1985, 36, 77-115.

Colmer, TD; Voesenek, LACJ. Flooding tolerance: suites of plant traits in variable environments. Functional Plant Biology, 2009, 36, 665-681.

Cracker, LE. Medicinal and Aromatic Plants—Future Opportunities. Reprinted from: Janick J; Whipkey A, editors. Issues in new crops and new uses. Alexandria, VA: ASHS Press;

2007.

Crosby, GW; Cracker, LE. Development of soilless culture methods for production of moringa (Moringa oleifera Lam.) root and leaf biomass. Acta Horticulturae, 2007, 756, 139-146.

Dall’Acqua, S; Aiello, N; Scartezzini, F; Albertin, V; Innocenti, G. Analysis of highly secondary-metabolite producing roots and flowers of two Echinacea angustifolia DC var.

angustifolia accessions. Industrial Crops & Products, 2010, 31(3), 466–468.

Del Baño, MJ; Lorente, J; Castillo, J; Benavente-Gacía, O; Del Río, JA; Ortuño, A; Quirin, KW; Gerard, D. Phenolicditerpenes, flavones, and rosmarinic acid distribution during the development of leaves, flowers, stems, and roots of Rosmarinus officinalis. Antioxidant activity. Journal of Agricultural and Food Chemistry, 2003, 51, 4247- 4253.

Dixon, RA; Paiva, NL. Stress-lnduced Phenylpropanoid Metabolism. The Plant Cell, 1995, 7, 1085-1097.

Domìnguez-Valdivia, MD; Aparicio-Tejo, PM; Lamsfus, C; Cruz, C; Martins-Loução, M.A;

Moran, JF. Nitrogen nutrition and antioxidant metabolism in ammoniumtolerant and -sensitive plants. Physiologia Plantarum, 2008, 132, 359-369.

Dorais, M; Papadopoulos, AP; Luo, X; Léonhart, S; Gosselin, A; Pedneault, K; Angers, P;

Gaudreau, L. Soilless greenhouse production of medicinal plants in North Eastern Canada. Acta Horticulturae, 2001, 554, 297-303.

Drew, MC. Plant injury and adaptation to oxygen deficiency in the root environment: a review. Plant and Soil, 1983, 75, 179-199.

Ehret, DL; Ng, K; Oates, B; Delaquis, P; Mazza, G. Production of specialty crops in greenhouses - wasabi as a case study. Acta Horticulturae, 2002, 633, 447- 452.

Ercal, N; Gurer-Orhan, H; Aykin-Burns, N. Toxic Metals and Oxidative Stress Part I:

Mechanisms Involved in Metal-induced Oxidative Damage. Current Topics in Medicinal Chemistry, 2001, 1(6), 529-539.

Ferrante, A; Incrocci, L; Maggini, R; Tognoni, F; Serra, G. Preharvest and postharvest strategies forreducing nitrate content in rocket (Eruca sativa). Acta Horticulturae, 2003, 628, 153-159.

Foyer, CH; Shigeoka, S. Understanding Oxidative Stress and Antioxidant Functions to Enhance Photosynthesis. Plant Physiology, 2011, 155, 93–100.

Geigenberger, P. Response of plant metabolism to too little oxygen. Current Opinion in Plant Biology, 2003, 6, 247-256.

Gill, SS, Tuteja, N. Reactive oxygen species and antioxidant machinery in abiotic stress tolerance in crop plants. Plant Physiology and Biochemistry, 2010, 48, 909-930.

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Giorgi, A, Licheri, GL, Mingozzi, M; Cocucci, M. 2007. Influence of light irradiance on total phenols content, antioxidant capacity, growth and photosynthetic pigments of yarrow

Giorgi, A, Licheri, GL, Mingozzi, M; Cocucci, M. 2007. Influence of light irradiance on total phenols content, antioxidant capacity, growth and photosynthetic pigments of yarrow