INTRODUCTION
Echinacea are herbaceous perennials of the Asteraceae family native to North America, with wild populations ranging from the eastern and central United States to southern Canada. They are widely used for wild flower establishment, perennial gardening and as a cut flower (Wartidiningsih and Geneve, Reference Wartidiningsih and Geneve1994). However, three species of Echinacea are used medicinally: E. purpurea, E. pallida and E. angustifolia (Mistríková and Vaverková, Reference Mistríková and Vaverková2007) because of their antiviral, antibacterial and immunostimulatory benefit to human (Barrett, Reference Barrett2003; Merali et al., Reference Merali, Binns, Paulin-Levasseur, Ficker, Smith, Baum, Brovelli and Arnason2003; Miller, Reference Miller2005; Murch et al., Reference Murch, Peiris and Shi2006). All three species of Echinacea have shown immune-modulating activity (Binns et al., Reference Binns, Livesey, Arnason and Baum2002). This activity appears to result from the combined effects of caffeoyl phenols, alkylamides and polysaccharides, which are present in all three Echinacea species but in different amounts (Briskin, Reference Briskin2000; Speroni et al., Reference Speroni, Govoni, Guizzardi, Renulli and Guerra2002; Randolph et al., Reference Randolph, Gellenbeck, Stonebrook, Brovelli, Qian, Bankaitis-Davis and Cheronis2003). Wild Echinacea populations are reportedly threatened by over-harvesting and anthropogenic modifications of habitats, and therefore, Echinacea species used medicinally are now being cultivated to meet the huge market demand (Li, Reference Li1998). Cultivated Echinacea populations are mainly located in the USA and Canada; nevertheless, Europe, Russia and Australia also have well-established Echinacea cultivation (Kreft, Reference Kreft2005; Letchamo et al., Reference Letchamo, Polydeonny, Gladisheva, Arnason, Liversey, Awang, Janick and Whipkey2002; Seidler-Lozykowska and Dabrowska, Reference Seidler-Lozykowska and Dabrowska2003; Willis and Stuart, Reference Wills and Stuart1999).
E. purpurea has been studied extensively in Europe and North America. Many types of phytomedicine are commercially produced from the aerial portions of E. purpurea for the prevention and treatment of the common cold and other upper respiratory infections, and the stimulation of immunomodulation (Goel et al., Reference Goel, Lovin, Chang, Slama, Barton, Gahler, Bauer, Goonewardene and Basu2005; Lindenmuth and Lindenmuth, Reference Lindenmuth and Lindenmuth2000; Mahady et al., Reference Mahady, Qato, Gyllenhaal, Chadwick and Fong2001; Vimalanthan et al., Reference Vimalanthan, Kang, Amiguet, Liversey, Arnason and Hudson2005). It has been recently introduced in to Taiwan and appears to grow well (Lin, Reference Lin2003). However, information regarding the effects of genetic diversity, growing climates and cultivation practices on active constituents (e.g. caffeoyl derivatives) and biomass production of E. purpurea is still very limited. It is known that the phytochemical traits of medicinal plants, depend on growing sites, climate conditions, cultural practices, vegetation phases and genetic modifications, and vary considerably within and between wild and cultivated populations (Millauskas et al., Reference Millauskas, Venskutonis and Van Beek2004). The objectives of the present study were to examine the biomass production of an E. purpurea population, which was selected in a previous study (Lin, Reference Lin2003), grown in different seasons. Several caffeoyl derivatives were also determined and compared between the E. purpurea populations harvested. The collected data may help us to select and breed a morphologically superior E. purpurea population with desirable active constituents that are adapted to the environmental conditions of Taiwan.
MATERIALS AND METHODS
Seeds of Echinacea purpurea selected from a consecutive mass selection (the spring crop of 2003) were used. In June 2003, selected seeds were soaked in running water for 8 h, and then planted in 104-plugs filled with a mixture of peat moss and vermiculite (3:1) at a depth of 1.5 cm, and watered as necessary (Figure 1A). The indoor-raised seedlings with four to five leaves were transplanted to the experimental farm of the Department of Agronomy, National Chung Hsing University in July 2003. The seedlings were planted on raised two-row beds (1 m wide and 6 m long with 30 cm bed spacing) covered with silver-black polyethylene sheets for weed control (Figure 1B). The plant spacing was 30 × 30 cm. Pre-plant fertilizers were applied at the rates of 100 kg N ha−1, 60 kg P2O5 ha−1 and 100 kg K2O ha−1.
The growth and development of E. purpurea plants. (A) seedlings plugs in nursery. (B) transplanted seedlings. (C) transplanted E. purpurea plants started to flower. (D) rhizome-regenerated E. purpurea plants started to flower. (E) the E. purpurea plants at full bloom. (F) 18-months-old rhizomes and roots.
For biomass determinations, plant samples, composed of two rows 3 m long, were taken at the full flower stage (Figure 1E). For 2003 autumn crops (Figure 1C), the plants were harvested in December 2003. The number of flowers produced per plant was counted and recorded, and the plants were then cut at 10 cm above ground level. The plots were irrigated after the completion of harvest. The plants regenerated from rhizomes in January 2004. The regenerated and developed plants (Figure 1D) were harvested again in June 2004 (2004 spring crop) using the same harvest procedures as for the previous crop. The rhizome re-generated plants resumed growth and development in July 2004 (Figure 1F) and harvested in December 2004 (2004 autumn crops). All the harvested plants were separated into leaves, stems and flowers for biomass determinations. All the sampled materials were dried in a forced hot air dryer at 43 °C to a moisture content of 10% after drying for 4–7 d, and weighed for biomass determinations.
The total phenol content was estimated by a colorimetric assay based on procedures described by Taga et al. (Reference Taga, Miller and Pratt1984). Fifty milligrams of dried ground tissue were extracted using 3 ml 60% (v/v) methanol containing 0.3% (v/v) HCl for 60 min, and then centrifuged at 18 000 g for 15 min. A 10 μl aliquot of tissue extract was dissolved in 200 μl of 2% (v/v) Na2CO3, and 10 μl of the Folin and Ciocalteu's phenol reagent (50%, v/v) were added. The mixture was left to stand at room temperature for 30 min. Absorbance measurements were taken at 725 nm using a spectrophotometer, and caffeic acid was used in the construction of the standard curve.
For caffeic acid derivatives determinations, the tissue extract used for total phenol determination (20 μl) was filtered through a 0.2 μm syringe filter (Minisart RC 15, Sartorius) and then analysed using a HPLC (Hitachi, Japan) consisting of a pump (L-7100), column oven (655A-52), UV-VIS detector (L-4200) (330 nm) and auto sampler (L-7200) (Hu and Kitts, Reference Hu and Kitts2000). The column used was Mightysil RP-18 GP 5 μm 150 × 4.6 mm (Kanto, Tokyo, Japan). Two eluents were used: acetonitrile/water 10:90 and acetonitrile/water 25:75. Various levels of caftaric acid, chlorogenic acid, cynarin, echinacoside and cichoric acid were used in the construction of standard curves.
The experimental design was a randomized complete block design with four replicates. All data were subjected to an analysis of variance and when a significant (p < 0.05) F ratio occurred for treatment effects, a least significant difference (LSD) was calculated.
RESULTS
Significant differences in daily mean temperature (Figure 2A) and photoperiod (Figure 2B) were found between spring and autumn crop seasons. Daily mean temperature peaked in July at about 29 °C and dropped to a minimum of 16 °C in December in 2003 (2003 autumn crop season). In the following year, daily mean temperature gradually increased from 17 °C in January to around 28 °C in July (2004 spring crop season), and then followed by another temperature decline (2004 autumn crop season). Similar patterns were also noted for daily photoperiods (Figure 2B).
The changes in (A) daily mean temperature and (B) photoperiod during the growth and sampling of E. purpurea plants.
Both seedling-transplanted (2003 autumn growing samples) (Figure 1C) and rhizome-regenerated (2004 spring and autumn growing samples) (Figure 1D) E. purpurea plants grew vigorously under natural conditions. As shown in Table 1, both morphological and agronomic traits in harvested plants were highly variable, as indicated by the relatively greater standard deviations of the samples examined. Crop season was found to affect the morphological traits of E. purpurea plants (Table 1). The spring-grown plants generally grew taller and produced more flowers than autumn-grown plants. As a result, spring-harvested E. purpurea produced more biomass in the aerial portion of the plants than autumn-harvested plants (Table 1). Moreover, the seedling-transplanted E. purpurea (2003 autumn season) grew better and produced more biomass than the E. purpurea regenerated from rhizomes (2004 autumn season) (Table 1).
The morphological and agronomic traits of Echinacea purpurea grown under different seasons. The morphologic traits (plant height and produced flowers) were expressed on per plant basis. The agronomic traits were expressed on g dry weight per plant basis.
The average content of total phenolics in harvested E. purpurea leaves and flowers is presented in Tables 2 and 3, respectively. As with the morphological and agronomic traits, the content of total phenolics in leaf and flower tissues also varied greatly (Tables 2 and 3). Both leaf and flower tissues showed that the content of total phenolics was higher from autumn-harvested plants than from spring-harvested plants.
The contents (mg g−1 dry weight) of total phenolics and caffeic acid derivatives in the leaves of Echinacea purpurea grown under different seasons.
The contents (mg g−1 dry weight) of total phenolics and caffeic acid derivatives in the flowers of Echinacea purpurea grown under different seasons.
Large variations were also found in the content of total caffeic acid derivatives in leaves and flowers (Tables 2 and 3). As shown in Table 2, the contents of caffeic acid derivatives in leaf tissues differed by harvest season. The leaves harvested in the autumn had more caffeic acid derivatives than leaves harvested in the spring. Among the five caffeic acid derivatives examined in the present study, leaves harvested in both autumn 2003 and 2004 had the highest cichoric acid content and followed by caftaric acid (Table 2). The cichoric acid and caftaric acid produced were at the same level for the leaf tissues harvested from the spring season crop, which were relatively lower than that of the leaf tissues harvested from the 2003 or 2004 autumn seasons (Table 2). In all cases, the contents of chlorogenic acid, cynarin and echinacoside were relatively low in comparison with those of cichoric acid or caftaric acid (Table 2).
The contents of total caffeic acid derivatives in flowers (Table 3) were consistently higher than those in leaves (Table 2). The contents of total caffeic acid derivatives in flowers also differed by harvest season (Table 3). The autumn-harvested flowers had more caffeic acid derivatives than spring-harvested flowers. In all three seasons, harvested flowers contained the highest cichoric acid content and followed by caftaric acid and chlorogenic acid. The contents of cynarin and echinacoside were relatively low compared to cichoric, caftaric and chlorogenic acids (Table 3).
DISCUSSION
The objective in commercial E. purpurea production is to produce high biomass with a high bioactive compound content (i.e. caffeoyl phenols). The content of bioactive compounds varies between E. purpurea plant organs, with the content of caffeoyl phenols in leaves and flowers considerably higher than in underground parts (Stuart and Wills, Reference Stuart and Wills2000; Thygesen et al., Reference Thygesen, Thulinn, Mortensen, Skibsted and Molgaard2007). That is why the majority of E. purpurea preparations in the commercial market, ranging from direct pressed juices to freeze-dried ethanolic or hydrophilic extracts are made from whole or powered dried leaves and flowers (Barrett, Reference Barrett2003; Perival, Reference Perival2000). Therefore, in the present study, only the aerial parts of the E. purpurea plant were sampled for biomass and caffeoyl phenols production.
In the present study, all the morphological, agronomic and biochemical traits in harvested plants were highly variable, as described in previous reports (Kreft, Reference Kreft2005; Wills and Stuart, Reference Wills and Stuart1999). Kreft (Reference Kreft2005) indicated that only a small portion of the large variability could be explained by environmental and cultural conditions, with the inter-individual differences being the main source of variability. Our data support his findings. E. purpurea is a cross-pollinated plant and tends to be self-incompatible (Li, Reference Li1998). Therefore, the large variability in its morphological and agronomic traits is not unexpected. It appears that a continuous mass selection is a necessity to reduce the heterogeneity in these morphological and agronomic traits within the cultivated E. purpurea populations.
As shown in Table 1, the spring-grown plants produced more flowers than autumn-grown plants. The greater flower setting in the spring crop is not surprising because E. purpurea is an intermediate-day plant and the flowering percentage is greater under photoperiods of 13–15 h (Runhle et al., Reference Runhle, Heins, Cameron and Carlson2001). The photoperiods for the 2004 spring season were around 13 h, but they dropped to about 11 h in the 2003 and 2004 autumn seasons (Figure 2B). The relatively lower mean daily temperatures recorded during the autumn in comparison with the spring might also limit the growth and development of E. purpurea plant to some extent (Figure 2A). However, it should be noted that the monsoon (May to June), and typhoons (July to September), which occur frequently in Taiwan, would be two environmental risks affecting the successful growth and development of E. purpurea, if the growers intend to cultivate these plants during the spring.
The data in Table 1 further demonstrated that the seedling-transplanted plants (2003 autumn season) appeared to grow better and produce more biomass than E. purpurea plants regenerated from rhizomes (2004 autumn season). However, this could also be a result of higher temperatures in 2003 autumn compared to 2004 autumn. Thus, for optimal production, the first harvest of the aerial parts of autumn-grown plants should be carried out at the end of the season (around December), and then allow the plants to regenerate from rhizomes in the coming spring. However, the rhizome-regenerated plants should be ploughed up and replaced with newly grown seedlings at the end of summer. In contrast, Kreft (Reference Kreft2005) recommended that plantations should be ploughed up and replanted every three years.
Phenolic substances extracted from aerial parts of E. purpurea plants are very efficient antioxidants, which have been suggested for the treatment of various types of illness (Thygesen et al., Reference Thygesen, Thulinn, Mortensen, Skibsted and Molgaard2007). In the present study, crop season was also found to affect the content of total phenolics in leaf and flower tissues (Tables 2 and 3). The environmental factors that might affect the accumulation of phenolics are still unknown. Because E. purpurea plants are native to temperate regions (Mistríková and Vaverková, Reference Mistríková and Vaverková2007), growing and developing in the relatively cool conditions (Figure 2B) and low humidity (autumn crop season) may be beneficial to the expression of phenolic compounds differentially, particularly in the leaf and flower portions of the plants.
Cichoric acid is one of the most important markers affecting the market quality of E. purpurea (Thygesen et al., Reference Thygesen, Thulinn, Mortensen, Skibsted and Molgaard2007). Kreft (Reference Kreft2005) found that E. purpurea grown in Slovenia contained cichoric acid at 11 and 16 mg g−1 dry weight (DW) in flower and leaf tissues of the plant. The contents of cichoric acid in E. purpurea grown in Germany have been reported to be 13 mg g−1 DW in leaves and flowers. The Australian-grown E. purpurea flowers and leaves contain 30–38 and 4–15 mg g−1 DW, respectively (Wills and Stuart, Reference Wills and Stuart1999). In all cases, the levels of cichoric acid pound in our study were much higher than their results. The higher cichoric content in leaves and flowers of E. purpurea would give commercial cultivation of this medicinal plant in Taiwan a greater competitive edge over other E. purpurea growing regions.
In conclusion, the present results indicate that the biomass and caffeoyl phenols production of E. purpurea plants are indeed influenced by the growing season. Autumn-grown plants produce more caffeoyl phenols, particularly cichoric acid and caftaric acid, in their leaves and flowers than spring-grown plants. Spring-grown E. purpurea plants produced more biomass possibly due to their greater flowers and stem yields than those of autumn-grown plants. Our results indicate that to grow E. purpurea at the end of the summer and subsequently harvest aerial parts of the plants in the autumn, and then harvest the rhizome-regenerated plants again in next spring is technically feasible in Taiwan. This unique cultural practice, which allowing the growers to harvest E. purpurea plants containing high caffeoyl phenols content twice annually, would give commercial cultivation of E. purpurea in Taiwan a great competitive advantage over other E. purpurea-growing regions. However, the introduction of E. purpurea on a commercial scale requires a more homogenous plant population. In this regard, a consecutive mass selection and purification programme to breed an environmentally suitable population should be continued in Taiwan.
Acknowledgements
We gratefully acknowledge financial support from the National Science Council of ROC (NSC93-2313-B-005-040).
