Tropane Alkaloids Elicitation of Black Henbane Parts with Calcium and Nitrogen Application Under Hydroponics Culture

Volume 17(4), 107- 113, 2013 JOURNAL of Horticulture, Forestry and Biotechnology www.journal-hfb.usab-tm.ro

Tropane alkaloids elicitation of black henbane parts with calcium and nitrogen application under hydroponics culture

Atena Alaghemand1, Mansour Ghorbanpour2*, Behnaz Moghaddasian1

1Department of Horticulture, Faculty of Agriculture, Saveh Branch, Islamic Azad University, Saveh, Iran; 2Department of Medicinal Plants, Faculty of Agriculture and Natural Resources, Arak University, Arak, 38156-8-8349, Iran.

*Corresponding author: [email protected]

Abstract Black henbane (Hyoscyamus niger L.) an species in Key words Solanaceae family has long been used as a medicinal plant, which is well known for their alkaloid secondary metabolites mainly hyoscyamine and Hyoscyamus niger, Tropane scopolamine. Industrial synthesis of these compounds is difficult due to their alkaloids, Hyoscyamine, complex chemical structure so the compounds are extracted from Solanaceae Scopolamine, Nitrogen, family plants. This study examined the effects of various calcium (Ca) and Calcium, Hydroponics -1 nitrogen (N) concentrations (0, 16.6, 33.3 and 50 mg.L as Ca0-Ca3 and N0- N3) on henbane roots and leaves dry weight and their two main alkaloids content under hydroponics culture. Results showed the highest hyoscyamine -2 -2 content (4.6 g/g.10 and 2.69 g/g.10 ) were obtained at N2Ca2 in total foliage and root dry weight, respectively. Also, the highest scopolamine content (14.51 g/g.10-2 and 7.77 g/g.10-2) were obtained at N1Ca2 and N2Ca2 in total foliage and root dry weight, respectively. In henbane plants, hyoscyamine content of leaves were increased significantly with increasing nitrogen and calcium concentration up to N3Ca3 treatment, while the largest leaf scopolamine content was observed in the N3Ca1 treatment. In the roots, biosynthesis location of alkaloids, maximum hyoscyamine and scopolamine content were observed under N3Ca2 and N3Ca3 treatments, respectively. Results also showed that hyoscyamine and scopolamine content of roots under N3Ca2 and N3Ca3 conditions were almost 67% and 64% higher than that of control plants, respectively. The largest plant total alkaloids accumulation was observed in fertilized plants under N3Ca1 treatment, which showed the highest elicitation index (65.70). It seems that calcium and nitrogen fertilizer are essential nutrient elements necessary for henbane growth and metabolism, which could be considered as abiotic elicitor or precursor in alkaloid biosynthesis pathway.

Plant alkaloids constitute the largest groups of natural the most obvious environmental factors to manipulate. products, providing many pharmacologically active In fact, a secondary metabolite pathway depends on compounds. Tropane alkaloids such as hyoscyamine numerous factors but nutritional status leads to major and scopolamine are among the most valuable drugs in effects, major minerals such as nitrates, calcium, medicine due to its higher pharmacological activities potassium, sulfur and also carbon hydrates, are among and fewer side effects, which are produced by many the most essential ingredients of the nutrient medium Solanaceous species such as hyoscyamus niger (Drager known to affect the growth and metabolism, such as and Chromatogr, 2002). These secondary metabolites alkaloids (Al-Humaid, 2004). are used in medicine as anticholinergic, antispasmodic, It is stated that the availability of essential nutrient hypnotic, and sedative (Waller and Nowacki, 1979). elements necessary for Datura growth and metabolism Hyoscyamine is used in the treatment of cause vigorous vegetation and high chemical gastrointestinal disorders and Parkinson’s disease, production. Alkaloids are groups of naturally occurring while scopolamine is employed against naupathia chemical compounds that contain mostly basic nitrogen (Pitta-Alarez et al., 2006). atoms. Because of the complex chemical structures of Although, plant heredity controls the biosynthesis of these alkaloids, industrial synthesis has been found to alkaloids, some environmental and agrotechnical be prohibitively expensive and therefore they are parameters (e.g., biological and abiological factors and mainly obtained from plant resources especially nutrition) could enhance or inhibit tropane alkaloids Solanaceae plant (Hashimoto and Yamada, 1986). production (De-Baricevic et al., 1999; Ghorbanpour et Nitrogen is one the most important essential nutrient al., 2013). It is reported that mineral nutrition is one of element in plants, which affects all levels of plant

107 function from metabolism to resource allocation, The plants were fertilized six times with six equal growth and development. The content of secondary portions of the proposed fertilizer dose in once every metabolites including tropane alkaloids (particularly five days intervals, starting after 40 days from scopolamine to hyoscyamine ratio) in cultivated plants germination time. The experiment was arranged in a can be considerably influenced by environmental randomized complete block design with six conditions and other factors such as soil, salt replications (n=6). Also, calculation of the weight of concentration and agrotechnical parameters (Drager chemical nutrients used to prepare stock solution under and Chromatogr, 2002). Nitrogen has been found to open hydroponic system is given in table 1. During the increase the content of alkaloids in some of the experimental period, all normal agricultural practices medicinal as well as non-medicinal plants, such as, were performed (no pesticide or herbicide was applied tobacco, lupines, barley, Atropa, Papaver (Waller and to the plants). After 4 month, the plants were harvested Nowacki, 1979). Also, reported that the fertilization of and their parts were separated into leaf and root, and medicinal plants causes an increase in the yield of then dried at ambient temperature in the shad. bioactive compounds (Hashimoto and Yamada, 1986). Thereafter, root and leaf dry weight and alkaloids Data on the effect of the ion-balance on yield and content were evaluated. alkaloid content in Datura stramonium were showed differences in alkaloid accumulation in the upper Alkaloid extraction vegetative plant parts when potassium or calcium were Root and shoot samples were air dried, grinded into the dominant cations within the interionic balance of fine powder and sieved with laboratory mesh (size 30, the six major elements (Cuneyt et al., 2004). mesh opening 545 μm). A subsample of two grams It is well established that that cell wall strength and from each samples was added to appropriate volume of thickness were increased by calcium addition. Calcium CHCl3: MeOH: NH4OH 25%, (15:5: 1), sonicated for is one the most important essential nutrient element in 20 min and then kept at water bath (40 ºC) for one plants, which affects all levels of plant function from hour. Subsequent sample preparation and alkaloids metabolism to resource allocation, growth and extraction were based essentially on the method development. However, not much information is described by Kamada (1986). available on the effect of nitrogen fertilization with urea and calcium on the content of leaf and root Alkaloid analysis and quantification alkaloids in Hyoscyamus niger under hydroponics Alkaloids extracted were identified by gas culture conditions. Therefore, purpose of the present chromatography (GC) and gas chromatography-mass study was to investigate the effect of various spectrometry (GC-MS) analysis. Gas chromatography concentrations of nitrogen and calcium fertilization on analysis was performed using a GC system equipped root and leaf dry weight and alkaloids content in black with a flame ionization detector (FID) and HP-5MS henbane. capillary column (30 m × 0.25 mm, film thickness 0.25 µm). Injector and detector temperatures were set at 220 Materials and Methods and 290 ºC, respectively. The column temperature was initially kept at 50 ºC for 5 min, then gradually Seed preparation and germination increased to 300 ºC at a rate of 3 ºC/min and Henbane seeds generally have low germination rate maintained for 3 min. The flow rate of gas helium was even under normal laboratory conditions. Therefore, 0.8 mL/min. Then 1 µL of extract was directly injected seeds were treated with 250 mg/l gibberellic acid into the gas chromatograph. Each extraction was (GA3) for 48 h at room temperature (22±2˚C) for replicated three times and the compound percentages accelerating germination. After that seeds were are the means of the three replicates. GC-MS analysis surface-sterilized in 70% ethanol for 2 min and then in was carried out on an Agilent 6890 gas chromatograph 25% commercial bleach for 10 min and finally rinsed (Agilent Technologies, Palo Alto, USA) fitted with a with sterile distilled water. Subsequently, seeds were fused silica HP-5MS capillary column (30 m × 0.25 placed in petri dishes on two layers of filter paper mm × 0.25 µm). Oven temperature was programmed moistened with 4 ml distilled water. After 3 days, 90% from 50 ºC to 285 ºC at 3 ºC/min, and helium was of seeds germinated steadily (with 1-2 mm radicle used as carrier gas (0.8 mL/min), Mass spectra were length). After 2 weeks later, seedling (with 2 leaves) obtained in an Agilent 5973 system operating in were planted in plastic pots and irrigated immediately electron impact mode (EIMS) at 70 eV, coupled to an for better establishment. During the whole experiment GC system. The identification of alkaloids was based they received 16-h light/8-h dark (day/night light on the comparison of their GC retention time and mass cycle) under greenhouse conditions. The plants were spectra (MS) data with their standards substances cultivated on cocopeat and perlite. (HYO. HCl and SCO. HBr, Merck). Elicitation index (EI) was calculated as: EI= alkaloid content with Nitrogen and Calcium application elicitation- alkaloid content without elicitation/ Nitrogen and Calcium were applied at the alkaloid content without elicitation× 100 (Amdoun et concentrations of 0 (control), 16.6, 33.3, 50 mg.L-1. al., 2009).

108 Statistical analysis analyzed using computer SAS software (version 9.1; Data were processed by the analysis of variance CoHort Software), and the means were compared by (ANOVA) on the basis of completely randomized Duncan’s multiple range test (P < 0.05). design (CRD) with six replications. The data were

Table 1 Stock solution used in the experiment Chemicals Mg.L-1 P 40 K 50 Fe 250 Zn 200 Cu 150 Mg 100 Br 5 Mo 5

Results and Discussions lowest leaf dry weight (1.10 g) was observed at the highest nitrogen combined with the lowest calcium Leaf and root dry weight treatment (N3Ca1). The results also showed that Results indicated that there were significant (P < 0.01) nitrogen treatments significantly (P < 0.05) influenced variations between fertilized plants compared to henbane root dry weight. The minimum (1.01 g) root untreated controls for the investigated traits. The effect dry weight was obtained at the highest nitrogen of different nitrogen and calcium fertilization doses on application (N3) irrespective calcium concentration, root, foliage and whole plant of henbane was shown in while maximum (1.88 g) root mass was gained under table 2. Results revealed that N1Ca3 treatment had N1Ca2 treatment. The highest root to leaf mass ratio significant and the highest influence on henbane leaf (1.169) was calculated at the equal amount of nitrogen dry weight and whole plant biomass (table 2). The and calcium concentration (N1Ca1) treatment.

Table2 Effect of different calcium and nitrogen fertilization on henbane leaf, root and plant (roo+leaf) dry weight (n=6)

Root/Leaf Plant biomass Leaf dry weight Root dry weight Treatment mass (g) (g) (g) fg efg g de N0Ca0 1.23 1.35 2.58 0.911 def bc e g N0Ca1 1.43 1.77 3.20 0.807 cde b cd h N0Ca2 1.54 1.96 3.50 0.785 bc ab c fg N0Ca3 1.64 2.01 3.65 0.815 a ab ab e N1Ca0 1.84 2.04 3.88 0.901 a cde d a N1Ca1 1.86 1.59 3.45 1.169 a bcd c b N1Ca2 1.88 1.72 3.60 1.093 cd a a k N1Ca3 1.58 2.34 3.92 0.675 ab ab b f N2Ca0 1.78 2.04 3.82 0.872 efg defg f c N2Ca1 1.35 1.4 2.75 0.964 efg bcd e h N2Ca2 1.34 1.72 3.06 0.779 h cdef f h N2Ca3 1.18 1.55 2.73 0.761 h cdefg g i N3Ca0 1.02 1.4 2.46 0.708 h g h de N3Ca1 1.01 1.1 2.11 0.918 h fg h fg N3Ca2 1.01 1.22 2.23 0.827 h g h f N3Ca3 1.02 1.2 2.22 0.850

N0-N3; refer to 0, 16.6, 33.3 and 50 mg/L nitrogen, As can be observed in table 2, high concentration of respectively. Ca0-Ca3; refer to 0, 16.6, 33.3 and 50 nitrogen (N3) reduced plant biomass. In our study, 50 mg/L calcium, respectively. Values with different mg.L-1 N affected the biomass; the diminution was of letter(s) were significantly different at P < 0.05 14.2 % compared to the biomass obtained with the best (Duncan's test). level of nitrogen (N1; 13.33).

109 It is well known that the N status of plants has a great increased significantly (up to 4.6 g/g.10-2) with impact on their root mass fraction (Ingestad and Agren, increasing nitrogen and calcium concentration up to 1991). Furthermore, biomass allocation to shoots and N3Ca3 treatment, while the largest leaf scopolamine -2 roots has been shown to be influenced by the content of content (14.51 g/g.10 ) was observed in the N3Ca1 leaves, suggesting that may act as a signal for shoot treatment (table 3). In the roots, however, biosynthesis allocation (Scheible et al., 1997) as well as root location of alkaloids, maximum hyoscyamine (2.69 branching (Zhang and Forde, 1998). It is also reported g/g.10-2) and scopolamine (6.77 g/g.10-2) content were that fertilization increases the availability of limited observed under N3Ca2 and N3Ca3 treatments, nutrient, and then could alter system properties, which respectively. Results also showed that hyoscyamine might be a potentially practical way to stimulate plant and scopolamine content of roots under N3Ca2 and growth (Baligar et al., 2001; Singh et al., 2005; Dang N3Ca3 conditions were almost 67% and 64% higher et al., 2006). In maize, for example, plants responded than that of control plants, respectively (table 4). to N deficiency by increasing root to shoot ratio, total Interestingly, alkaloids (hyoscyamine + scopolamine) root length, the elongation of individual axial roots, content of henbane in both leaves and roots were and enhancing lateral root growth, but with a reduction increased with nitrogen application up to N3 amount, in the number of axial roots (Feil et al., 1990; Eghball but the calcium concentration determined the best and Maranville, 1993; Chun et al., 2005). On the other treatment. On the other hand, the highest leaf and root hand, high N supply decreases lateral root density (Guo alkaloids content (18.91 and 9.05 g/g.10-2) were et al., 2005). obtained in N3Ca1 and N3Ca2 treatments, respectively (table 5). Hyoscyamine and scopolamine accumulation The largest plant total alkaloids accumulation (27.06 -2 g/g.10 ) was observed in fertilized plants under N3Ca1 There was a significant interaction in hyoscyamine and treatment, which showed the highest elicitation index scopolamine production in root and leaf of henbane (65.70) (Table 5). plants under fertilization treatments. In the fertilized henbane plants, hyoscyamine content of leaves were

Table 3 Effect of nitrogen and calcium fertilization on leaf hyoscyamine and scopolamine content of henbane (n=6)

Leaf scopolamine Leaf Treatment ( g/g.10-2) hyoscyamine (g/g.10-2) i m N0Ca0 2.8 7.81 j l N0Ca1 2.55 8.9 h k N0Ca2 2.96 10.61 g j N0Ca3 3.41 11.3 f i N1Ca0 3.67 11.77 k de N1Ca1 2.2 12.97 k b N1Ca2 2.14 13.56 e h N1Ca3 3.96 12.29 d j N2Ca0 4.11 11.49 e fg N2Ca1 3.86 12.59 f c N2Ca2 3.59 13.26 cd ef N2Ca3 4.13 12.81 c cd N3Ca0 4.24 13.13 b a N3Ca1 4.4 14.51 g gh N3Ca2 3.35 12.50 a cde N3Ca3 4.6 13.03

N0-N3; refer to 0, 16.6, 33.3 and 50 mg/L nitrogen, letter(s) were significantly different at P < 0.05 respectively. Ca0-Ca3; refer to 0, 16.6, 33.3 and 50 (Duncan's test). mg/L calcium, respectively. Values with different

110 Table 4 Effect of nitrogen and calcium fertilization on root hyoscyamine and scopolamine content of henbane (n=6)

Root scopolamine Root Treatment ( g/g.10-2) hyoscyamine (g/g.10-2) h h N0Ca0 1.61 4.11 i g N0Ca1 1.41 4.77 def g N0Ca2 2.19 4.64 ab f N0Ca3 2.55 5.28 g e N1Ca0 1.88 5.89 cde f N1Ca1 2.31 5.24 a g N1Ca2 2.74 4.82 g g N1Ca3 1.79 4.6 i bc N2Ca0 1.41 6.49 f f N2Ca1 2.01 5.35 bc cd N2Ca2 2.47 6.34 ab d N2Ca3 2.55 6.16 i b N3Ca0 1.27 6.65 cd e N3Ca1 2.33 5.82 a cd N3Ca2 2.69 6.36 cde a N3Ca3 2.03 6.77

Table 5 Comparison of the different levels of nitrogen and calcium fertilization on elicitation index (EI), total alkaloids (Hyoscyamine + Scopolamine) and their ratio in henbane root and leaf

Leaf Root Elicitation Plant Total Leaf Root Treatment Sco/Hyo Sco/Hyo Index Alkaloids Hyo+ Sco Hyo+ Sco ( g/g.10-2) ( g/g.10-2) (g/g.10-2) i h i c gh N0Ca0 5.72 10.61 16.33 - 2.55 2.78 h g h i c cde N0Ca1 6.18 11.45 17.63 7.9 3.38 3.49 h f g h d cd N0Ca2 6.83 13.57 20.4 24.92 2.11 3.58 ef e f g de cde N0Ca3 7.83 14.71 22.54 38.02 2.05 3.31 ef d e f c cde N1Ca0 7.77 15.44 23.21 42.13 3.13 3.20 f ed f g d b N1Ca1 7.55 15.17 22.72 39.13 2.26 5.89 f d e f ef a N1Ca2 7.56 15.70 23.26 42.43 1.75 6.33 h c f g d def N1Ca3 6.39 16.25 22.64 38.64 2.56 3.10 e c d e b gh N2Ca0 7.90 16.55 24.45 49.72 4.60 2.79 g d f fg d cde N2Ca1 7.36 15.60 22.96 40.60 2.66 3.26 b c c c d cd N2Ca2 8.81 16.85 25.66 57.13 2.56 3.69 c c c c d def N2Ca3 8.71 16.94 25.65 57.07 2.41 3.10 e b c d a def N3Ca0 7.92 17.37 25.29 54.86 5.23 3.09 d a a a d cde N3Ca1 8.15 18.91 27.06 65.70 2.49 3.29 a d d de d cd N3Ca2 9.05 15.85 24.90 52.4 2.36 3.73 b b b ab c g N3Ca3 8.80 17.90 26.70 63.5 3.33 2.83

N0-N3; refer to 0, 16.6, 33.3 and 50 mg/L nitrogen, Because secondary metabolism in the whole field- respectively. Ca0-Ca3; refer to 0, 16.6, 33.3 and 50 grown plants is highly regulated in time and space (as mg/L calcium, respectively. Values with different problems associated with climate), and with regarding letter(s) were significantly different at P < 0.05 that hyoscyamine and scopolamine are synthesized in (Duncan's test). the pericycle of roots (Flores et al., 1999), considerable efforts have been devoted to develop another ways in

111 order to produce these valuable alkaloids using hairy roots could be enhanced synergistically by hydroponics technology. limiting phosphate in the culture medium. As previously reported by Poovaiah et al., the calcium The effect of nitrogen as has been reported by ions seem to have a role of secondary messenger in Nussbaumer et al., (1998) plays a metabolic role on transduction of signal into the cells after stimulus tropane alkaloids biosynthesis, especially on (Poovaiah et al., 1987). The environmental signals hyoscyamine. In our current study, the highest alkaloid stimulated the calcium ion fluxes between cell -2 rate (27.06 g/g.10 ) was found with cultures grown on membrane (Rincon et al., 1986). -1 medium containing 50 mg.L nitrogen combined with The positive effect of calcium on tropane alkaloid -1 13.33 mg.L calcium. This treatment can lead to a very production was already described by Gontier et al. positive effect on plant (root+leaf) total alkaloids (Gontier et al., 1994) on a Datura innoxia Mill. (hyoscyamine+scopolamine). suspension cell line. These authors hypothesized that Tropane alkaloids such as hyoscyamine and calcium would stimulate putrescine N-methyltransfe scopolamine are plant natural compounds used as rase (PMT), one of the first enzymes in the pharmaceuticals ingredients. These molecules are biosynthetic pathway of hyoscyamine and abundant in many Solanaceae species (Parr et al., scopolamine. This hypothesis was confirmed on the D. 1990). It is well established that hyoscyamine and stramonium hairy root, the loss of hyoscyamine scopolamine, derived from phenylalanine, ornithine production with lower concentration of Ca2+ was and arginine (N containing molecules), are synthesized concurrent with the loss of PMT activity (Pinol et al., almost exclusively in the plant roots. Scopolamine is 1999). synthesized from hyoscyamine via 6b- The use of elicitors and precursors are the effective hydroxyhyoscyamine. The hyoscyamine 6b- means employed to increase the production of hydroxylase catalyzes the two-step epoxidation of important alkaloids. From the current study results, it hyoscyamine to scopolamine (Facchini, 2001). can be concluded that in hydroponic system plants can In our study, plant above ground parts (leaf) showed grow with high biomass production and an increase of more alkaloids than the roots. In general hyoscyamine the secondary metabolites can be obtained through and scopolamine are synthesized in the pericycle of addition of precursors or elicitors. Hydroponic plants branch roots and the then transferred or accumulated in elicit their compounds without loose of viability. the aerial plants (Flores et al., 1999). 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