Acmella oleracea (L.) R.K. Jansen: alkylamides and phenolic compounds in aerial parts and roots of in vitro seedlings

Introduction Acmella oleracea (L.) R.K. Jansen (Asteraceae), popularly known as jambu and widely cultivated in the Brazilian Amazon region, represents an economically important medicinal and edible plant crop. The different anatomical parts of jambu, such as flowers, leaves and stems, are commonly used as food ingredients in typical Amazon cuisine, and as a traditional medicinal herb. It is a rich source of several bioactive compounds, notably alkamides, flavonoids and other phenolic compounds, used for the treatment of different human disorders [1]. A good approach to obtain a great number of plants, in a short period of time under selected standardized conditions of cultivation, is the use of in vitro plant cultures which allows rapid improvement of raw material and metabolite production [2]. This research involved the study of aerial parts and roots of Acmella seedlings grown in vitro and developed starting from two different techniques: from single seed and from regenerating lines derived by organogenesis. The study aimed to characterize the phytochemical profile of the extracts obtained from the two parts of the plants, focusing on the evaluation of its characteristic secondary metabolites, i.e. alkylamides and phenolic compounds, by using an HPLC-DAD-MS method. The variability of the quantitative results was evaluated by analysing more batches collected over time. The optimization of the extraction procedure and the analytical method was also performed. Experimental The samples are aerial parts and roots obtained from in vitro seedlings plants from seed and by regenerating lines derived by organogenesis of Acmella oleracea, according to Maggini et al. [2]. Three different extractive procedures were tested to evaluate the most efficient one for the recovery of phytochemicals. The most efficient was then applied to all the samples. A fractionation step with hexane was then performed allowing the distribution of the recovered compounds in two extracts: a hydroalcoholic (HAE) and a lipophilic (HE). The chromatographic analyses were conducted using an HP 1260 Infinity II liquid chromatograph equipped with DAD detector and MS detector with an API/electrospray interface (Agilent Technologies, Palo Alto, CA, USA). A RaptorTM ARC-18 column (150 × 3 mm, 5 µm, Restek, USA) was used, applying a flow rate of 0.4 mL min−1. Water at pH 3.2 by formic acid (solvent A) and acetonitrile (solvent B) were used. The MS analyses were carried out in positive and negative ionization mode with fragmentors 150 V and 200 V. Alkylamides were quantified at 229 nm by a five-point calibration curve of spilanthol, phenolic acids with a five-point calibration line of chlorogenic acid at 330 nm and flavonoids were expressed as quercitrin (quercetin-3-L-rhamnoside) used to build a five-point calibration line at 350 nm. The proposed analytical method was evaluated in terms of linearity, accuracy and precision (in terms of repeatability) from stock standard solutions. Results The proposed method allowed a satisfactory chromatographic resolution for all the target analytes in a short time. A total of 30 compounds (15 phenols and 15 alkylamides) were detected. The chromatographic profiles of aerial parts and roots extracts obtained from seed and regenerating lines presented the same pattern of secondary metabolites. Aerial parts. The HAEs from seed showed the lowest content of phenolic compounds with a mean value of 3.40 mg/g DM. A significant higher concentration was found for the samples from regenerating lines, with a mean amount of 4.93 mg/g DM. The content of alkylamides in the HAEs ranged from 1.5 to 2.1 mg/g DM. In the HEs the average alkylamides content was below 2.4 mg/g DM. The spilanthol content in the aerial parts represented about 70-80% of the total alkylamides both in HAE amd HE extracts. Roots. The HAEs showed a higher average phenolic content than that measured in the aerial parts, with a maximum value of 11.19 mg/g DM. Also for this tissue, seedlings obtained from seed showed the lowest phenolic content (below 8.0 mg/g DM). The total amount of alkylamides in the HAEs and HEs was less than 0.29 mg/g DM and 0.46 mg/g DM, respectively. The spilanthol content was approximately 36-60% of the total alkylamides, with values about ten to fifteen times lower than the concentration measured in the aerial parts. Conclusions In this study the content of alkylamides and phenols in the aerial parts and roots of Acmella oleracea plant obtained in vitro was evaluated for the first time. The chromatographic method allowed determining all the target molecules in a single step and resulted a useful tool for evaluating the natural variability of the secondary metabolites content in seedlings produced over time. The same pattern of secondary metabolites was observed in aerial parts and roots extracts obtained from seed and regenerating lines. The hexane fractionation step effectively recovered spilanthol and its derivatives in a concentrated extract. The aerial parts were confirmed to be richer in alkylamides and spilanthol, while low amount of spilanthol was found in the roots. The data set collected for the first time for Acmella oleracea in vitro seedlings can be useful to define future steps focused on a highly controlled and reproducible raw material production in terms of phytochemical content.