Medios de transmisión de datos

At present, a lot of studies have been undertaken relating to the hydroponic growing of medicinal plants. Since the year 2000, about 185,000 works have been published concerning medicinal species and about 8,000 have been published concerning hydroponics (source:

Scopus; www.scopus.com/home.url; accessed 13^th June 2013); only 430 papers report about the hydroponic culture of medicinal plants. The viability and the advantages of this growing system for the production of secondary metabolites from medicinal plants have been demonstrated for a lot of species (e.g.; Dorais et al., 2001; Léonhart et al, 2002; Hyden, 2006;

Azarmi et al., 2012). For example, Stewart and Lovett-Doust (2003) pointed out that greenhouse hydroponic cultivation under controlled environmental conditions in Calendula officinalis could ensure pesticide-free conditions, lacking environmental contaminants, resulting in superior product quality and consistency. Brechner et al. (2007) emphasized that growing Hypericum perforatum in controlled environments, such as the greenhouse or growth chamber, can remove wide variations of common variables such as temperature, insect and disease pressures, and water status. Recently, Prasad et al. (2012) reported that the hydroponic systems can be an effective platform for the production of clean and good quality Centella asiatica herb for the pharmaceutical companies. It was also observed that greenhouse hydroponics could help to overcome germination and establishment problems which may arise with the soil cultivation of medicinal species that are difficult to grow in open field (e.g.: Canter et al., 2005; Crosby and Cracker, 2007; Dall'Acqua et al., 2010).

Tabatabaie et al. (2007) reported that hydroponics could be used for the production of both valerian (Valeriana officinalis var common) and lemon verbena (Lipia citriodora var.

Verbena) under glasshouse. These authors employed different types of soilless culture for both species, and obtained the highest fresh biomass production using the floating raft system.

The same technique was successfully applied also to the cultivation of Camptotheca acuminata, which is used for the production of the anticancer molecule camptothecine (Li and Liu, 2005).

In general, hydroponics ensures a high biomass production, because the nutrient elements are readily available at the root zone and can be easily taken up by the plants. Therefore, higher production of plant material can be obtained with hydroponics (particularly, with water culture) compared to that of soil-grown crops (e.g.: Dorais et al., 2001; Letchamo et al., 2002).

Complimentary Contributor Copy

Hydroponic Production of Medicinal Plants 99

Generally, plant growth is also much faster in hydroponic culture than in open field. For example, Léonhart et al. (2002) reported that Tanacetum parthenium, Achillea millefolium, Taraxacum officinale and Calendula officinalis were all well adapted to greenhouse hydroponic growing conditions and provided abundant yield and high produce quality in a short time period. Dorais et al. (2001) evaluated the growth of several medicinal plants in a floating raft system and found that, after 50-120 days, both the root and the shoot dry weight of Achillea millefolium, Artemisia vulgaris, Inula helenium, Stellaria media, Taraxacum officinale and Valeriana officinalis were much higher in the floating system compared to those of field-grown plants. With the exception of Taraxacum officinale, in all the species under examination the rate of biomass accumulation was faster in the aboveground parts than in the roots; Artemisia vulgaris showed the fastest growth rate. This result was in agreement with those of a previous study carried out with 31 species belonging to the Asteraceae family and grown hydroponically (Almeida-Cortez et al., 1999). Among them, Artemisia vulgaris exhibited the fastest growth rate (0.226 g g^-1 day^-1). Similarly, Echinacea spp., which is traditionally cultivated two to four years in open field, provided high biomass yields in hydroponics in a much shorter time period (a few months only). For example, in Echinacea angustifolia the root yield harvested in nearly eight months from two consecutive hydroponic cultures was comparable with the yield reported in the literature for field cultivations lasting two years or more (Maggini et al., 2012). Moreover, the production yield in Echinacea purpurea was found to increase 2.3 times compared to the average soil cultivation in North America (Letchamo et al, 2002). All these studies evidenced that hydroponics could really offer the opportunity to shorten the growing cycle used in conventional field cultivation and increase at the same time the biomass production.

Together with a higher biomass yield, a higher concentration of secondary metabolites has also been obtained with hydroponics for a lot of medicinal species. Among secondary metabolites, essential oils have been often found in higher amounts in plants grown hydroponically compared to those grown in open field. For some officinal plants such as Pelargonium roseum, Cymbopogon citratus, Ocimum gratissimum, Vetiveria zizanioides e Nepeta transcaucasica, the hydroponic system provided 5-6 times more essential oil than traditional cultivation. Moreover, hydroponically produced essential oil of Pelargonium roseum had a higher geraniol content and was therefore of better quality (Mairapetyan, 1999).

Recently, Azarmi et al. (2012) indicated the floating system as an efficient method to produce large biomass of Aloysia citriodora L with high content of volatile oil.

In addition to essential oils, other classes of secondary metabolites have been found at higher concentrations in hydroponically-grown than in soil-grown medicinal plants.

Tadevosyan et al. (2005) reported about the hydroponic cultivation of Humulus (a species used in Armenian traditional medicine) as an efficient and prospective technique in the Ararat Valley conditions. Humulus plants grown in hydroponics contained higher concentrations of alkaloids, tannins and essential oil than those cultivated in the soil. The content of hypericin, hyperforin and pseudohypericin in the flower tissues of hydroponically-grown Hypericum perforatum was similar or higher than those previously reported for field-grown plants (Murch et al., 2002). It was found that, under outside hydroponic conditions, Celandine poppy (Chelidonium majus L.) presented higher contents of alkaloid, tannins and vitamin C, and lemon catmint (Nepeta cataria L. var. citriodora) contained remarkably higher concentrations of essential oil, tannins and vitamin C compared to field cultivated plants (Manukyan, 2005).

Complimentary Contributor Copy

In contrast with these results, some studies on the production of secondary metabolites in Echinacea angustifolia reported much lower root concentrations of caffeic acid derivatives (especially of the marker compound echinacoside) in hydroponically-grown plants (Zheng et al., 2006b; Maggini et al., 2010, 2012; Sabra et al., 2012) than in field-grown crops (Berti et al., 2002). This was probably the consequence of plant harvesting after only a few months of hydroponic cultivation, whereas field-grown Echinacea plants are commonly harvested after a few years (ontogenetic effect).

Anyhow, all these studies indicated that, although a lot of medicinal species are easily adapted to greenhouse hydroponic conditions and have been successfully cultivated by this growing system, other species still require further work for the development of profitable growing protocols, based on the knowledge of their specific growing needs.

Manipulation of Growing Conditions

Several studies have shown that in greenhouse hydroponic culture the accumulation of secondary metabolites of pharmaceutical interest can be stimulated by modifying the composition of the nutrient solution (e.g. Briskin, 2000; Maia et al., 2001; Zheng et al., 2006b; Montanari et al. 2008; Kiferle et al., 2013) or the climate inside the greenhouse, such as temperature (McChesney, 1999) or light conditions (Giorgi et al., 2007; Hou et al., 2010).

Some authors reported that treating hydroponically-grown plants with growth regulators (Wikremesinhe and Arteca, 1996) or bio-stimulants (Parađiković et al., 2011) resulted in larger production of secondary metabolites.

Plant mineral nutrition may affect both plant growth and secondary metabolism (Briskin, 2000; Zheng et al., 2006a). Nitrogen (N) is the most important nutrient for plants. As a consequence, N starvation is a primary cause for growth reduction, as it limits primary metabolism, thus reducing the production of biomass. However, N deficiency may have an opposite effect on secondary metabolism. The C/N balance hypothesis proposed by Bryant et al. (1983), assumes that the C/N ratio within the plant regulates the concentration of C-based secondary metabolites. A limitation of N supply which restricts growth more than photosynthesis, results in over-production of carbohydrates. These compounds are in part allocated to C-based secondary metabolites. At the same time, N deficiency enhances the formation of reactive oxygen species (Kováčik et al., 2007). For these reasons, phenolic compounds, which are a major group of C-based antioxidant molecules, play a central role in plants’ adaptation to N starvation. In chamomile (Matricaria chamomilla) an increase in the production of phenolics was observed in N-deficient plants (Kováčik et al., 2007). In several medicinal plants, lowering the concentration of N in the nutrient solution resulted in an increase of the content of bioactive secondary metabolites. For example, feeding licorice (Glycyrrhiza glabra Linn.) plants with dilute nutrient solution (approximately equivalent to a quarter unit of Hoagland solution) provided the highest glycyrrhizin content in root tissues and the highest plant growth (Sato et al., 2004). In Camptotheca acuminata, decreasing N concentration in the hydroponic nutrient solution increased the content of the secondary metabolite camptothecine (Li and Liu, 2005).

In soilless culture, N is usually supplied as nitrate (NO₃^-; Pardossi et al., 2006). Some authors (e.g. Munoz et al., 2008; Massa et al., 2010) proposed to lower the NO₃ -concentration in the nutrient solution to reduce the environmental impact of soilless culture

Complimentary Contributor Copy

Hydroponic Production of Medicinal Plants 101

associated with NO₃^- leaching. Decreasing the concentration of NO₃^- in the nutrient solution also reduces the accumulation of NO₃^- in leafy vegetables (Santamaria et al., 1998), which is potentially toxic to human health.

Like N, phosphorus (P) is an essential nutrient for plants. Stewart and Lovett-Doust (2003) reported that Calendula officinalis showed promise as a medicinal greenhouse crop that requires low P levels for optimal production of inflorescence, which is the target tissue containing bioactive compounds. Moreover, due to the xerophytic characteristics of this species, the best results in terms of flower-head tissues production were obtained when relatively low ratios of P relative to N and potassium (K) were associated to intermittent watering regime. The authors suggested that discontinuous water and nutrient supply in hydroponic culture may be widely applicable to medicinal plants, since a lot of species share Calendula’s xerophytic characteristics. Nutrient solutions differing in concentrations and ratios of N, P, and K were reported to influence also the synthesis of various pharmaceutical compounds such as alkaloids, essential oils, tannins, and vitamin C in Chelidonium majus L.

and Nepeta cataria L. (Manukyan, 2005).

Sodium (Na⁺) and chloride (Cl^-) are the most common non-nutrient ions dissolved in irrigation water. The induction of a salt stress by addition of sodium chloride (NaCl) to the nutrient solution determines a rise in the electrical conductivity and results in osmotic stress, as well as ion (Na⁺ or Cl^-) cytotoxicity (Saleh and Maftoon, 2008; Silva et al., 2008; Munns and Tester, 2008; Dashti et al., 2010), and oxidative damage to macromolecules and cell structure (Neto et al., 2006; Eraslan et al., 2007).

Depending on the species, salt stress may have different effects on the production of plant secondary metabolites. For example, salinity was reported to decrease the production of essential oils in Matricaria chamomilla (Razmjoo et al., 2008) and Melissa officinalis (Ozturk et al., 2004), and to have no significant effect on the content of echinacoside per plant in Echinacea angustifolia (Maggini et al., 2013). Mehrizi et al. (2012) observed that salinity induced oxidative stress in hydroponically-grown rosemary, resulting in lipid peroxidation and increase in cell membrane permeability to toxic ions, which in turn reduced plant growth.

As a response to oxidative damage, the total phenolic content in medicinal plants was often reported to be influenced by salinity (Mehziri et al 2012¸ Navarro et al., 2006; Ksouri et al., 2007). A strong correlation between salt tolerance and antioxidant capacity was found in several plant species (Gill and Tuteja, 2010). In particular, higher levels of phenolics were reported in salt tolerant species compared to non tolerant ones.

Together with NO3-, ammonium (NH₄⁺) is a main source of N and is readily absorbed by plants. However, likewise excess Na⁺ or Cl^-, excess NH₄⁺ may have a toxic effect on plants, although the biochemical mechanisms of NH₄⁺ toxicity remain to be further elucidated (Britto and Kronzucker, 2002). The concentrations at which the toxic effects are observed depend on plant species. Several studies have been conducted on the effect of NH₄⁺ on the growth of some crop species (e.g.: Britto and Kronzucker, 2002; Savvas et al., 2006; Cárdenas-Navarro et al., 2006; Cao et al., 2011). One of the main effects of NH₄⁺ toxicity is a lower root/shoot ratio (Kiferle et al., 2013), although the opposite was observed in some species (Britto and Kronzucker, 2002).

On the other hand, the presence of NH₄⁺ along with NO₃^- could have also favorable implications, as NH₄⁺ may reduce NO₃^- absorption. In addition, in hydroponics NH₄⁺ may help in managing the pH of the nutrient solution, as it controls the alkaline drift in pH determined by NO3- assimilation (Savvas, 2001). The pH of the nutrient solution is known to

Complimentary Contributor Copy

affect plant growth and metabolism, as reported for the hydroponic culture of Artemisia afra Jacq. (Koehorst et al. 2010).

At the same time, NH₄⁺ absorption may alter intracellular pH gradients, which affect a lot of metabolic pathways (Dixon and Paiva, 1995). Little information has been reported concerning the response of plant secondary metabolism to N form. Anyway, the use of nutrient solutions supplemented with both NH₄⁺ and NO₃^- at different ratios was reported to affect the production of bioactive compounds in medicinal species grown in hydroponics. It was observed that the supply of 50% total N as NH4+ enhanced the accumulation of the alkaloids catharanthine and vinblastine in Catharanthus roseus (Guo et al., 2012). In contrast, the supply of a mixture of NH₄⁺ and NO₃^- in Echinacea angustifolia decreased the concentration of some caffeic acid derivatives (Montanari et al., 2008). At the same time, a decrease was also observed in the activity of phenylalanine ammonia lyase, a key enzyme of the phenylpropanoid pathway involved in the biosynthesis of these secondary metabolites (Montanari et al., 2008). In sweet basil irrigated with a nutrient solution containing 10.0 mM NH₄⁺, the total content of essential oil was markedly reduced as compared to the plants fed exclusively with NO₃^- (Adler et al., 1989).

A scarce oxygen (O₂) level in the root zone (hypoxia) is a further cause of metabolism imbalance. Although the effect of hypoxia on the secondary metabolism of medicinal plants has been scarcely investigated, in floating system this condition may occur in the stagnant nutrient solution, especially in warm season, as high temperatures may reduce O2 solubility while increasing root respiration (Gorbe and Calatayud, 2010). An adequate O₂ level is necessary to ensure root functionality, whereas O₂ deficiency reduces the uptake of both water and nutrients such as NO₃^- (Horchani et al., 2010; Ferrante et al., 2003). Moreover, O₂ deficit enhances the formation of reactive oxygen species (Colmer and Voesenek, 2009).

Anyway, a large part of the literature on the effects of hypoxia concerns plant growth with little attention paid to secondary metabolism. Growth reduction is considered one of the first adaptive plant responses to hypoxia, as this allows to conserve energy, inhibiting a wide range of ATP-consuming processes to decrease O₂ demand (Geigenberger, 2003). The detrimental effect of low O₂ in the root zone of plants grown in hydroponics was observed in several crop species (e.g.: Ferrante et al., 2003; Shi et al., 2007). Plant sensitivity to hypoxia conditions depends on plant species and may vary even among different cultivars of the same species. In some cultivars of Medicago sativa the growth of both roots and shoots was limited by waterlogging, while in other cultivars only root growth was severely restricted, whereas shoot biomass was unaffected (Smethurst and Shabala, 2003). Under root zone hypoxia conditions, a differential response between the root system and the aerial organs may be associated to ethylene entrapment in submerged plant tissues, as a consequence of the much lower gas diffusion rate in water than in air (Visser and Vosenek, 2004). Ethylene plays a key role in the mechanisms of plant adaptation to hypoxia, for instance by regulating the formation of adventitious roots and aerenchyma (Licausi, 2011). On the other hand, this hormone is known to inhibit root growth, even at low concentration (Abeles et al., 1992).

In addition to a change in the composition of the nutrient solution, a proper modification of the growing conditions could also result effective in stimulating the secondary metabolism.

For example, it was found that: low temperatures increased the accumulation of morphine in Papaver somniferum (McChesney, 1999); water stress increased the concentration of flavonolignans in primary blooms of Silybum marianum (L.) Gaertn. (Belitz and Sams,2007);

low irradiance favored the accumulation of glycyrrhizic acid and liquiritin in the roots of

Complimentary Contributor Copy

Hydroponic Production of Medicinal Plants 103

Glycyrrhiza uralensis Fisch. (Hou et al., 2010). Several experiments demonstrated that supplemental lighting on medicinal plants grown hydroponically under greenhouse accumulated more bioactive molecules compared to field-grown crops (Pedneault et al., 2002;

Brechner et al., 2007). In contrast, an opposite effect of supplemental lighting was reported on other medicinal species. For example, the concentration of phenolic compounds from Tarassacum officinale was 6.2 times higher in field-grown plants compared to those cultivated in hydroponic culture. In Inula helenium, sesquiterpene lactones were more concentrated in field-grown root compared to hydroponically-grown root and parthenolide was more concentrated in field-grown flowers and leaves than in the same organs of hydroponically-grown plants (Pedneault et al., 2002).