Optimization of Pressurized Liquid Extraction of Lycopodiaceae Alkaloids Obtained from Two Lycopodium Species

Abstract

Alkaloids of the Lycopodiaceae family are of great interest to researchers due to their numerous properties and wide applications in medicine. They play a very important role mainly due to their potent antioxidant, antidepressant effects and a reversible ability to inhibit acetylcholinesterase (AChE) enzyme activity. This property is of immense importance due to the growing problem of an increasing number of patients with neurodegenerative diseases in developed countries and a lack of effective and efficient treatment for them. Numerous studies have shown that Lycopodiaceae alkaloids are a rich source of AChE inhibitors. In the obtaining of new therapeutic phytochemicals from plant material, the extraction process and its efficiency is crucial. Therefore, the aim of this work was to optimize the conditions of modern PLE to obtain bioactive alkaloids from two Lycopodium species: L. clavatum L. and L. annotinum L. Five different solvents of different polarity were used for prepared plant extracts in order to compare the alkaloid content in and thereby effectiveness of the entire extraction. PLE parameters were used based on multiple studies conducted that gave the highest alkaloids recovery. Crude extracts were purified using solid-phase extraction (SPE) on Oasis HLB cartridge and examined by HPLC/ESI-QTOF–MS of the highly abundant alkaloids. To the best of our knowledge, this is the first time such high recoveries have been obtained for known Lycopodiaceae alkaloids. The best extraction results of alkaloid-lycopodine were detected in the dichloromethane extract from L. clavatum, where the yield exceeded 45%. The high recovery of annotinine above 40% presented in L. annotinum was noticed in dichloromethane and ethyl acetate extracts. Moreover, chromatograms were obtained with all isolated alkaloids and the best separation and quality of the bands in methanolic extracts. Interestingly, no alkaloid amounts were detected in cyclohexane extracts belonging to the non-polar solvent. These results could be helpful for understanding and optimizing the best conditions for isolating potent AChE inhibitors.

Fractionation of Lycopodiaceae Alkaloids and Evaluation of Their Anticholinesterase and Cytotoxic Activities

“…The purified alkaloid samples obtained from all experiments (104 in total) from the three plant species were analyzed using HPLC/ESI-QTOF-MS. This is a reliable tool for the efficient determination of unknown compounds present in plant extracts, as documented in many of our works [23,26]. This technique has numerous advantages such as high sensitivity, high mass accuracy (below 1 ppm) and high reproducibility of the analysis.…”

“…Three plant species belonging to the Lycopodiaceae family: L. clavatum, L. annotinum, H. selago, were subjected to extraction using the PLE method. The conditions of this process were previously carefully selected through many trials and studies in our laboratory, and the results were published [26]. Based on these studies, it was found that the highest percentage of Lycopodium alkaloids were isolated under high pressure and temperature (100 bar, 80 • C) from methanolic extracts, so these conditions were used in this approach.…”

“…The samples were placed in a 34 mL stainless steel extraction cell and extracted at an elevated temperature of 80 • C and 100 bar pressure, using methanol as a solvent. This extraction procedure and conditions have been previously tested and optimized, and the results have been described in detail [26]. The extracts obtained were transferred into 100 mL volumetric flasks and diluted to the mark with the solvent used for extraction.…”

“…The novel pressurized liquid extraction (PLE) used proved to be a rapid, reproducible and very efficient method for alkaloid extraction [23]. No previous studies carried out on plant materials of the Lycopodium species have achieved such high recoveries of desired compounds [26]. The percentage of major alkaloids such as lycopodine (in L. clavatum extract) or annotinine (in L. annotinum extract) exceeded even 40%.…”

Methods of isolation and bioactivity of alkaloids obtained from selected species belonging to the Amaryllidaceae and Lycopodiaceae families

“…Thus, the Amaryllidaceae alkaloids (such as galanthamine or lycorine) were eluted with 80-100% recovery [22]. The same procedure was used for the extraction and purification of Lycopodiaceae alkaloids [25]. These methods allowed gaining results with high accuracy and ascertaining the influence of types of solvents on the obtained amount of alkaloids.…”