Supercritical CO2 extracts rich in palmitoylethanolamide from Glycine max (L.) Merr. waste and Medicago sativa L. aerial parts

Glycine max (L.) Merr. (soy) and Medicago sativa L. (alfalfa) (Fabaceae family) have long-standing traditional uses and hold significant importance in the fields of food and health. Soy is renowned for its nutritional properties and versatile applications in food products, while alfalfa is highly regarded as a nutritious forage crop for livestock (1). However, it is worth mentioning that soybean processing generates considerable waste which pose challenges in terms of disposal and environmental impact. Moreover, both soy and alfalfa contain beneficial substances such as N-acylethanolamines (NAEs), saponins, polyphenols, and phytosterols, further contributing to their nutritional value and potential health benefits. In particular, NAEs are minor components of plant lipids with anti-inflammatory activity by acting on the endocannabinoid system. The potent biological activity of NAEs has led to the exploration of therapeutic applications, highlighting their anti-inflammatory, antitumoral and antiviral properties. Among these NAEs, palmitoylethanolamide (PEA), is present in significant quantities in both alfalfa and soy (2). PEA is a bioactive lipid, derived from palmitic acid and ethanolamine, that mediates anti-inflammatory, neuroprotective and immunosuppressive activity through the activation of peroxisome proliferator receptor (PPAR α). The objective of this study was to extract the lipophilic fraction of G. max waste and M. sativa aerial parts to obtain an extract rich in PEA, considering its reported activities in the literature (3). The environmentally sustainable and clean technology of supercritical fluid extraction was employed for both plant materials, utilizing CO2 under three different experimental conditions including high-pressure extraction (HPE) with 96% ethanol as a co-solvent, HPE without a co-solvent, and low-pressure extraction (LPE) without a co-solvent. This technique avoids the use of toxic organic solvents, ensures high-quality and pure extracts, and prevents degradation of active compounds by extracting the lipophilic fraction of plant matrices. Quantification of PEA was conducted on the three extracts of M. sativa and the three extracts of G. max by using an external standard to determine the amount present in each sample. Solutions at known concentrations of PEA (concentration range 1- 0.0625 mg/mL) were prepared and injected in triplicate into the UHPLC-ESI-HR-Orbitrap/MS system for constructing a calibration curve. Based on the results, the highest amounts of PEA were found in the alfalfa and soy waste extracts obtained through HPE using 96% ethanol as co-solvent, yielding 65.5 ± 3.1 and 40.5 ± 2.1 mg/100 g of dry extract, respectively. PEA could protect the central nervous system during viral infections, keeping the immune response under control, and exerting a direct action towards viral replication, enhancing pathways and changing the vesicular pH. For this reason, PEA-rich extracts were evaluated against the Zika virus, a + ssRNA neurotropic virus belonging to the Flaviviridae family, both exhibiting a potential antiviral activity against the virus. 1) Y. Li, H. Yang, Z. Li, S. Li, J. Li (2023) Int. J. Mol. Sci., 24, 2620 2) B. J. Venables, C. A. Waggoner, K. D. Chapman (2005) Phytochemistry, 66, 1913-1918 3) R. Fonnesu, V. B. S. C. Thunuguntla, G. K. Veeramachaneni, J. S. Bondili, V. La Rocca, C. Filipponi, P. G. Spezia, M. Sidoti, E. Plicanti, P. Quaranta, G. Freer, M. Pistello, M. L. Mathai, M. Lai (2022) Viruses, 14, 1080