Plant Soil (2017) 417:117–126 DOI 10.1007/s11104-017-3245-6

REGULAR ARTICLE

Methyl jasmonate treatment increases production in hexandrum roots under glasshouse conditions

Christel L. C. Seegers & Rita Setroikromo & Pieter G. Tepper & Peter Horvatovich & Ron Peters & Wim J. Quax

Received: 29 November 2016 /Accepted: 30 March 2017 /Published online: 4 April 2017 # The Author(s) 2017. This article is published with open access at Springerlink.com

Abstract methyl jasmonate on the podophyllotoxin production Background and aim The endangered Podophyllum was determined. hexandrum is an important industrial source of Results More root formation was observed in peat-perlite podophyllotoxin, which is a precursor for the anticancer soil than in sand soil. Furthermore, root formation was drugs and . Attempts to obtain higher at 15 °C than at 25 °C. This resulted in the highest podophyllotoxin through cell cultures or chemical syn- podophyllotoxin production per in peat-perlite at thesis have still a long way to go before being economical 15 °C (160 ± 22 mg/plant d.w.). Furthermore, methyl feasible. The objective of this study was to increase the jasmonate treatment of the increased the root formation and podophyllotoxin production of podophyllotoxin production in the roots by 21%. P. hexandrum cultivated in a glasshouse. Conclusion We were able to cultivate P.hexandrum in a Methods Root formation and podophyllotoxin produc- glasshouse in the Netherlands and improve the root tion of P. hexandrum in sand or peat-perlite soil at 15 °C formation and podophyllotoxin production. This paves or 25 °C was determined. Furthermore, the influence of the way for large-scale cultivation of P. hexandrum in the temperate latitudes for the production of the phar- maceutical interesting podophyllotoxin. Responsible Editor: Hans Lambers. Keywords Podophyllum hexandrum . Electronic supplementary material The online version of this . . . . article (doi:10.1007/s11104-017-3245-6) contains supplementary Podophyllotoxin Etoposide Soil Temperature material, which is available to authorized users. Methyl jasmonate

C. L. C. Seegers : R. Setroikromo : P. G. Tepper : * W. J. Quax ( ) Introduction Department of Chemical and Pharmaceutical Biology, Groningen Research Institute of Pharmacy, University of Groningen, Antonius Deusinglaan 1, 9713 AV, Groningen, The Netherlands The high demand for podophyllotoxin as precursor for e-mail: [email protected] the synthesis of important anticancer drugs (etoposide and teniposide) has led to the search for alternative P. Horvatovich Department of Analytical Biochemistry, Groningen Research sources (Imbert 1998). The commercially exploited nat- Institute of Pharmacy, University of Groningen, Groningen, ural sources of podophyllotoxin are Podophyllum The Netherlands hexandrum and Podophyllum peltatum (Guerram et al. 2012). Other podophyllotoxin producing in this R. Peters Proeftuin Ron Peters, Gantel 12, 7891 XAKlazienaveen, genus are Podophyllum sikkimensis and Podophyllum The Netherlands pleianthum (Jackson and Dewick 1985;Pauletal. 118 Plant Soil (2017) 417:117–126

2013). The highest concentration of podophyllotoxin was done on increasing root formation of mature plants was found in P. hexandrum roots, which varies between or increasing podophyllotoxin production in vivo. 0.025% and 9.53% (d.w.) depending on the geographic Several researchers reported that the plant morphology location (Purohit et al. 1999;Alametal.2009; Kitchlu and the geographic location, especially the altitude, are et al. 2011;Sharmaetal.2012; Liu et al. 2015;Pandey important factors for podophyllotoxin production et al. 2015). Podophyllotoxin production is not restricted (Purohit et al. 1999;Alametal.2009; Kitchlu et al. to the roots as low amounts of podophyllotoxin (0.003– 2011;Pauletal.2013;Pandeyetal.2015). Alam and 0.229% d.w.) are also found in P. hexandrum leaves Naik found that high podophyllotoxin production was (Pandey et al. 2013). Furthermore, several researchers correlated to low pH, high organic content and high reported podophyllotoxin production in the leaves of nitrogen levels in the soil (Alam and Naik 2009). P. peltatum (Bastos et al. 1996; Moraes et al. 2000, Another factor important for the production of 2002; Cushman et al. 2006; Zheljazkov et al. 2011). podophyllotoxin is temperature as some lignan biosyn- The most recent population study was by Zheljazkov thesis genes were found to exhibit a higher expression and coworkers who found up to 2.53% (d.w.) level at 15 °C rather than at 25 °C (Kumari et al. 2014). podophyllotoxin (Zheljazkov et al. 2011). The leaves Until now, the influence of soil composition and culti- of P. peltatum can be an attractive alternative source for vation temperature on podophyllotoxin production in P. podophyllotoxin due to its renewable properties. hexandrum roots has not been investigated in a P. hexandrum is an endangered species according glasshouse. to the Convention of International Trade in Besides cultivation conditions, hormone induction Endangered Species of Wild Fauna and Flora can be used to increase production of secondary metab- (https://www.cites.org/eng/app/appendices.php#hash2), olites. Elicitation of medicinal plant species by because of its excessive harvesting. Therefore, several jasmonates activates transcription factors followed by researchers focused on production of podophyllotoxin upregulation of the production of structurally divergent by chemical synthesis or in vitro cell cultures. The secondary metabolites, such as nicotine and artemisinin chemical synthesis is difficult due to the presence of (De Geyter et al. 2012). Most elicitation studies have four contiguous chiral centers, and the presence of a been performed on cell suspension and hairy root cul- base sensitive trans-lactone moiety (Canel et al. 2000). tures (van der Fits 2000;Häkkinenetal.2004; Baldi and Therefore, at least five chemical synthesis steps are Dixit 2008;Toddetal.2010;Shojietal.2010; necessary to convert the commercially available Suttipanta et al. 2011). Also the podophyllotoxin pro- bromopiperonal, a building block of the GPR30 recep- duction in P. hexandrum suspension cultures was in- tor antagonist (Dennis et al. 2009), into podophyllotoxin creased by seven to eight-fold after stimulation with or epipodophyllotoxin (Ting and Maimone 2014). As an methyl jasmonate (Bhattacharyya et al. 2012). alternative, podophyllotoxin production in cell suspen- Furthermore, methyl jasmonate treatment is also effec- sion cultures has been explored. This approach, howev- tive in vivo, as treatment of Nicotiana attenuata leaves er, provides a low yield with the highest production rate increased nicotine production in the roots (Baldwin of podophyllotoxin reaching 0.65% (d.w.) (Petersen and 1996). Whether methyl jasmonate can increase the Alfermann 2001; Ionkova et al. 2010). Untill now nei- amount of podophyllotoxin in vivo, and whether this ther the chemical synthesis nor the in vitro production of effect can be obtained by spraying the leaves, has not podophyllotoxin is economically competitive with the been reported up to now. extraction of podophyllotoxin from P. hexandrum. Our aim was to enhance the root formation and Therefore, we focused in this study on improving the podophyllotoxin production in P. hexandrum in a glass- cultivation conditions of P. hexandrum to ensure a sus- house in the Netherlands to meet the high demand of tainable supply of P. hexandrum roots for isolation of podophyllotoxin. The influence of soil, temperature and podophyllotoxin. Until now, most conservation studies methyl jasmonate was investigated in a systematic ap- focused on enhancing seed germination or the proach. In order to compare the podophyllotoxin pro- propagation/transplantation of the plants (Nadeem duction between conditions, a novel quick extraction et al. 2000; Guo et al. 2012). However, no research method was designed. Plant Soil (2017) 417:117–126 119

Materials and methods arrived in pots with peat soil enriched by addition of calcium and NPK (nitrogen, phosphorous, and potassi- Experimental design um) and were stored at 7–8 °C in the dark to prevent shoot formation. Plants were cultivated in the glass- P. hexandrum plants were cultivated under various house of Proeftuin Ron Peters (Klazienaveen, the growth conditions (soil, temperature and jasmonate treat- Netherlands) in sand soil or peat-perlite soil ment) to investigate root formation and podophyllotoxin (Table S1). The peat and perlite were mixed in a ratio production. The temperature study was done with 150 2:1 (w/w). For every condition and time point fifteen plants; 75 plants cultivated at 15 °C and 75 plants culti- plants were randomly harvested. vated at 25 °C. The soil study (sand versus peat-perlite) The soil and temperature study was done with the was performed at both temperatures with 30 plants for batch from Borculo (150 plants). The plants cultivated at each soil type. Sand and peat-perlite were previously 15 °C were grown in March and April of 2015 and the described to be successful for P. hexandrum, respectively plants cultivated at 25 °C in May and June 2015. The for the germination of seeds and in transplanting of root- average temperatures were 15 ± 2 °C and 22 ± 4 °C, lets into soil (Kharkwal et al. 2008; Guo et al. 2012). For respectively, and the average radiation sums were statistical analysis, a factorial design was used with three 1158 ± 450 joules/cm2 and 1554 ± 531 joules/cm2 factors: soil, temperature and time. Soil was analyzed at (Table S2). The root biomass and podophyllotoxin con- two levels: sand and peat-perlite soil; temperature at two tent of fifteen plants of each temperature group were levels: 15 °C and 25 °C; and time at three levels: 0, 20 and analyzed at the beginning. The other plants were har- 40 days. The podophyllotoxin content was determined vested after 20 or 40 days of cultivation. per three plants; therefore, plant identifiers were included The batch from Gilde (75 plants) was used for the as additional factor in the podophyllotoxin data analysis. methyl jasmonate experiment from March until April The methyl jasmonate study was performed with 75 2015. All plants were first cultivated for 20 days before plants, from which 30 plants were treated with methyl methyl jasmonate treatment; 15 plants were immediate- jasmonate and 45 plants were treated with water. ly harvested for baseline control, 30 plants were sprayed with water (control) and 30 plants were sprayed with 5 l Chemicals of 1.5 mM methyl jasmonate. After nine days, fifteen plants in each group were harvested for analysis. The Technical methanol (98.5% (v/v)) and acetonitrile (99.9% plants in the treatment group were sprayed again for (v/v)) were purchased from VWR, Fontenay-Sous-Boris, three consecutive days, five liters of 3 mM methyl France. Podophyllotoxin (≥ 98% (v/v)), methyl jasmonate jasmonate each day. The next day all plants were har- (95% (v/v)) and ammonium formate (> 97% (v/v)) were vested for analysis. purchased from Sigma-Aldrich, St. Louis, USA. Other chemicals were methanol absolute AR (99.8% (v/v), Plant processing Biosolve, Valkenswaard, The Netherlands) and formic acid (98–100% (v/v), Merck, Darmstadt, Germany), peat Roots were collected from each plant and rinsed with tap (Horticoop, Klazienaveen, The Netherlands) and perlite water. The roots were dried for 18 h at 40 °C (Table S3). (Pull Rhenen, Rhenen, The Netherlands). Plant roots were pooled per three plants and ground to 1mmsizewithanMF10basicgrinderofIKA, Plant cultivation 3000 rpm, and stored at room temperature in closed containers in the dark. Two batches of isogenetic P. hexandrum plants were obtained from plant breeder Heutinck (Borculo and Extraction of podophyllotoxin from plant roots Gilde, The Netherlands). The first batch contained 150 plants that had been grown for 12 to 24 months in The amount of podophyllotoxin in the roots was deter- Borculo. The second batch contained 75 plants that mined by extraction of podophyllotoxin by two different had been grown for 24 months in Gilde. All plants extraction methods: quick methanol and Soxhlet. 120 Plant Soil (2017) 417:117–126

Quick methanol extraction Clara, USA) and an Eclipse XDB-C18 guard column containing cartridges (4.6 id. × 12.5 mm, 5 μm, Agilent, A quick methanol extraction method was designed to SantaClara,USA)wereused.ThemobilephaseAwas process the large number of samples in this study. All [H2O:ACN (95:5)] and B [ACN:H2O (95:5)], both sup- extractions were done in triplicate. One gram of plant plementedwith0.1%formicacidand2mMammonium material was weighed and 10 ml methanol was added. formate. The injection volume was 10 μl with a flow rate The sample was vortexed for thirty seconds on a of 1 ml min−1 using a time program of 40 min consisting Heidolph Reax top, at 2500 rpm (Heidolph, Essex, of (A:B) 10 min 70:30 (v/v) isocratic; gradient UK), and incubated in a 65 °C water bath for ten minutes. 8 min to 50:50 (v/v); gradient 7 min to 10:90 (v/v); The sample was centrifuged at 2400 g for ten minutes at 5 min 10:90 (v/v) isocratic; gradient 5 min to 70:30 (v/ 4 °C and the supernatant was transferred to a clean tube. v) and equilibration of the LC column with 5 min 70:30 This extraction was repeated five times. The volume of (v/v) isocratic elution prior to the next analysis. The each extraction was separately adjusted in volumetric column temperature was held constant at 25 °C and flasks, 50 ml for the first three extractions and for detection a wavelength of 289 nm was used. A 20 ml for the last three extractions. The podophyllotoxin calibration curve was used to determine the concentration was determined by HPLC analysis. podophyllotoxin concentration (10–320 μg / ml, corre- Samples were stored at 4 °C before analysis. lation coefficients >0.999). Podophyllotoxin is stable in the refrigerator at 4 °C for at least three months and at 25 ° in the autosampler of Confirmation of podophyllotoxin by LC-ESI-MS/MS the HPLC for at least 30 h. The presence of podophyllotoxin in the extracted sam- Soxhlet extraction ples was confirmed by LC-ESI-MS/MS (Fig. S1). The analysis was performed using a Shimadzu LC system, The Soxhlet method has been previously used for consisting of two LC-20 AD gradient pumps and a SIL- podophyllotoxin extraction (Gupta et al. 2013). 20 AC auto sampler. The LC system was coupled to an Soxhlet extraction was done in a Tecator Soxtec API 3000 triple quadrupole mass spectrometer (Applied System HT2 that consisted of two 1045 extraction units Biosystems/MDS Sciex) via a TurboIonSpray source. connected to one 1046 service unit (Gemini, Apeldoorn, Data were collected and analyzed by Analyst 1.5.2 ac- The Netherlands). One gram of plant material was quisition software (Applied Biosystems/MDS Sciex). weighed and transferred to a cellulose thimble (Fisher The same column, guard column, buffers and gradient Scientific, Pittsburgh, USA). The sample was extracted program were used as for the HPLC analysis. Samples three times for one hour. The first two extractions were were diluted 50 times and 20 μl was injected. The pooled and the volume was adjusted to 100 ml in a ionization was performed by electrospray in the positive + volumetric flask. The volume of the third extraction mode ((M + H) and/or (M + NH4) adduct ions). The was adjusted to 20 ml. The podophyllotoxin concentra- source temperature was set to 450 °C. The instrument tion was determined by HPLC analysis. Samples were was operated with an ionspray voltage of 5.2 kV. stored at 4 °C before analysis. Nitrogen was used both for curtain gas and nebulizing gas. Full scan mass spectra were acquired at a scan rate Assessment of podophyllotoxin concentration by HPLC of 1 scan/4 s with a scan range of 100–1300 amu and a step size of 0.1 amu. To determine the amount of podophyllotoxin in the extracted samples, HPLC analysis was performed as Statistics previously described by Hendrawati and coworkers with some modifications (Hendrawati et al. 2011). A Statistical analysis was performed with SPSS 23 soft- Shimadzu-VP system (Shimadzu, ‘s-Hertogenbosch, ware and Matlab R2013b. For temperature and soil The Netherlands) was used, consisting of a LC-10AT study, two-way ANOVAwas used for significance test- pump, a SIL-20A auto sampler and a diode array detec- ing of the root biomass and multiple-way ANOVAwith tor SPD-M10A. For analysis a Zorbax Eclipse XDB- plant as nested factor for the podophyllotoxin content C18column(4.6id.×150mm;5μm, Agilent, Santa (mg/g or mg/plant). ANOVA analysis assumes normal Plant Soil (2017) 417:117–126 121 distribution and homoscedasticity of the residuals. The Table 1 Podophyllotoxin extraction by quick methanol and Soxhlet extraction data were loge transformed before ANOVA analysis. Outliers were discarded with 5% cumulative distribution Extraction method Extraction round PPT (mg/g) cut-off for the root biomass data and 2.5% cumulative distribution cut-off for the podophyllotoxin data (two- Methanol (n =4) 1–2 16.0 ± 0.4 sided). Factors were considered significant for p-values 3 2.4 ± 0.1 <0.05. Bonferroni multiple testing correction was per- 4 0.90 ± 0.02 formed for post hoc analysis and for the methyl 5 0.35 ± 0.03 jasmonate study. 6 0.15 ± 0.03 Soxhlet (n =3) 1–2 18.1 ± 0.6 3ND Results Podophyllotoxin content was determined by HPLC. Values pres- ent means ± standard deviation (d.w.). PPT: podophyllotoxin, ND: Validation of the quick methanol extraction method not detectable for podophyllotoxin extraction biomass was observed; therefore, we focused on the A novel, quick methanol extraction method was applied effect of soil and temperature after 40 days of cultivation in this study to process the large number of samples. To (Fig. 1a,Table4). Soil has an effect on root biomass validate this method the amount of podophyllotoxin in production. In peat-perlite soil (7 ± 2 g d.w.) higher root the roots was determined by the traditional Soxhlet biomass was observed than in sand soil (4 ± 2 g d.w.) for extraction method earlier described by Gupta and co- cultivation at 25 °C. No differences were observed at workers (Gupta et al. 2013). Furthermore, the presence 15 °C. Furthermore, after 80 days of cultivation the of podophyllotoxin was confirmed by LC-ESI-MS/MS. average root weight was increased three times more on The podophyllotoxin yield after two rounds of methanol peat-perlitesoil(13±2g)thanonsandsoil(3.6±0.2g, extraction was 16.0 ± 0.4 mg/g, after three rounds Fig. 1b). Next, the effect of temperature on root biomass 18.1 ± 0.6 mg/g and after six rounds 19.4 ± 0.5 mg/g production was determined. At 15 °C (8 ± 1 g d.w.) more (Table 1). On the contrary, only 18.1 ± 0.7 mg/g was root biomass was observed than at 25 °C (4 ± 2 g d.w.) for extracted by Soxhlet extraction. For the quick methanol cultivation in sand soil. However, this difference was not extraction method the intraday and interday variation observed in peat-perlite soil. Increase in root formation in were lower than 4% (Table 2). As the majority of time was only observed for peat-perlite at 15 °C (5 ± 2 g podophyllotoxin was extracted from P. hexandrum to 10 ± 3 g d.w.). roots in the first three fractions, the final extraction Besides root formation, the effect of soil and temper- protocol included three consecutive extraction rounds. ature on the podophyllotoxin production was also deter- The quick methanol extraction method is able to mined. Soil and temperature had no effect on extract podophyllotoxin from P. hexandrum roots podophyllotoxin production if normalized for biomass in high quantities. Moreover, this method uses less (mg/g d.w.) (Fig. 2a, Table 3). Higher podophyllotoxin solvent and is quicker, easier and more suitable to ex- production per plant (Fig. 2b, Table 3) was observed for tract multiple samples simultaneously than the Soxhlet cultivation in peat-perlite (126 ± 40 mg/plant d.w.) than extraction method.

Influence of soil and temperature on root formation Table 2 Intra-day and inter-day variation in quick methanol and podophyllotoxin production extraction Coefficient of variation (%) Root formation and podophyllotoxin production of P. hexandrum roots cultivated in a glasshouse were de- Conc. (μg/ml) Intra-day Inter-day termined for two soil types and two temperatures. Soil Podophyllotoxin 14.2 ± 0.6 3.1 3.8 type, temperature and cultivation time have effect on the Podophyllotoxin was extracted from five samples on each of the root formation as shown by two-way ANOVA (Fig. 1a, three analysis days (n = 15). Podophyllotoxin content was deter- Table 3). After 20 days of cultivation no increase in mined by HPLC 122 Plant Soil (2017) 417:117–126

Fig. 1 Influence of soil type and a temperature on the root biomass * of Podophyllum hexandrum * roots. a Root biomass production after 20 days (left) and 40 days (right) was determined. Means are shown by horizontal lines. Podophyllum hexandrum was cultivated in sand soil at 15 °C (●) or 25 °C (■) or in peat-perlite soil at 15 °C (▲)or25°C(▼). Post hoc analysis p <0.0042*is shown in the figure. b Visual comparison of P. hexandrum b roots cultivated for 80 days in sand soil (left, n = 2) and peat- perlite (right, n =3)

in sand soil (107 ± 26 mg/plant d.w.). Cultivation at Overall, we can conclude that peat-perlite soil and 15 °C (138 ± 29 mg/plant d.w.) had significantly higher 15 °C are the best conditions for root formation and production than at 25 °C (72 ± 25 mg/plant d.w.). podophyllotoxin production per plant.

Table 3 The influence of cultivation time, temperature and soil type on root biomass and podophyllotoxin production

Cultivation time (days) Temperature (°C) Soil type= Root Biomass (g) PPT (mg/g) PPT (mg/plant)

0 15 Pot 8±3 12±2 66±12 25 Pot 5 ± 3 15 ± 2 71 ± 11 20 15 Sand 5 ± 2 12 ± 2 62 ± 9 Peat-perlite 5±2 13±4 64±24 25 Sand 3±2 12±3 45±12 Peat-perlite 5±2 16±4 85±23 40 15 Sand 8 ± 1 13 ± 1 115 ± 12 Peat-perlite 10 ± 3 15 ± 2 160 ± 22 25 Sand 4±2 12±3 56±16 Peat-perlite 7±2 12±3 92±17 Soil *** ns *** Temperature *** ns ** Time *** ns *** Soil x Temperature x Time ns *** ***

Values present means ± standard deviation (d.w.) and ** and *** denote significance at 0.01 and 0.001 probability level, respectively. ns: non-significant, PPT: podophyllotoxin Plant Soil (2017) 417:117–126 123

Table 4 Bonferroni multiple testing correction for root biomass and podophyllotoxin production

Factor 1 vs. Factor 2 Root biomass (g) Podophyllotoxin (mg/plant) t =0,15°C t =0,25°C 0.0113 0.2990 t = 20, 15 °C, Sand t = 20, 15 °C, Peat-perlite 0.3163 0.7632 t = 20, 25 °C, Sand t = 20, 25 °C, Peat-perlite 0.0236 <0.0001 ** t = 20, 15 °C, Sand t = 20, 25 °C, Sand 0.0736 0.0007 * t = 20, 15 °C, Peat-perlite t = 20, 25 °C, Peat-perlite, 0.634 0.0643 t = 40, 15 °C, Sand t = 40, 15 °C, Peat-perlite 0.1173 <0.0001 ** t = 40, 25 °C, Sand t = 40, 25 °C, Peat-perlite 0.0011 * <0.0001 ** t = 40, 15 °C, Sand t = 40, 25 °C, Sand 0.0015 * <0.0001 ** t = 40, 15 °C, Peat-perlite t = 40, 25 °C, Peat-perlite, 0.0487 <0.0001 ** t = 20, 15 °C, Sand t = 40, 15 °C, Sand 0.0112 <0.0001 ** t = 20, 15 °C, Peat-perlite t = 40, 15 °C, Peat-perlite 0.0018 * <0.0001 ** t = 20, 25 °C, Sand t = 40, 25 °C, Sand 0.16 0.0363 t = 20, 25 °C, Peat-perlite t = 40, 25 °C, Peat-perlite 0.0251 0.2916

* and ** denote significance at 0.0038 and 0.0001 probability level, respectively

Influence of methyl jasmonate treatment treatment (1.5 mM solution) was insufficient to enhance on the production of podophyllotoxin the podophyllotoxin production after 9 days (Fig. 3a,p- value 0.39). Increasing the dosage to 2-fold (3 mM In literature, enhancement of certain secondary solution) and treatment on three consecutive days re- metabolites was obtained through chemical induc- sulted in 21% higher podophyllotoxin production tion by methyl jasmonate treatment. Therefore, we stud- (14±3mg/gto17±3mg/gd.w.,Fig.3b,p-value ied whether spraying of methyl jasmonate on 0.01). In conclusion, methyl jasmonate treatment on the P. hexandrum leaves can increase the amount of leaves can enhance the podophyllotoxin production in podophyllotoxin in the roots. A single methyl jasmonate the roots of P. hexandrum.

Fig. 2 Influence of soil type and a temperature on the podophyllotoxin production in Podophyllum hexandrum roots. a Biomass normalized podophyllotoxin production after 20 days (left) and 40 days (right), b Podophyllotoxin production per plant after 20 days (left)and 40 days (right). Means are shown by horizontal lines. P. hexandrum was cultivated in sand soil at b ** 15 °C (●)or25°C(■)orinpeat- ** ** perlitesoilat15°C(▲)or25°C (▼). Post hoc analysis: p-value ** ** <0.0042 (*) and p-value <0.0001 * (**) are shown in the figure 124 Plant Soil (2017) 417:117–126

2009;Kumarietal.2014). Therefore, root biomass and podophyllotoxin production were determined for two soil types (sand and peat-perlite) and two temperatures (15 °C and 25 °C). As expected controlling soil aeration and water drainage by perlite in combination with the properties of peat to absorb moisture and nutrients re- sulted in the highest root biomass per plant. Lower temperatures are favorable for root formation as the root biomass production was higher at 15 °C than at 25 °C. However, the podophyllotoxin production normalized to the root biomass was neither effected by various soils nor temperatures. This is in contrast to the report of Kumari and coworkers who observed a decreasing trend of podophyllotoxin accumulation at 25 °C in growth chambers under controlled artificial light (Kumari et al. 2014). This contrast in findings can be explained by the importance of sunlight duration on the production of podophyllotoxin (Cushman et al. 2005; Liu et al. 2015). Kumari’s study was performed under constant artificial Fig. 3 Influence of methyl jasmonate on the amount of light and our study was done in natural daylight, which podophyllotoxin in Podophyllum hexandrum roots. a Single methyl varied at 15 °C (1158 ± 450 joules/cm2) and 25 °C jasmonate treatment (1.5 mM, harvested after 9 days), b additional 2 treatment on three consecutive days (3 mM, harvest on the fourth (1554 ± 531 joules/cm ). day), Means are shown by horizontal lines and the baseline by the Methyl jasmonate is known to upregulate various dotted line. Post hoc analysis p-value <0.025 (*) is shown in the transcription factors, such as R2R3 Myb. This transcrip- figure. Treatment symbols: ● = water; ■ = methyl jasmonate tion factor upregulates the rate-limiting steps in the phenylpropanoid biosynthesis, which is followed by the Discussion lignan pathway were podophyllotoxin is produced (Gális et al. 2006). Treatment of the leaves of the P. hexandrum Podophyllotoxin is the precursor for high value anti- plant with methyl jasmonate indeed increased the cancer drugs, such as etoposide and teniposide. The podophyllotoxin production in the roots by 21%. This scarcity of the podophyllotoxin source, P. hexandrum, observation is in line with the findings of Bhattacharyya in nature has led to the need of controlled cultivation. in cell cultures of P. hexandrum (Bhattacharyya et al. Furthermore, increase of podophyllotoxin production per 2012). As previously observed by Baldwin, treatment plant will reduce the number of plants necessary for of the leaves triggers the secondary metabolite production podophyllotoxin extraction. Therefore, the influences of (nicotine) in the roots (Baldwin 1996). In our study, the soil, temperature and methyl jasmonate treatment on the podophyllotoxin production was increased from 1.4 to root biomass formation and podophyllotoxin production 1.7% (d.w.). These percentages are also observed for were determined by cultivation of P. hexandrum in a P. hexandrum in the natural habitat (Purohit et al. 1999; glasshouse in the Netherlands. First, a novel quick meth- Alam et al. 2009; Kitchlu et al. 2011;Sharmaetal.2012; anol extraction method was designed for the extraction of Liu et al. 2015;Pandeyetal.2015). Our study describes podophyllotoxin from P. hexandrum roots in order to the ability to improve the root formation and the process the large number of samples. The extraction yield podophyllotoxin production in isogenic P. hexandrum of this method is comparable to Soxhlet. However, the cultivated in a glasshouse in the Netherlands. The sea- benefit of the quick methanol extraction method is a sonal variations in temperature in the Netherlands are shorter and easier extraction procedure and usage of less optimal for cultivation of P. hexandrum in glasshouses organic solvent. without extra heating expenses. The advantage of grow- Environmental conditions like soil composition and ing in a glasshouse is the ability to control cultivation temperature are important parameters for P. hexandrum conditions, such as light and water. Furthermore, pest cultivation and podophyllotoxin production (Alam et al. control can be applied in order to create stable and robust Plant Soil (2017) 417:117–126 125 conditions for growth of this medicinal plant. This study Cushman KE, Moraes RM, Gerard PD et al (2006) Frequency and paves the way for large-scale cultivation of P. hexandrum timing of removal affect growth and podophyllotoxin content of Podophyllum peltatum in full sun. Planta Med 72: in the temperate latitudes for the production of the phar- 824–829. doi:10.1055/s-2006-946675 maceutically important podophyllotoxin. De Geyter N, Gholami A, Goormachtig S, Goossens A (2012) Transcriptional machineries in jasmonate-elicited plant second- Acknowledgements We thank Filipa Bico for her support in the ary metabolism. Trends Plant Sci 17:349–359. doi:10.1016/j. glasshouse. We thank C.M. Jeronimus-stratingh of the Mass tplants.2012.03.001 Spectrometry Core Facility of the University of Groningen for the Dennis MK, Burai R, Ramesh C et al (2009) In vivo effects of a LC-ESI-MS/MS analysis. This research was financially supported GPR30 antagonist. Nat Chem Biol 5:421–427. doi:10.1038 by EU regional funding: the PhytoSana project in the INTERREG /nchembio.168 IV A Deutschland-Nederland program (34- INTERREG IVA I-1- Gális I, Simek P, Narisawa T et al (2006) A novel R2R3 MYB 01 = 193 PhytoSana). transcription factor NtMYBJS1 is a methyl jasmonate- dependent regulator of phenylpropanoid-conjugate biosyn- Open Access This article is distributed under the terms of the thesis in tobacco. Plant J 46:573–592. doi:10.1111/j.1365- Creative Commons Attribution 4.0 International License (http:// 313X.2006.02719.x creativecommons.org/licenses/by/4.0/), which permits unrestrict- Guerram M, Jiang Z-Z, Zhang L-Y (2012) Podophyllotoxin, a ed use, distribution, and reproduction in any medium, provided medicinal agent of plant origin: past, present and future. Chin you give appropriate credit to the original author(s) and the source, J Nat Med 10:161–169. doi:10.3724/SP.J.1009.2012.00161 provide a link to the Creative Commons license, and indicate if Guo Q, Zhou J, Wang Z, Yang H (2012) In vitro rooting of changes were made. Podophyllum hexandrum and transplanting technique. Engineering 4:142–145. doi:10.4236/eng.2012.410B037 Gupta DK, Verma MK, Lal S et al (2013) Extraction studies of Podophyllum hexandrum using conventional and nonconven- References tional methods by HPLC-UV-DAD. J Liq Chromatogr Relat Technol 37:259–273. doi:10.1080/10826076.2012.745134 Häkkinen ST, Rischer H, Laakso I et al (2004) Anatalline and Alam MA, Gulati P, Gulati AK et al (2009) Assessment of genetic other methyl jasmonate-inducible nicotine alkaloids from diversity among Podophyllum hexandrum genotypes of the Nicotiana tabacum cv. By-2 cell cultures. Planta Med 70: north-western Himalayan region for podophyllotoxin pro- 936–941. doi:10.1055/s-2004-832620 – duction. Indian J Biotechnol 8:391 399 Hendrawati O, Woerdenbag HJ, Hille J et al (2011) Seasonal Alam MA, Naik PK (2009) Impact of soil nutrients and variations in the deoxypodophyllotoxin content and yield of environmental factors on podophyllotoxin content among Anthriscus sylvestris L. (Hoffm.) grown in the field and under 28 Podophyllum hexandrum populations of northwest- controlled conditions. J Agric Food Chem 59:8132–8139. ern Himalayan region using linear and nonlinear ap- doi:10.1021/jf200177q proaches. Commun Soil Sci Plant Anal doi:10.1080 Imbert TF (1998) Discovery of . Biochimie 80: /00103620903111368 207–222. doi:10.1016/S0300-9084(98)80004-7 Baldi A, Dixit VK (2008) Yield enhancement strategies for Ionkova I, Antonova I, Momekov G, Fuss E (2010) Production of artemisinin production by suspension cultures of Artemisia podophyllotoxin in Linum Linearifolium in vitro cultures. annua. Bioresour Technol 99:4609–4614. doi:10.1016/j. Pharmacogn Mag 6:180–185. doi:10.4103/0973-1296.66932 biortech.2007.06.061 Jackson DE, Dewick PM (1985) Tumour-inhibitory aryltetralin Baldwin IT (1996) Methyl jasmonate-induced nicotine production lignans from . Phytochemistry 24: in Nicotiana attenuata: inducing defenses in the field without 2407–2409. doi:10.1016/S0031-9422(00)83052-6 wounding. Entomol Exp Appl 80:213–220. doi:10.1111 Kharkwal AC, Kushwaha R, Prakash O et al (2008) An efficient /j.1570-7458.1996.tb00921.x method of propagation of Podophyllum hexandrum:anen- Bastos JK, Burandt CL, Nanayakkara NPD et al (1996) dangered medicinal plant of the western Himalayas under ex Quantitation of aryltetralin lignans in plant parts and among situ conditions. J Nat Med 62:211–216. doi:10.1007/s11418- different populations of Podophyllum peltatum by reversed- 007-0217-9 phase high-performance liquid chromatography. J Nat Prod Kitchlu S, Ram G, Koul S et al (2011) Podophyllum lignans array 59:406–408. doi:10.1021/np960155d of Podophyllum hexandrum Royle populations from semi- Bhattacharyya D, Sinha R, Ghanta S et al (2012) Proteins differ- desert alpine region of Zanskar valley in Himalayas. Ind Crop entially expressed in elicited cell suspension culture of Prod 33:584–587. doi:10.1016/j.indcrop.2010.12.010 Podophyllum hexandrum with enhanced podophyllotoxin Kumari A, Singh HR, Jha A et al (2014) Transcriptome sequenc- content. Proteome Sci 10:34. doi:10.1186/1477-5956-10-34 ing of tissue of hexandrum at two Canel C, Moraes RM, Dayan FE, Ferreira D (2000) temperatures. BMC Genomics 15:871. doi:10.1186/1471- Podophyllotoxin. Phytochemistry 54:115–120. doi:10.1016 2164-15-871 /S0031-9422(00)00094-7 Liu W, Liu J, Yin D, Zhao X (2015) Influence of ecological factors Cushman KE, Maqbool M, Lata H et al (2005) Podophyllotoxin on the production of active substances in the anti-cancer plant content and yield of American mayapple leaves in sun and Sinopodophyllum hexandrum (Royle) T.S. Ying. PLoS one shade. Hortscience 40:60–63 10:e0122981. doi:10.1371/journal.pone.0122981 126 Plant Soil (2017) 417:117–126

Moraes RM, Bedir E, Barrett H et al (2002) Evaluation of populations of Podophyllum hexandrum. Curr Sci 77:1078– Podophyllum peltatum accessions for podophyllotoxin pro- 1079 duction. Planta Med 68:341–344. doi:10.1055/s-2002-26740 Sharma TR, Singh BM, Sharma NR, Chauhan RS (2012) Moraes RM, Burandt C, Ganzera M et al (2000) The American Identification of high podophyllotoxin producing biotypes of mayapple revisited—Podophyllum peltatum—still a potential Podophyllum hexandrum royle from north-western Himalaya. cash crop? Econ Bot 54:471–476. doi:10.1007/BF02866546 J Plant Biochem Biotechnol 9:49–51. doi:10.1007 Nadeem M, Palni LMS, Purohit AN et al (2000) Propagation and /BF03263084 conservation of Podophyllum hexandrum Royle: an impor- Shoji T, Kajikawa M, Hashimoto T (2010) Clustered transcription tant medicinal herb. Biol Conserv 92:121–129. doi:10.1016 factor genes regulate nicotine biosynthesis in tobacco. Plant /s0006-3207(99)00059-2 Cell 22:3390–3409. doi:10.1105/tpc.110.078543 Pandey H, Kumar A, Palni LMS, Nandi SK (2015) Suttipanta N, Pattanaik S, Kulshrestha M et al (2011) The tran- Podophyllotoxin content in rhizome and root samples of scription factor CrWRKY1 positively regulates the terpenoid Podophyllum hexandrum Royle populations from Indian indole alkaloid biosynthesis in Catharanthus roseus. Plant Himalayan region. J Med Plant Res 9:320–325. Physiol 157:2081–2093. doi:10.1104/pp.111.181834 doi:10.5897/JMPR2014.5627x Ting CP, Maimone TJ (2014) C-H bond arylation in the synthesis of Pandey HK, Nandi SK, Palni LMS (2013) Podophyllotoxin con- aryltetralin lignans: a short total synthesis of podophyllotoxin. tent in leaves and stems of Podophyllum hexandrum Royle Angew Chem Int Ed Eng 53:3115–3119. doi:10.1002 from Indian Himalayan region. J Med Plant Res 7:3237– /anie.201311112 3241. doi:10.5897/JMPR2013.4491 Todd AT, Liu E, Polvi SL et al (2010) A functional genomics Paul S, Nandi SK, Palni LMS (2013) Assessment of genetic screen identifies diverse transcription factors that regulate diversity and interspecific relationships among three species alkaloid biosynthesis in Nicotiana Benthamiana. Plant J 62: of Podophyllum using AFLP markers and podophyllotoxin 589– 600. doi:10.1111/j.1365-313X.2010.04186.x content. Plant Syst Evol 299:1879–1887. doi:10.1007 van der Fits L (2000) ORCA3, a jasmonate-responsive transcrip- /s00606-013-0844-4 tional regulator of plant primary and secondary metabolism. Petersen M, Alfermann W (2001) The production of cytotoxic Science. 289:295–Scie297. doi:10.1126/science.289.5477.295 lignans by plant cell cultures. Appl Microbiol Biotechnol 55: Zheljazkov VD, Cantrell CL, Astatkie T (2011) Variation in 135–142. doi:10.1007/s002530000510 podophyllotoxin concentration in leaves and of Purohit MC, Bahuguna R, Maithani UC et al (1999) Variation in American mayapple (Podophyllum peltatum L.) Ind Crop podophylloresin and podophyllotoxin contents in different Prod 33:633–637. doi:10.1016/j.indcrop.2010.12.025