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Letchamo, W., J. Livesey, T.J. Arnason, C. Bergeron, and V.S. Krutilina. 1999. Cichoric acid and isobutylamide content in Echinacea purpurea as influenced by flower developmental stages. p. 494–498. In: J. Janick (ed.), Perspectives on new crops and new uses. ASHS Press, Alexandria, VA.


Cichoric Acid and Isobutylamide Content in Echinacea purpurea as Influenced by Flower Developmental Stages

W. Letchamo, J. Livesey, T.J. Arnason, C. Bergeron, and V.S. Krutilina


  1. METHODOLOGY
    1. The Plant Material, Selection and Harvesting
    2. Sample Preparation
    3. HPLC Analysis
  2. RESULTS
    1. Identification and Naming of the Cultivar 'Magical Ruth'
    2. Cichoric Acid
    3. Alkamide (Isobutylamide)
    4. Echinacoside and Chlorogenic Acid
  3. REFERENCES

Echinacea purpurea (L.) Moench, Asteraceae, a native of North America is among the most frequently utilized Echinacea spp. for medicinal, cosmetic, and beverage preparations. Most of the echinacea preparations in the industry originate from wild harvesting and few from non-standardized (unregulated) cultivation and harvesting. According to the recent reports (Brevoort 1996), echinacea preparations are among the best selling herbal immunostimulant products in health food stores in the US. Currently more than 800 echinacea-containing drugs, including homeopathic preparations, are available on the German market. Most of the products are derived either from the aerial or underground parts of Echinacea.

Figure 1
Fig. 1. Some of the hydrophilic components of E. purpurea extract.

Within several herbs, Echinacea species are among the main investigation targets, because of their value as immunostimulants (Bauer and Wagner 1991). The therapeutic effect of Echinacea has been assigned to the presence of caffeic acid derivatives such as cichoric acid, echinacoside, chlorogenic acid (Fig. 1), and lipophilic polyacetylene-derived compounds, such as alkylamides, constituting isobutylamides (Fig. 2), and various other components found in the hydroalcoholic extracts. The low molecular weight constituents of hydroalcoholic tinctures can be determined by HPLC or TLC analysis.


Figure 2
Fig. 2. Some of the major alkamides (isobutylamides) of E. purpurea extracts.

Cichoric acid has been shown to possess phagocytosis stimulatory activity in vitro and in vivo, while echinacoside has antibacterial and antiviral activity (Bauer and Wagner 1991). Cichoric acid was also recently been shown to inhibit hyaluronidase and to protect collagen type III from free radical induced degradation, while lipophilic alkamide (isobutylamides), polyacetylenes, and glycoproteins/polysaccharides have been shown to possess immunomodulatory activity (Bauer and Wagner 1991). When testing the alcoholic extracts obtained from the aerial parts and from the roots for phagocytosis stimulating activity, the lipophilic fraction alkamide (isobutylamides) showed the highest activity (Bauer and Wagner 1991). Most of the alkamides are reported to be responsible potent inhibitors of cyclooxygenase, while the requirement for inhibition of 5-lipoxigenase depends more on the particular structure of the single compound. The inhibitory properties of the alkamides on arachidonic acid metabolism are in accordance with the traditional use of the herbal drugs in the therapy of inflammatory diseases. Alkamide (isobutylamides) is also responsible for local tongue anaesthesis (tingling effect) when ingested and used as an informal test for the quality of Echinacea (Bauer and Wagner 1991). Purified alkamide fractions (isobutylamides) from E. purpurea and E. angustifolia were recently shown to enhance phagocytosis in the Carbon-Clearance-Test by a factor of 1.5 to 1.7 thereby contributing to the immunstimulatory activity of echinacea tinctures (Bauer and Wagner 1991). It is possible, therefore, to consider that cichoric acid, echinacoside, and alkamide (isobutylamides) are parts of the active principles and suitable for standardization of E. purpurea row material and finished products.

There is no published scientific information to date on the relationship between flower developmental stages and the accumulation of some of the above mentioned components of E. purpurea. Recent phytochemical investigation shows that there is a tremendous variation in the quality of echinacea products that were made available from various international sources (Letchamo et al. 1999). Furthermore, there is a dire need for genetic improvement and precise knowledge on the influence of ecological and developmental factors on yield and quality of Echinacea. The objective of this investigation was therefore to study the pattern of the accumulation of the active ingredients during different developmental stages of the flower heads of E. purpurea as part of the, introduction, selection, genetic improvement and agronomic programs of medicinal herbs in Trout Lake Farm since summer 1996.

Figure 3
Fig. 3. A promising E. purpurea that had been selected from among two million plants for further propagation, Trout Lake WA, summer 1996.
Figure 4
Fig. 4. Organoleptic evaluation of E. purpurea, Trout Lake, WA, summer 1996.

METHODOLOGY

The Plant Material, Selection and Harvesting

A new selection of E. purpurea named 'Magical Ruth' (Fig. 3) was propagated by root division and organically grown under sandy loam soil of volcanic origin (pH = 6.5), at Trout Lake Farm, Washington. The selection procedure and identification of the promising E. purpurea cultivars was based on yield potentials, better morphological traits, and freedom from diseases and pest infections. Morphological traits such as suitability for mechanical harvesting (erect and non-lodging), medium plant height, number of tillers, branches and flower heads, flower size, uniformity in flowering, possession of large, smooth green leaves with minimum leaf-hairs (less potential to carry dirt and microbes) were seriously considered and recorded. In addition to this, the plants ornamental values such as possession of attractive flower heads with deep purple color, higher production of pollen grains, speed and intensity of "tongue tingling" effects of the stems based on the organoleptic tests were included (Fig. 4).

Several workers with multiple years of practical experience in the organic production of herbs volunteered to participate in the preliminary field evaluation in the summer of 1996. From a field with two million plants, about 420 plants which fulfilled most of the above mentioned criteria were selected, numbered, and flagged for further organoleptic tests and phytochemical analysis. Preliminary field organoleptic tests ("tongue tingling") were carried out on 400 plants. About 360 different lines that meet the preliminary selection criteria were selected and propagated by root divisions (Fig. 5). In autumn 1996, all the selected lines were transplanted (about 80 plants from each line with 20–30 plants planted at three different locations). Evaluation continued as above during the 1997–1998 growing seasons.


Figure 5 left Figure 5 right
Fig. 5. Left: Preparation of uprooted echinacea for root crown division. Right: Root crown divisions ready for an immediate field planting.

Figure 6 top
Figure 6 bottom
Fig. 6. Top: flower developmental stages of E. purpurea, cv. Magical Ruth. Bottom: variations in the content of cichoric acid and isobutylamide in flower heads at different developmental stages (1= stage I, 2= stage II, 3= stage III, 4= stage IV).

For phytochemical investigation, the aerial parts of 20 plants replicated three times were harvested in July–August, 1997 and 1998. Immediately after harvest, the flower heads were separated by hand from the stems and divided into four different developmental stages as described below (Fig. 6). Stage I (small or early stage): flower buds starting to develop until the beginning of the first appearance of ligulate florets without rolling out (expansion), and dehiscence of disc florets. Stage II (medium stage): the first two to three lines of the disc florets open (dehisced), while the ligulate florets just starting to roll out (expansion) and attain their typical purple color. Stage III (large or mature stage): more than three quarter of the disc florets open, with fully expanded ligulate florets and intensive release of the pollen grains. Stage IV (old or overblown stage): Ligulate florets starting to droop and slightly turning to dark-brown color, with visible seed development and sharp decline in pollen grain formation. This classification was slightly modified from earlier work for Chamomilla recutita (Letchamo 1996). After the separation, the samples were dried at 38°C for 72 h and sent to the University of Ottawa for phytochemical analysis.

Sample Preparation

The dried samples were milled to powder and aliquots of each (ca 0.5 g) were extracted three times in 8 mL 70% ethanol using ultrasound (5 min), followed by centrifugation and collection of the subsequent supernate. The extract volume was adjusted to 25 mL, and filtered through a 0.45 µm PTFE membrane prior to injection of 5 µL into HPLC system. The liquid extracts were centrifuged to pellet any particulate matter, and the supernate was filtered as above prior to injection. The oil sample was diluted with ethanol (ca 1/20 for the lipophilic analysis, and 1/5 for the hydrophilic analysis), and filtered as above prior to injection.

HPLC Analysis

Separation and quantitative analysis of the components was based on a modified method of Livesey et al. (1998), based on Bauer and Remiger (1989). HPLC (C-18 column, 75 × 4.6 mm; 3 mm) was used to determine the lipophilic and hydrophilic constituents.

RESULTS

Identification and Naming of the Cultivar 'Magical Ruth'

The new E. purpurea cultivar 'Magical Ruth' (Fig. 3) was selected because of its relatively unique agronomic traits, freedom from pests and diseases, excellent frost resistance, attractive ornamental qualities, uniformity in growth, flowering, high yielding potentials, and better "tongue numbing" effect compared with other E. purpurea lines under investigation. There were also a dozen of other interesting lines that were simultaneously propagated in the experimental fields and meet the selection criteria.

Cichoric Acid

The highest content of cichoric acid was found at stage I (4.67%), with content declining to stage IV (1.42%) (Fig. 6).

Alkamide (Isobutylamide)

The concentration of isobutylamide was higher in stage III and IV than stage I and II with the peak at stage III, (Fig. 6).

Echinacoside and Chlorogenic Acid

Echinacoside content was maximal at stage 2 (Table 1). Maximum content of chlorogenic acid was recorded at the 1st stage, while the lowest concentration was measured at the 4th stage (Table 1).

Table 1. Variations in the content of echinacoside and chlorogenic acid in flower heads of the new Echinacea purpurea cultivar 'Magical Ruth'. The results are obtained from 20 plants replicated three times.

Flower stage

Hydrophilic components (%)

Chlorogenic acid

Echinacoside

I

(early)

0.060

0.012

II

(medium)

0.024

0.022

III

(mature)

0.023

0.015

IV

(overblown)

0.020

0.016

Our results indicate that the quality of echinacea is strongly influenced by the floral developmental stage. To obtain optimum yield levels of both hydrophilic and lipophilic components, echinacea flowers should be harvested at the 3rd (mature) developmental stage. It is important that floral developmental stages be considered during screening and selection.

REFERENCES