Lignan Formation in Hairy Root Cultures of Edelweiss (Leontopodium Nivale Ssp

Fitoterapia 97 (2014) 219–223

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Lignan formation in hairy root cultures of Edelweiss (Leontopodium nivale ssp. alpinum (Cass.) Greuter)

Christoph Wawrosch a,⁎, Stefan Schwaiger b, Hermann Stuppner b, Brigitte Kopp a a Department of Pharmacognosy, University of Vienna, Althanstr. 14, A-1090 Vienna, Austria b Institute of Pharmacy/Pharmacognosy, University of Innsbruck, Josef-Moeller-Haus, Innrain 52c, A-6020 Innsbruck, Austria article info abstract

Article history: A hairy root line of Edelweiss (Leontopodium nivale ssp. alpinum (Cass.) Greuter) was obtained Received 14 April 2014 upon transformation with Agrobacterium rhizogenes strain ATCC15834. Elicitation of this line Accepted in revised form 5 June 2014 with silver nitrate, sucrose, methyl jasmonate and yeast extract at various concentrations in Available online 14 June 2014 most cases resulted in a stimulation of lignan biosynthesis. Through elicitation with 6% sucrose the roots accumulated the pharmacologically active lignans leoligin and 5-methoxy-leoligin at Keywords: levels of 0.0678% and 0.0372%, respectively, without significant growth inhibition. These lignan Leontopodium nivale ssp. alpinum levels were comparable to those found in intact roots of cultivated Edelweiss. The biotechnological Edelweiss production of leoligin could be an attractive option for the continuous, field culture-independent Leoligin production of the valuable secondary metabolites leoligin and 5-methoxy-leoligin. 5-Methoxy-leoligin © 2014 Published by Elsevier B.V. Lignan Agrobacterium rhizogenes Hairy roots Elicitor

1. Introduction In the latter class the metabolite, leoligin, has recently been isolated [10]. This lariciresinol-type lignan (see Fig. 1) has The genus Leontopodium (R.Br. ex Cassini) belongs to the been shown to inhibit in vitro leukotriene biosynthesis and Asteraceae family and comprises about 30 species [1]. The intimal hyperplasia of venous bypass grafts, seemingly without well-known alpine Edelweiss (Leontopodium nivale ssp. toxic side effects [11]. It inhibits in vivo neointima formation alpinum (Cass.) Greuter or L. alpinum Cass.) is found in the without causing endothelial damage, and it is not thrombo- mountain regions of Europe, mostly in the Alps, Carpathians, genic [11]. Vein graft disease, i.e. the progressive degeneration Pyrenees, the Tatra and the Balkan Peninsula [2]. Edelweiss of veins used in surgical bypass operations, is characterized by has been used in traditional medicine since centuries against endothelial damage, smooth muscle cell proliferation and ailments like diarrhea, abdominal aches, bronchitis or fever pro-inflammatory signaling [11,12]. Drug eluting stents are [3,4]. Recent studies revealed that extracts from aerial parts considered to be the most likely approach in the prevention of and roots possess anti-inflammatory [5,6] and analgesic [6] graft failure, but currently there is a lack of drugs with specific activities. Phytochemical research has revealed the occur- activity. Leoligin is considered to have a great potential for the rence of various secondary compounds like diterpenes [7], treatment of vein graft disease [11]. The structurally related sesquiterpenes [8,9], benzofuranoids [10] and lignans [7,10]. compound 5-methoxy-leoligin (see Fig. 1) has recently been shown to be a very promising candidate for the development of the first low molecular weight pro-angiogenic and pro- Abbreviations: LG, leoligin; MLG, 5-methoxy-leoligin; MS, nutrient medium arteriogenic drug for the treatment of myocardial infarction after Murashige and Skoog (1962); MeJa, methyl jasmonate; YE, yeast extract ⁎ Corresponding author. Tel.: +43 1 4277 55286; fax: +43 1 4277 9552. [13]. Myocardial infarction is a major cause of mortality E-mail address: [email protected] (C. Wawrosch). worldwide. Recent treatment strategies in the therapy of

properties isolated from the fruits of the milk thistle plant, Silybum marianum. While hairy roots contained more isosilybin A and B than untransformed root cultures, the content of four other flavonolignans was higher in the latter culture type [24]. It has been demonstrated previously that L. nivale ssp. alpinum can be transformed with A. rhizogenes, and resulting hairy root lines were shown to produce anthocyanins, hy- droxycinnamic acid esters, and essential oil [25]. In the present study a hairy root clone of Edelweiss was investigated specifically in respect of its leoligin and 5-methoxy- leoligin content and the influence of the treatment with elicitors, molecules that stimulate defense or stress-induced responses in plants [26],onproductformation.

2. Experimental

Fig. 1. Structure of leoligin (R = H) and 5-methoxy-leoligin (R = OCH3). 2.1. Plant material

Seeds of L. nivale ssp. alpinum were purchased from Austrosaat AG (Vienna, Austria) and were surface sterilized myocardial infarction aim at the improvement of ventricular for 30 min with an aqueous NaOCl solution (3.4% active function, among others by stimulating angiogenesis and chlorine). They were then aseptically germinated on mod- arteriogenesis, namely the induction of artery growth to ified semisolid half-strength Murashige and Skoog (MS) bypass occluded arteries [13]. In this, there is an urgent need medium [27] supplemented with 1% sucrose and 0.3% Gelrite® to find new drugs, and 5-methoxy-leoligin has shown to (Carl Roth, Karlsruhe, Germany). Shoots of 4 weeks old possess corresponding pro-angiogenic and pro-arteriogenic seedlings were subsequently transferred to modified semisolid activities. MS medium with 3% sucrose, 0.4 mg/L thiamine hydrochloride, Edelweiss is a protected plant in many countries. While it 80 mg/L myo-inositol, 100 mg/L caseine hydrolysate, 0.7% agar is cultivated in large quantities in Switzerland [14],the (Merck,Darmstadt,Germany),0.55μM 1-naphthaleneacetic isolation of relevant amounts from Edelweiss roots remains acid and 0.25 μMkinetin[28] for further multiplication. Every a laborious task due to the low content [10,15] and the thin 4 weeks shoot clusters were divided into single shoots which and fibrous nature of the roots of cultivated plants [16]. were transferred to fresh medium. All cultures were kept at As the chemical synthesis has not yet been described the 25 ± 1 °C under a 16 hour photoperiod with a light intensity of biotechnological production might be an alternative ap- 40 μM·m−2 ·s−1 (Sylvania Gro-Lux® fluorescent tubes). A proach to the procurement of relevant amounts of these number of seedlings of this seed batch were also potted into lignans. Hairy root cultures, i.e. in vitro cultured roots which gardening soil and grown to the flowering stage. Voucher result from the infection (transformation) of higher plants specimens and living plants are kept at the Department of with the soil-born bacterium Agrobacterium rhizogenes,have Pharmacognosy, University of Vienna. been investigated for a few decades as biological systems for Three normal root samples of cultivated L. nivale ssp. the production of secondary compounds from medicinal alpinum plants were obtained from the Mediplant Swiss plants [17]. Hairy roots can in many cases produce the same Research Centre on Medicinal and Aromatic Plants, Conthey, compounds found in normal roots of the parent plant, but Switzerland (two samples) and from W. Faulhammer, while in callus or cell suspension cultures this productivity Innsbruck, Austria (one sample). Vouchers are stored at the frequently diminishes over time it remains stable in herbarium of the Institute of Pharmacy/Pharmacognosy, transformed roots [18]. Furthermore, in the recent past University of Innsbruck. hairy root technology has been significantly improved concerning accumulation and excretion of secondary me- 2.2. Hairy root culture tabolites after elicitation, and scale-up of the culture process [17]. With regard to the production of lignans from hairy A. rhizogenes strain ATCC15834 was grown in liquid YMB roots a number of studies have focused on podophyllotoxin medium [29]. The middle veins of leaves of in vitro grown and its derivatives, an important lead for anticancer drugs. Edelweiss shoots were gently scratched with a scalpel dipped For example, hairy root lines of Linum album produced in the bacterial suspension and the shoots where then 105 μg/L [19] or 5.12 mg/L [20] of the lignan while a content cultivated on modified semisolid half-strength MS medium of 14.11 mg/L was measured in the roots of wild growing with 3% sucrose in the light. Single roots which emerged from plants [19].InLinum flavum, hairy roots were reported to the wounded sites after 4 weeks in average were dissected contain up to 3.5% 5-methoxypodophyllotoxin [21] which is from the leaves and treated twice for 10 days each with comparable to the amount of 3.68% described for roots of liquid modified MS medium containing 3% sucrose and greenhouse grown plants [22]. Another arylnaphthalene 500 mg/L of the antibiotic, cefotaxim. Subsequently the lignan, justicin B, is found in normal root cultures (12.5 mg/L) hairy roots were routinely transferred to liquid modified MS and hairy root cultures (16.9 mg/L) of Linum austriacum[23]. medium with 3% sucrose every 4 weeks: An inoculum of ca. Silymarin is a flavonolignan complex with hepatoprotective 0.5 g was transferred to 50 mL of medium in a 250 mL C. Wawrosch et al. / Fitoterapia 97 (2014) 219–223 221

Table 1 Final biomass (g dry wt.) and contents (% dry wt.) of leoligin and 5-methoxy-leoligin in hairy root clone K8A of Leontopodium nivale ssp. alpinum treated with various elicitors.

⁎ ⁎⁎ ⁎⁎ Sample/treatment Final biomass (g dry wt.) Leoligin (w%) 5-methoxy-leoligin (w%)

K8A control 0.62 ± 0.03ade 0.0062 ± 0.0021a 0.0049 ± 0.0014ab bc bc e 15 μM AgNO3 0.44 ± 0.03 0.0321 ± 0.0094 0.0260 ± 0.0075 bc ab bcd 30 μM AgNO3 0.43 ± 0.02 0.0183 ± 0.0021 0.0153 ± 0.0019 b ab cde 60 μM AgNO3 0.36 ± 0.03 0.0217 ± 0.0015 0.0177 ± 0.0014 5% Sucrose 0.64 ± 0.04ae 0.0142 ± 0.0032ab 0.0083 ± 0.0018abc 6% Sucrose 0.59 ± 0.03ade 0.0678 ± 0.0042d 0.0372 ± 0.0025f 7% Sucrose 0.70 ± 0.05a 0.0221 ± 0.0062ab 0.0101 ± 0.0027abcd 1 g/L YE 0.69 ± 0.04a 0.0192 ± 0.0054ab 0.0118 ± 0.0028abcd 2 g/L YE 0.61 ± 0.05ade 0.0337 ± 0.0094bc 0.0204 ± 0.0050de 5 g/L YE 0.59 ± 0.05ade 0.0035 ± 0.0016a 0.0019 ± 0.0008a 50 μM MeJa 0.50 ± 0.05cd 0.0316 ± 0.0097bc 0.0116 ± 0.0028abcd 100 μM MeJa 0.45 ± 0.05bc 0.0498 ± 0.0152cd 0.0188 ± 0.0050cde 200 μM MeJa 0.54 ± 0.04cde 0.0249 ± 0.0037ab 0.0098 ± 0.0013abcd 300 μM MeJa 0.59 ± 0.03ade 0.0173 ± 0.0035ab 0.0113 ± 0.0021abcd

⁎ Values are mean ± S.E. (n = 5) per culture flask with 50 mL nutrient medium after 4 weeks of culture; means followed by the same letter are not significantly different (p = 0.05) according to Duncan's multiple range test. ⁎⁎ Values are mean ± S.E. (n = 2, each measured 3 times); means followed by the same letter are not significantly different (p = 0.05) according to Duncan's multiple range test.

Erlenmeyer flask and cultivated at 25 ± 1 °C in the dark on a HPLC-UV. The efficacy of the extraction protocol was proved rotary shaker at 100 RPM. The root line K8A was used for the by a fourth extraction with 10 mL dichloromethane. The elicitation experiments. extract was evaporated separately, dissolved in 1.00 mL MeOH and analyzed by HPLC-UV. Since no signals of leoligin 2.3. Elicitor treatments and 5-methoxy-leoligin were found, extraction was found to be exhaustive. Two extracts of each hairy root culture sample Elicitors were added to 3 weeks old hairy root cultures were prepared. Both were analyzed three times, and the six except for the treatments with elevated sucrose concentra- values of the peak area were used to perform the quantifi- tions which occurred for the whole culture period. Silver cation of leoligin and 5-methoxy-leoligin in the samples. nitrate (Merck, Darmstadt, Germany, analytical grade) was dissolved in distilled water and the filter sterilized (0.22 μm) 2.4.2. HPLC-UV method and quantiﬁcation parameters solution was added to hairy root cultures at final concentra- An HP 1050 system (Agilent, Waldbronn, Germany) tions of 15, 30 and 60 μM AgNO , respectively. Similarly, filter 3 equipped with auto sampler, DAD and column thermostat sterilized solutions of methyl jasmonate (MeJa; Sigma– was used. The stationary phase was a Phenomenex Kinetex Aldrich, St. Louis, USA; 95% purity) in 96% ethanol were 2.6 μ C18 100 A (100 mm × 2.1 mm) with 2.6 μm particles added to achieve final concentrations of 50, 100, 200 and equipped with the corresponding guard column. The tem- 300 μM. Yeast extract (YE; Sigma–Aldrich, St. Louis, USA) was perature was set to 40 °C. The mobile phase consisted of purified by dual precipitation with ethanol as described by two solvents: A = water, B = acetonitrile. The composition Hahn and Albersheim [30]. After autoclaving (20 min at during run was set as following: 0 min: 65% A, 35% B; 121 °C) of the resulting aqueous solution aliquots were 20 min: 50% A, 50% B; 25 min: 1% A, 99% B; stop: 35 min; added to hairy root cultures at final concentrations of 1, 2 post time: 15 min. The flow rate was 0.250 mL/min and the or 5 g/L. For osmotic treatment, hairy roots were inoculated detection wavelength was 205 nm. As standard for quanti- in liquid modified MS media supplemented with 5, 6 or 7% fication pure leoligin and 5-methoxy-leoligin were dissolved sucrose. Root materials were always harvested after a total in 1.00 mL of methanol and diluted to obtain five reference cultivation period of 4 weeks and were dried at room solutions (73.1 μg/mL to 4.6 μg/mL; 54.4 μg/mL to 3.4 μg/mL), temperature. which were analyzed to afford a calibration curve of y = 230818.83405x + 496.66528; R2 = 0.99819 for leoligin as 2.4. HPLC-UV analysis well as y = 190969.00754x − 47.98333; R2 = 0.9995219 for 5-methoxy-leoligin. 2.4.1. Sample preparation A small amount of ground root sample was weighted (100.0 mg). The plant material was sonicated with 20 mL of 2.4.3. Chemicals and reagents dichloromethane for 10 min in the ultrasonic bath before All solvents (dichloromethane, methanol and acetonitrile) being filtrated over cotton wool. This manipulation was were of analytical grade (99.9%) provided from Merck repeated two times with 10 mL of dichloromethane each. (Darmstadt, Germany). The used water was produced via After evaporation of the solvent of the combined extracts at reverse-osmosis. Leoligin and 5-methoxy-leoligin had a reduced pressure the dry residue was dissolved in 1.00 mL of purity level higher than 98%, according to LC-DAD/MS and methanol, again filtered over cotton wool and analyzed by NMR examination. These references were prepared at the 222 C. Wawrosch et al. / Fitoterapia 97 (2014) 219–223

Institute of Pharmacy/Pharmacognosy of the University of affected. In cell cultures of S. miltiorrhiza 100 mg/L but not 50 Innsbruck [7]. or 200 mg/L YE led to the highest tanshinone yield [41].In hairy root cultures of the same plant species rosmarinic acid 3. Results and discussion formation was enhanced similarly irrespective of the concentration of YE elicitor, while cryptotanshinone biosynthesis The hairy roots line K8A which was investigated in this was improved in a dose dependent manner [42]. study was chosen due to its fast growth. When cultivated As a second biotic elicitor we chose methyl jasmonate under standard conditions, i.e. in liquid modified MS medium (MeJa) which has been shown to stimulate secondary with 3% sucrose, it yielded 0.0062% LG and 0.0049 MLG metabolite formation in a wide variety of plant in vitro cultures. (Table 1). In many cases, secondary metabolite accumulation In particular, the production of lignans was shown to rise in cell in hairy root cultures can be enhanced by elicitation, i.e. suspension cultures of Forsythia x intermedia[43] and L. album treatment of the culture with biotic and abiotic elicitors [31]. [44] upon treatment with MeJa. Elicitation of Edelweiss hairy We therefore treated our hairy root line with the two abiotic root clone K8A with 50 or 100 μM MeJa resulted in accumula- elicitors silver nitrate and sucrose, and the two biotic tion of up to 0.0498% LG and 0.0188% MLG (8-fold and 3.8-fold elicitors yeast extract and methyl jasmonate. As elicitation higher than in the control, respectively), although at 100 μM can, despite an improved product yield, also result in MeJa root growth was significantly suppressed. Higher concen- decrease of biomass accumulation [32], hairy root growth trations (200 or 300 μM MeJa) showed no influence on biomass was assessed in terms of final dry weight, too. Silver nitrate production but enhanced lignan formation only to a moderate at a concentration of 15 μM led to a ca. 5-fold increase of the non significant extent. levels of both LG and MLG, but on the other hand root The leoligin content of wild growing Edelweiss has been growth was negatively influenced (about 30% less biomass). described to account for 0.005–0.010% [15]. Within the

Elicitation with 30 or 60 μMAgNO3 had a less pronounced present study, analysis of two samples of cultivated L. nivale and not significant impact on lignan biosynthesis. Similarly, ssp. alpinum from Switzerland (Mediplant Swiss Research enhanced production of silymarin in hairy root cultures of Centre on Medicinal and Aromatic Plants) revealed a content S. marianum has also been achieved through the use of silver of 0.0155 and 0.0547% LG and 0.1293 and 0.0672% MLG, nitrate [33]. A treatment with 30 or 60 μMofthiselicitor respectively. Another sample from field culture in Innsbruck, lowered the total phenolics content in hairy roots of Salvia Austria yielded 0.0632% LG and 0.0421% MLG. Although no miltiorrhiza, but slightly stimulated the formation of these further data concerning the lignan content in Edelweiss are metabolites at a concentration of 15 μM [31].But,inthe available so far, a variability regarding both the content and sameculturetypeGeandWu[34] found Ag+ to stimulate the ration of LG to MLG seem to be likely. Furthermore, it is the production of tanshinones, and silver nitrate significantly not clear whether the growing conditions (wild vs. controlled increased the accumulation of the tropane alkaloids, scopol- cultivation) have an influence on lignan production. Anyhow, amine and hyoscyamine in hairy roots of Brugmansia candida the basic LG content of the hairy root clone K8A used in the [35]. present study was found to be of the order of wild growing Beside its role as carbon source in in vitro cultures, Edelweiss [15]. Similarly, untransformed roots and hairy sucrose at elevated concentrations can act as abiotic elicitor roots of L. flavum accumulated comparable amounts of the due to increased osmotic pressure. We therefore investigated lignan, 5-methoxypodophyllotoxin [21,22]. As delineated the effect of this sugar at concentrations of 5, 6 or 7%. While above, lignan contents in hairy roots have been described to lignan formation was not significantly enhanced when 5 or be higher, lower or equal to that in normal roots depending 7% sucrose was used, at 6% the content of LG and MLG on factors like e.g. plant genotype or Agrobacterium strain increased to 0.0678% and 0.0372%, respectively, which was used for transformation. The production of LG could be 10.9-fold and 7.6-fold higher than that in the control roots significantly increased nearly 11-fold through elicitation with with the standard sucrose concentration of 3%. Hairy root 6% sucrose. Similarly, the MLG content of clone K8A could be growth was not significantly impaired at any of the tested enhanced 7.6-fold, and elicitation had no significant impact sucrose concentrations. An elevated sucrose concentration of on biomass accumulation. 6% also stimulated glycyrrhizin production in hairy roots of Glycyrrhiza inflata[36]. In hairy roots of Withania somnifera, 4. Conclusions accumulation of the steroidal lactone, withaferin A was enhanced by treatment with 4% but not with 6% sucrose [37]. Only recently the highly active lignan compounds Plant cell suspension cultures of Melastoma malabathricum leoligin and 5-methoxy-leoligin have been isolated from produced more anthocyanins with 4.5–7.5% sucrose than the roots of the alpine Edelweiss, casting new light on this with 3% [38], and using 5% sucrose the formation of rosmarinic tradition-steeped plant. As an alternative to the laborious acid in suspension cultures of Ocimum sanctum was higher than extraction of the low amounts found in the plant, we could with 4 or 7% of the sugar [39]. show that a transformed hairy root line of Edelweiss Yeast extract (YE) and preparations thereof have been accumulates the valuable lignans in concentrations which, widely used as elicitors in plant tissue culture [40]. When upon elicitation with 6% sucrose, resembles the content in purified YE [30] was added to cultures of the Edelweiss hairy normal roots. Analyses of three samples of normal roots root clone K8A, 1 or 5 g/L did not significantly influence revealed a degree of variation in both total lignan content neither lignan formation nor root growth. At a concentration and ratio of the two compounds. Future work will therefore of 2 g/L a 5-fold (LG) and 4-fold (MLG) promotion of product focus on the establishment of further hairy root clones formation was observed, while biomass increase was not starting from high yielding Edelweiss genotypes, and using C. Wawrosch et al. / Fitoterapia 97 (2014) 219–223 223 additional A. rhizogenes strains. In any case, as a continuous, [20] Baldi A, Srivastava AK, Bisaria VS. Improved podophyllotoxin production by transformed cultures of Linum album. Biotechnol J 2008;3:1256–63. sustainable and renewable production system independent of [21] Oostdam A, Mol JNM, van der Plas LHW. Establishment of hairy root climatic or environmental effects [45] the biotechnological cultures of Linum flavum producing the lignan 5-methoxypodophyllotoxin. production of leoligin and 5-methoxy-leoligin would be of Plant Cell Rep 1993;12:474–7. [22] Wichers HJ, Versluis-De Haan GG, Marsman JW, Harkes MP. advantage over the extraction from field grown plants. 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