Natural distribution and alkamides production in Acmella radicans

Abstract

Alkamides comprise over 200 related compounds. There are many studies revealing much useful information about the structure, chemistry and biological activity of this novel class of compounds. However, there is a lack of information about the natural distribution and their synthesis. Here we reported the synthesis of the main alkamides during the growth of Acmella radicans. A. radicans seed were surface-sterilized and sowed in soil. One week after placement in the soil, the seed began germination with 90% of germination. Alkamide content was evaluated every week during six months. We found six alkamides in this plant. N-isobutyl-(2E,6Z,8E)-decatrienamide (affinin) biosynthesis initiated, just before seedling. In the third week appeared the N-(2-methylbutyl) -(2E,6Z,8E)-decatrienamide and the N-(2-methylbutyl)- (2E,4Z,8E,10E)-dodecatetraenamide; N-(2-phenylethyl)- (2Z,4E)-octadienamide; N-(2-phenylethyl)-nona-2E-ene-6,8-diynamide and 3-pheny-N-(2-phenylethyl)-2-propenamide appeared in the fifth week. The profiles of these six alkamides in A. radicans vary with the time and developmental stage. Affinin and other alkamides have been found to alter the architecture of the root system and to regulate cell division and differentiation process in Arabidopsis thaliana. The dependence of the alkamides concentration with plant age, found in this study suggests a possible plant hormones role of the alkamides in this plant. [PUBLICATION ABSTRACT]

Headnote

Abstract: Alkamides comprise over 200 related compounds. There are many studies revealing much useful information about the structure, chemistry and biological activity of this novel class of compounds. However, there is a lack of information about the natural distribution and their synthesis. Here we reported the synthesis of the main alkamides during the growth of Acmella radicans. A. radicans seed were surface-sterilized and sowed in soil. One week after placement in the soil, the seed began germination with 90% of germination. Alkamide content was evaluated every week during six months. We found six alkamides in this plant. N-isobutyl-(2E,6Z,8E)-decatrienamide (affinin) biosynthesis initiated, just before seedling. In the third week appeared the N-(2-methylbutyl) -(2E,6Z,8E)-decatrienamide and the N-(2-methylbutyl)- (2E,4Z,8E,10E)-dodecatetraenamide; N-(2-phenylethyl)- (2Z,4E)-octadienamide; N-(2-phenylethyl)-nona-2E-ene-6,8-diynamide and 3-pheny-N-(2-phenylethyl)-2-propenamide appeared in the fifth week. The profiles of these six alkamides in A. radicans vary with the time and developmental stage. Affinin and other alkamides have been found to alter the architecture of the root system and to regulate cell division and differentiation process in Arabidopsis thaliana. The dependence of the alkamides concentration with plant age, found in this study suggests a possible plant hormones role of the alkamides in this plant.

Key words: Alkamides, affinin, developmental stage, synthesis

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Introduction

The most general structure of the alkamides originates from the condensation of an unsaturated fatty acid and one amine (Hofer et al., 1986). There is great interest in these secondary metabolites because of their biological activity. The distribution of alkamides in plants seems to be limited to ten plant families. Decadienoic isobutylamide is widely distribution in the Asteraceae, Rutacea, Piperacea and Aristolochiaceae (Greger and Werner, 1990).

Many of the species containing alkamides have been used in traditional medicine of different civilizations. They are known for their pungent taste, and for causing itching and salivating. Some species have been used to the treatment of toothaches and sore throats. Other biological activities are: insecticidal (Kadir et al., 1989), antifungal (Molina-Torres et al., 2004), mulluscicidal (Johns et al., 1982), antimicrobial (Gutierrez-Lugo et al., 1996; Molina-Torres et al., 1999), and more recently Ramirez-Chávez et al., (2004) reported that some alkamides display an hormonal like activity during the growth and development of Arabidopsis thaliana.

Alkamides are compartmentalized in different organs of the plant. In Echinacea species, roots alkamides are mainly isobutylamides highly unsaturated with olefinic and/or acetylenic bonds (Greger, 1984), However in Acmella species roots, leaves, stems and flower heads besides the aliphatic alkamides, present aromatic (Martin and Greger, 1985; Rios-Chavez et al., 2003). Alkamides in the aerial parts of three Echinacea species do not show characteristic differences among them. However in the case of E. angustifolia and E. pallida the alkamides in the aerial parts differed significantly from the alkamides in the roots (Bauer and Remiger, 1989).

Despite the potential importance of the alkamide, few reports are available about the natural distribution during the development of the plant. The objective of this study was to analyze the distribution of the alkamides in Acmella radicans during development until flowering period, and the evaluation of different tissue alkamide contents.

Material and Methods

Plant Material and Growth Conditions

The Acmella radicans plants were collected in Queserías, Colima, México. Voucher specimens were deposited at the Instituto de Ecología AC, Patzcuaro, Michoacán, Mexico, under number IEB126. Seeds of Acmella radicans were collected from ripe floral heads of plants grown in the green house. Seeds were surface sterilized in 0.02% (w/v) HgCl2 for 1 min, then with 70% (v/v) ethanol for 1 min, and finally by a 20 min soak in a 20% (v/v) bleach solution. Seeds were rinsed in distilled water and placed at 4°C during 2 days to facilitate germination. Seed were put in small plastic pots containing a mixture of previously sterilized sand and soil (1:1 w/w). In order to maintain high humidity, the pots were covered with plastic bags and leftin a greenhouse. Plants were grown under ambient light in a greenhouse, at 22-24°C, with daily watering.

To determine the time and location of alkamide biosynthesis or accumulation, the plants were harvested at the same time of the day from the first to 24th weeks after germination during growth and flowering. Harvested plants were used for alkamide content assay.

Extraction of alkamides

Each sample (from 1 to 2 g of green tissue) was ground in 10 mL ethanol for 5 min with a mortar and pestle. The resultant suspension was transferred to a capped tube, vortexed for 30 s, then heated at 70°C for 15 min, sonicated for 30 min, and centrifuged for 3 min at 3,000 rpm. The pellet was discarded, the ethanol extract evaporated under a stream of nitrogen, and the residue re-suspended in 200 µL of ethanol. The samples were analyzed by GCMS with the aid of the MSChem from Agilent Technologies, processed with the Automated Mass Spectral Deconvolution and Identification System, and the Mass Spectral Search Program for the NIST/EPA/NHI Mass Spectral Library v 2.0 2008 from the National Institute of Standards and Technology.

GC-MS analysis

These were performed in an Agilent Technologies 7890A GC System equipped with a capillary column [HP-5MS (30 m x 0.25 mm; 0.25 µm)] coupled to a mass selective detector (Hewlett Packard 5973N MSD). An HP 7683B autoinjector was used. The sample (1 µL) was injected with a split ratio of 5:1. Operating conditions were: injector temperature 250°C; oven temperature programmed as: initial temperature 150°C for 3 min, then increasing at a rate of 4°C/ min to a final temperature of 280°C, which was maintained for 25 min. Helium was used as carrier gas with a constant flow of 1 mL/ min. MS temperatures were: EI source 230°C, and MS Quadrupole 150°C. Individual alkamides were identified by comparison of their MS and retention times with their standards and NIST, as described above.

Statistical analysis

Data are expressed as average of two different experiments with three replicates each. The bars indicate standard deviation from the mean of each replicate treatment.

Results and discussion

Synthesis of alkamides

As opposed to Echinacea purpurea and E. angustifolia having alkamides in the achene (Schulthess et al., 1991; He et al., 1998), Acmella radicans does not have alkamides in seeds and the content in the roots is low (Rios- Chavez et al., 2003).

Acmella radicans is an annual plant whose distribution is primarily in moist weedy habitats especially along stream banks, widespread throughout Mexico and Central America. Figure 1 shows the development at different ages after germination.

Production of alkamides in A. radicans plants was found to start at the end of the first week after seed germination. In this stage just N-isobutyl-(2E,6Z,8E)-decatrienamide (affinin) was detected, during the third and fifth weeks there was a two-fold of this compound, however the highest level of affinin was found at the nine-tenth week (0.32 mg/g fresh weight) (Figure 2).

This plant presents two 2-methylbutyl alkamides, the synthesis of N-(2-methylbutyl)- (2E,6Z,8E)-decatrienamide started in the third week (Figure 2A), and its level increase slowly until the twentieth week, with a content of 0.16 mg/g fresh weight. The level of N-(2- methylbutyl)- (2E,4Z,8E,10E) dodeca tetraenamide was detected in the fifth week in low concentration; near the fifteenth week its level started to increase (0.15 mg/g fresh weight) in similar proportion to the other 2- methylbutyl alkamide, however the level of N- (2-methylbutyl)-(2E,4Z,8E,10E)- dodecatetraenamide decreased rapidly in the twenty week (Figure 3B).

Acmella radicans var. radicans contains aliphatic and aromatic alkamides (Rios-Chavez et al., 2003). As seen in figure 3, the aromatic alkamide, N-(2-phenylethyl)- (2Z,4E)- octadienamide biosynthesis started in the fifth week, this alkamide showed the same pattern of synthesis than affinin, the highest alkamide content were determined in the ninetenth week after seed germination, and then declined to the initial level.

The biosynthesis of N-(2-phenylethyl)- nona-2E-en-6,8-diynamide (Figure 5A) and 3- phenyl-N-(2-phenylethyl)-2-propenamide (Figure 5B) were similar. Both of them started from the fifth week after seed germination, although the concentration of the first was twice times higher than the last, in this stage these aromatic alkamides had the highest level, and then declined gradually their levels, as the plants entered the flowering phase.

Studies of the distributions of alkamides in different organs of E. angustifolia indicate they are present mainly in roots, lower content in flower and not detectable in leaves (Wu et al., 2004). Here we found the highest concentrations of alkamides in the flower heads (Figure 6), which have significantly higher amount of two alkamides N-isobutyl- (2E,6Z,8E)-decatrienamide (0.69 mg/g fresh weight) and 3-phenyl-N-(2-phenylethyl)-2- propenamide (0.78 mg/g fresh weight).

Plant produces a broad spectrum of secondary metabolites, each with different purposes, such as antifeedant, antimicrobial, attractant and signal compounds. Based on the different functions, the content of the metabolite will be different. It is well known that some secondary metabolites have a constitutively content in a certain tissue. This can be presented in young tissues for protection against insects, or in flowers to attract pollinators by colour or volatile attractants. Now a days the function of the alkamides in the plants is still unknown, however among the numerous alkamides already reported, Echinacea roots generally accumulated more N-isobutyl- 2E,4E,8Z,10E/Z-dodecatraenamide with age. Some other species accumulated the most in younger cultivated roots (Binns et al., 2002). Recent research has shown that especially the accumulation of different olefinic and acetylenic alkamides represents a characteristic biogenetic capacity of the genus Achillea (Greger and Werner, 1990).

In this study we demonstrated that alkamides production was different along the development of A. radicans. Affinin was the first alkamide synthesis and predominant during all development stages of the plant, this alkamide presented three peaks. In the aerial parts and the flower heads the main alkamides were N-isobutyl-(2E,6Z,8E)-decatrienamide (affinin) and 3-phenyl-N-(2-phenylethyl)-2- propenamide.These alkamides levels were 10 fold higher in the flower heads than in aerial parts. The alkamides present in Acmella radicans var. radicans have been previously reported in other species. However, the specific distribution of these amides has not been reported previously. These data can help to understand the biosynthesis of the alkamides.

References

References

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