Effect of Harvest Season, Nitrogen, Phosphorus and Potassium on Root Yield, Echinacoside and Alkylamides in Echinacea angustifolia L.

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1 Effect of Harvest Season, Nitrogen, Phosphorus and Potassium on Root Yield, Echinacoside and Alkylamides in Echinacea angustifolia L. in Chile M. Berti, R. Wilckens and S. Fischer Facultad de Agronomía Departamento de Producción Vegetal Universidad de Concepción Casilla 537, Chillán Chile F. Hevia Facultad de Ingeniería Agrícola Departamento de Agroindustria Universidad de Concepción Casilla 537, Chillán Chile Keywords: fertilisation, phenology, phenological stages, development, active principles, biomass, yield Abstract Echinacea angustifolia L., originally from North America, is one of the three Echinacea species that traditionally have been used to stimulate the body's immune system. The active principles of the species are mainly echinacoside, alkylamides and cynarin. The amount of these components in the roots depends on many factors, which have to be investigated. The objectives of this research was to determine the influence of phenological stage and N, P and K on root yield, echinacoside and alkylamides content on a one year old E. angustifolia crop. Experimental design was a randomised complete block with 4 replicates and 12 treatments with a factorial arrangement of three levels of nitrogen (, 15, and 3 kg ha -1 ), two of P (.1 kg ha -1 ) and two of K (.1 kg ha -1 ). Echinacoside and alkylamide contents were determined using an HPLC. Results indicate that echinacoside and alkylamides content are strongly influenced by the phenological stage. Highest contents of echinacoside were observed before immature buds elongate 1 to 2 cm above the crown (stage = R 2 ) and echinoside drops rapidly after this phenological stage, except at one location. Also, a negative correlation between root yield and echinacoside and alkylamides content was observed. N, P, K or their interactions did not significantly influence root yield at the first harvest date. A N x P significant interaction for alkylamides was detected. Also K had a significant effect on echinacoside content. When the supply of K was increased the amount of echinacoside became higher at both harvest dates. However, individuals of E. angustifolia show very high variability for root yield and active principles, which made difficult to detect statistical differences among treatments. INTRODUCTION Echinacea angustifolia L. occurs originally in North America and it is one of the three Echinacea species that traditionally have been used to stimulate the body's immune system (Blumenthal et al., 2, Alonso, 1998, Bergner, 1997). Most of E. angustifolia used to produce phytopharmaceuticals in the United States is wild grown. However, there is a concern for Echinacea species being over-harvested in some locations in the US (Hulburt, 1999, Bourne, 2). E. angustifolia cultivation is more difficult that its relatives E. purpurea and E. pallida. Production information has been developed for E. purpurea mainly and there are only few reports on E. angustifolia crop management. E. angustifolia active principles are echinacoside, alkylamides and cynarin. A high variability in plant size, flowering and echinacoside and alkylamides content among individuals in E. angustifolia populations has been observed (Franke and Schenk, 1999). And it is also known that the cultivated E. angustifolia has a lower echinacoside content than wild E. angustifolia Moreover, active principles in the roots may be influenced by factors like phenological stages, nutrient supply, water supply, soil ph, soil texture, etc. Research conducted in E. purpurea indicates that alkylamides and chicoric acid may vary with the phenological stage and botanical structure (Stuart and Wills, 2). E. Proc. Int. Conf. on MAP Eds. J. Bernáth et al. Acta Hort. 576, ISHS 22 33

2 purpurea roots had approximately 7 % of the total plant alkylamides decreasing after flowering. Conversly, chicoric acid in the roots increased after flowering (Stuart and Wills, 2). Fertilisation of E. purpurea plants indicated that in absence or at low levels of nitrogen fertilisation ( and 1 kg acre -1 ) the addition of 5 and 1 kg acre -1 of potassium increased aerial parts, flower heads and root yield. However, influence of nitrogen and potassium on active principles was not reported in this study (Shalaby et al., 1997). Another report indicated that highest aerial biomass and root yield in E. purpurea was obtained with 1 kg ha -1 of nitrogen at constant rates of phosphorus and potassium. Polyphenol content was not influenced by nitrogen fertilisation and values fluctuated between 2.4 and 5.4 % in the aerial part at flowering and between 1.6 and 3.5 % in the roots (Kucharski, 1997). Franke and Schenk (1999) reported a slight increase in root yield of E. pallida when nitrogen was added. Fertilisation with nitrogen caused a decrease in the concentration of echinosides. Echinoside content was 1.16 % without nitrogen fertilisation, and.94 % with nitrogen fertilisation. In order to get more information on E. angustifolia cultivation, this study s objective was to determine the influence of phenological stages, nitrogen-, phosphorousand potassium fertilisation on root yield, echinacoside and alkylamides content in E. angustifolia cultivated at four locations in Chile. MATERIALS AND METHODS Study of the Phenological Stages This trial was conducted at four locations in southern Chile: El Carmen, Talca, Sta. Juana and Chillán, Chile. Before planting E. angustifolia seeds were pre-treated with a.1 um Etherphon solution for 5 minutes and then kept at 4 C for 15 days in a 5 % soil and 5 % sand mixture. After this the seeds were set at 2 C and when radicles emerged seeds were set in a 162 round cell TLC Pro-Tray. The previous treatment was done to break seed dormancy. When plantlets had four true leaves they were transplanted to raised beds in the field. Plant density at planting was 5.5 plants m -2 at all locations. Results of soil analyses made before planting at all locations are shown in Table 1. Planting dates were: September 2 th in Chillán, September 27 th through 3 th in El Carmen, November 2 th through 25 th in Santa Juana and December 1 st through 3 rd in Talca, all in Fertilisation applied at each location was 15 kg ha -1 N, 1 kg P 2 O 5 ha -1, 1 K 2 O ha -1 and 5 kg B ha -1. At all locations Echinacea plants were irrigated during the summer and weed control was done by hand weeding. A plant development staging system was made to easily compare results among locations and dates. A modification of the system described for sunflower by Schneiter and Miller (1981) was used for these trials. Developmental stages recorded were as follows: 1. Vegetative stages. From seed: Ve: Seedling has emerged, but the first leaf is less than.5 cm long, cotyledons are not counted; V 1, V 2,...V n These are determined by counting the number of true leaves completely expanded. From crown: V 1.1 Latent bud: Bud is still dormant and less than 1 mm long; V 1.2 Expanding bud: Bud is longer than 1 mm and leaves are not fully expanded; V 1.3 Expanded leaves: At least one leaf is fully expanded; V 1.n Expanded leaves: Number of expanded leaves before flower head appears in the apex of the stem. Reproductive stages: R 1 : The terminal bud of each stem forms a miniature floral head less than 1 cm above the apex. When viewed from directly above, the immature bracts form a star-like structure; R 2 : The immature bud elongates 1 to 2 cm above the nearest leaf of the crown; R 3 : The immature bud elongates to a distance more than 2 cm above the nearest leaf; R 4 : The inflorescence opens showing the white to pink coloured ligulated flowers; R 5.1 : Inflorescence is completely open, ligulated flowers are less than 1 cm long; R 5.2 : inflorescence is completely open, ligulated flowers are longer than 1 cm 34

3 long, but anthesis has not started; R 5.3 : Pollen is visible on disk flowers in less than 3% of the disk; R 5.4 : Over 3% of the disk flowers have pollen visible; R 5.5 : Anthesis has been completed in the whole inflorescence and seeds are being developed. This plant staging system is applicable to E. angustifolia, E. pallida and E. purpurea from seed and after the first year from crown. Evaluations: Each sample had 2 roots at a similar plant developmental stage that was recorded at sampling. Roots were cut 4 cm if longer than that and dried at 4 ºC. Root yield and aerial biomass were recorded for each sample. 2. Fertilisation trial. Trial was conducted at the Experimental Station of the University of Concepción in Chillán (36 26 South, 72 6 W at 144 m.o.s.l.). The soil is a "typical haploxerand" and soil analysis made before planting is shown in Table 1. Experimental design was a randomised complete block with 4 replicates and 12 treatments with a factorial arrangement of three levels of nitrogen (, 15, and 3 kg ha -1 ), two of phosphorus (, 1 kg P 2 O 5 ha -1 ) and two of K (, 1 kg K 2 O ha -1 ). Source of nitrogen was urea, source of phosphorous was triple phosphate, and source of potassium was potassium sulphate. The trial was planted on September of The stand was hand weeded and irrigated during summer. The trial was harvested at two different times, November 21 st and 22 nd, and April 2 nd 4 th. Roots were hands harvested and dried at 4 C. Dry aerial biomass, root yield, echinacoside and alkylamide content were recorded. 3. Analysis of alkylamides and echinacoside. E. angustifolia roots were washed with water and then dried to 4 C in an oven with air convection. Roots were milled to a 5 mesh, samples were extracted for 16 hours with methanol in a Soxhlet apparatus. The echinacoside and alkylamides content were determined according to the method described by Stuart and Wills (2) in a Merck-Hitachi L-62 HPLC cromatograph with an L- 42UV-visible detector and a 1 RP-18 5 µm Licrosphere column with a pre-column LichroCart 4-4, Flow: 1mL/min, injection volume 2 µl. Detections were made at 254 nm for alkylamides and at 235 nm for echinacoside with 6 minutes of detection. Solubilization solvent was methanol (HPLC grade) and as a reference standard for echinacoside, an INDENA standard was used. Analysis conditions were Solvent A: Water HPLC grade, Solvent B: Trifluoracetic acid.1% in acetonitrilo (HPLC). A linear gradient as shown in Table 2 was used. 4. Statistical analysis. The ANOVA procedure was used for fertilisation trial and analysis was done with the SAS statistical package. RESULTS Study of the Phenological Stages The results of the experiments done in three different locations are presented in Fig. 1, 2 and 3. Echinacea remains dormant during wintertime and buds in the crown start to elongate as soil temperatures increase early in the Spring. Root yield decreased in the first stages of bud elongation until leaves were fully expanded. Root dry mass decreased until R 2 and R 4 stages in Chillán and Talca (Fig. 3), respectively. Both Chillán and Talca are warmer and dryer locations than the rest. After these stages root dry mass increased until the end of reproductive stage indicating that plant starts to translocate photosynthetates to the root in these stages. Echinacoside and alkylamide content fluctuated following the same trend through the season. The highest echinacoside and alkylamide content was observed at the multiple expanded leaves V 1.1 stage, with values of 1.492% and 1.43%, respectively (Fig. 1). Echinacoside and alkylamides decreased after reproductive structures appear in the plant, until seed set. In Santa Juana, a similar fluctuation of echinacoside to the one at Chillán was observed, but at this location alkylamides variation was not parallel to the echinacoside curve. Echinacoside content, however, was much higher than at Chillán at the beginning of the season reaching the highest value at 2.49% (Fig. 2). There is a negative correlation between echinacoside content and root yield that 35

4 may explain somewhat the difference of echinacoside content at different locations. The results at El Carmen location were quite different because echinacoside remained above 1.9 % for a longer period of time until R 5.2 stage reaching its highest value of 2.342% on January 18 th at R 4. It is important to notice that at this last location the stage R 5.3 occurred almost two months later than in Chillán, however both were planted with only two weeks difference (Fig. 4). Fertilisation Trial The ANOVA procedure did not detect significant differences for N, P, and K main effects and their interactions for the first harvest date. For the second harvest date a significant effect was observed for K for echinacoside content and for the N x P interaction for alkylamides (Tables 3 and 4). Although main effect of N was not significant, a clear trend to reduce aerial biomass and root yield was observed as N rate increased, especially in November (Table 2 and 3). For the second harvest date both aerial and root yield decreased with 15 kg N ha -1 supply, but increased again with the higher rate. Echinacoside content did not show difference at the time of first harvest, but its amount decreased in April as a result of higher N dosage. In both harvests dates the amount of alkylamides increased parallel to N dosages. Phosphorous and potassium did not have remarkable effect on aerial biomass, root yield, echinacoside or alkylamides. However, potassium had a special influence on echinacoside content and a significant difference was detected at the time of second harvest. A significant interaction between nitrogen and phosphorus for alkylamides content was detected by ANOVA for the second harvest date, which can be observed in Table 4. DISCUSSION It is well known phenomenon that the wild-collected E. angustifolia has higher echinacoside content than the cultivated one. It is also interesting that in the majority of countries where the cultivation of E. angustifolia is going on, the roots are harvested in the fall. In spite of this our data indicates that echinacoside and alkylamides content decrease considerably in the fall. In our experiment the amount of echinacoside decreased in three locations, continuously, after multiple expanded leaves stage V 1.1. The plant at this stage utilises all its energy to grow and form floral buds. Translocation of photosynthetates are accelerated in this stage from roots to the aerial part. It might be possible, but needs more detailed investigations, that both echinacoside and alkylamides can be used by the plant as sources of energy to synthesize amino acids. Root yield is inversely correlated with echinacoside and alkylamide content, probably due to a dilution effect of the metabolites. Temperature during development may effect the fluctuation of echinacoside and alkylamides content, too. The plants grown at El Carmen, which can be characterised by cooler climate, accumulated more echinacoside. The effect of fertilisation on production and active agent content of Echinacea was proved as well. Nitrogen, in spite of literature data (Shalaby et al., 1997 and Kucharski, 1997), had a negative influence on root and aerial biomass of the plant. However, the reduction of echinacoside content as a result of increasing nitrogen supply, which had been reported by Franke and Schenk (1999), was observed in our trial only at the time of the second harvest. Phosphorus did not have remarkable effect on any parameters of the plants at the time of the first harvest. It must be due to the good P supply of the local soils, which is proved by the data of soil analysis performed (Table 1). At the time of the second harvest only alkylamides were affected by the N x P interaction, which may mean that alkylamides are negatively influenced by large amount of phosphorus available in the soil. Potassium had a remarkable effect on echinacoside content increasing its value in 36

5 both harvest dates, although the soil had plenty of potassium supply. This nutrient has an important role on sugar translocation to the root, which is probably utilised for synthesis of echinacoside. Literature Cited Alonso, J Tratado de Fitomedicina, Bases Clínica y Farmacológicas. ISIS Ediciones, Argentina Bergner, P The Healing Power of Echinacea and Goldenseal and Other Immune System Herbs. Prima Publishing, California, United States. Blumenthal, M., Goldberg, A. and Brinckmann, J. 2. Herbal Medicine. American Botanical Council, Integrative Medicine Communications, Newton, MA Bourne, J. 2. On the trail of sang poachers. Audubon. March-April Franke, R. and Schenk, R Echinacea influences of cultivation method on yield and content of active principles. Echinacea Symposium. Kansas City, USA Hulburt, D Endangered Echinacea- what threat, which species and where? United Plant Savers Newsletter, Summer 4-6. Kucharski, W. A Anbautechnology und Pflanzenschutz von Echinacea purpurea. Drogenreport 16: Schneiter, A.A. and Miller, J.F Description of sunflower growth stages. Crop Sci. 21: Shalaby, A.S, El-Gengaihi, S.E., Agina, E.A., El-Khayat, A.S. and Hendawy, S.F Growth and yield of Echinacea purpurea L. as influenced by plant density and fertilisation. J.l of Herbs, Spices and Medicinal Plants 5(1): Stuart, D.L. and Wills. R.B.H. 2. Alkylamide and chicoric acid levels in Echinacea purpurea tissues during plant growth. J. Herbs, Spices and Medicinal Plants. 7(1): Tables Table 1. Result of soil analysis made at different locations Soil characteristics Location El Carmen Talca Santa Juana Chillán ph N-NO 3 mg kg P Olsen mg kg Organic Matter % K (cmol + kg -1 ) Ca (cmol + kg -1 ) Mg (cmol + kg -1 ) Na (cmol + kg -1 ) Fe mgkg Mn mgkg Zn mgkg Cu mgkg B mgkg

6 Table 2. Effect of N, P and K on aboveground biomass of Echinacea angustifolia Nutrient (kg ha -1 ) First harvest (November 21-22) Second harvest (April 2-4) Aerial biomass (dry yield, kg ha -1 ) N P 2 O K S y C.V. % Table 3. Effect of N, P, and K on root yield, echinacoside and alkylamide content in Echinacea angustifolia Nutrient kg ha -1 First harvest (November 21-22) Second harvest (April 2-4) Echinacoside Alkylam Echinaco (%) ides (%) side (%) Root yield dry weight kg ha -1 Root yield dry weight kg ha -1 Alkylam ides (%) N P 2 O K a b.67 S y C.V. % S y : Standard error. Values within columns followed by different letters are significantly different (P >.5). N x P interaction for the second harvest was significant Table 4. Interaction effect of N and P for alkylamide content in the second harvest date Alkylamide content (%) N supply (kg ha -1 ) P (kg ha -1 ) P1 (kg ha -1 )

7 Figures Root yield (kg ha -1 ) Expanding bud Expanded leaves Multiple expanded leaves R2 R5.3 R5.5 8/28/2 9/21/2 9/29/2 11/6/2 12/7/2 1/11/21 Phenological stage and harvest date Fig. 1. Root yield, echinacoside and alkylamides content at Chillán, Chile Active Principles (%) Fresh weight (kg ha-1) Dry weight (kg ha-1) Echinacoside Total AA root yield kg ha Fresh weight kg ha Active Principles (%) Latent bud Emerging leaves Expanded leaves R2 8/3/29/21/21/18/211/8/212/5/212/26/22/1/21 R4-R5.1 Phenological stage and harvest date R5.1 R5.5 Fresh weight kg ha-1 Dry weight kg ha-1 Echinacoside total AA Fig. 2. Root yield, echinacoside and alkylamides content at Santa Juana, Chile 39

8 4 2 Root yield kg ha Active principles (%) Fresh weight Dry weight Echinacoside Expanded leaves R4 R5.2 and R5.3 R5.5 Total AA 1/6/2 11/8/2 1/19/21 3/15/21 Phenological stages and harvest dates Fig. 3. Root yield, echinacoside and alkylamides content at Talca, Chile Root yield kg ha Latent buds Expanded leaves in buds R1and R2 R4 R5.2 R5.4 y R Active Principles (%) 8/29/2 1/5/2 11/7/2 1/18/21 2/7/21 4/1/21 Fresh weight Phenological stages and harvest dates Dry weight Echinacoside Total AA Fig. 4. Root yield, echinocoside and alkylamides content at El Carmen, Chile 31