Abstract
This study focused on optimizing a Natural Deep Eutectic Solvent (NADES)-based extraction for recovering bioactive compounds, particularly phenolic and terpenic compounds, from orange by-products, aimed at neuroprotection. CO₂-assisted pressurized NADES extraction enhanced both yield and bioactivity compared to more conventional approaches. After the extraction, the encapsulation of the extract obtained with pressurized betaine:glycerol (Bet:Gly, 1:2) was optimized through a Box-Behnken design using soy phosphatidylcholine as encapsulating agent; optimum process conditions were: concentration of the extract, 4199 ppm; orbital agitation time, 99.1 min; temperature, 55.1 °C. The encapsulation process allowed retaining 66 %, 34 %, and 80 % of carotenoids, terpenes, and phenolic compounds, respectively. Once the optimum conditions were stablished, sunflower lecithin was also tested as a new encapsulating agent. The use of sunflower lecithin improved terpene and phenolic compounds encapsulation by 18 and 15 %, respectively, significantly enhancing potential neuroprotective capacity. Moreover, the chemical characterization of the extract further supported these findings. Stability tests revealed that pressurized NADES extracts maintained their bioactivity for 15 days, whereas the microencapsulation process improved stability and preserved bioactivity for over four months. Despite some losses in terpene and phenolic content under accelerated conditions, microencapsulation significantly extended the shelf-life and functionality of the bioactive compounds.
Highlights
- At 10 MPa, CO₂ and NADES do not form a single homogeneous phase.
- CO₂-pressurized NADES shows high extraction efficiency.
- Non-refined sunflower lecithin-based microencapsulation enhances extract stability.
- Sunflower lecithin showed better encapsulation efficiency than soy phosphatidylcholine.
Introduction
Citrus crops are globally significant, with citrus juices being the primary processed product. However, only about 40 % of the fruit’s weight is used for juice, leaving the majority as by-products such as peel, pulp, and seeds [1]. These by-products are rich in bioactive compounds such as carbohydrates, essential oils, terpenes, vitamins, and phenolic compounds, which have shown potential in mitigating age-related chronic diseases [2], [3].
Among chronic illnesses, Alzheimer’s disease (AD) and other neurodegenerative disorders are increasing worldwide and currently lack an effective cure [4]. AD is characterized by memory loss and cognitive decline linked to β-amyloid plaques, tau protein hyperphosphorylation, cholinergic deficits, oxidative stress, and neuroinflammation, which exacerbate neuronal damage [5]. Existing treatments focus on symptom management, but bioactive natural compounds with antioxidant, anti-inflammatory, and cholinesterase-inhibitory activities (often found in citrus by-products) are gaining interest for their preventive potential [6].
Terpenes and phenolic compounds, abundant in citrus by-products, show promising neuroprotective effects, making orange by-products valuable for extracting bioactive substances while reducing food waste [7].
raditionally, green solvents like ethanol and water have been used for such extractions, but natural deep eutectic solvents (NADES) have recently emerged as a superior alternative due to their unique hydrogen bonding networks, thermal stability, low volatility, and higher efficiency in extracting phenolics [8]. Hydrophilic NADES composed of sugars, amino acids, and carboxylic acids are particularly attractive due to their customizable properties and GRAS status, making them environmentally friendly compared to conventional organic solvents [9], [10]. Combining NADES with pressurized extraction methods offers a novel means to obtain high-purity bioactive extracts [11].
However, the high viscosity of hydrophilic NADES limits solvent penetration and mass transfer, negatively impacting extraction efficiency and operational ease [12]. To overcome this, biphasic systems combining supercritical CO₂ with NADES have been proposed, as CO₂ can reduce NADES viscosity and enhance the recovery of natural compounds [13].
Another limitation of natural extracts is their poor shelf-life, due to the instability of bioactive compounds. Encapsulation techniques, such as liposomal microencapsulation, improve stability by protecting these compounds from environmental degradation and digestive processes, allowing controlled release at the target site [14].
This study aims to explore the recovery of neuroprotective bioactive compounds from orange by-products using hydrophilic NADES pressurized with CO₂ under gas-expanded liquid conditions. The performance of this method is compared with traditional pressurized solvents and NADES extraction at atmospheric pressure. Additionally, liposomal microencapsulation of the best extracts to enhance their stability over time was optimized. To the authors’ knowledge, this is the first study to combine these approaches to produce stable, bioactive extracts from food by-products.
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2. Materials and methods
2.1. Samples and sample preparation
2.2. Reagents and materials
For the extraction, food-grade premium quality CO2 (purity 99.9 %) was supplied by Carburos Metálicos (Madrid, Spain). Ultrapure water was produced using an instrument designed to meet ASTM specifications for type I quality (Milaboratorio Web, Colmenar Viejo, Spain). HPLC-grade ethanol, methyl tert-butyl ether, and LC-MS grade methanol, chloroform, sulfuric acid (98 %), and ethyl acetate (EtAc) and glycerol (Gly) were purchased from VWR (Barcelona, Spain). Acetylcholinesterase (AChE) Type V1-S from Electrophorus electricus, butyrylcholinesterase (BuChE) from equine serum, acetylthiocholine iodide (ACth), butyrylcholinesterase iodide (BCth), linoleic acid (LA), 2,2’-azinobis (3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), 2,2’-diphenyl-1-picrylhydrazyl (DPPH), 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), Trizma hydrochloride (Tris-HCl), disodium phosphate (Na2HPO4), monopotassium phosphate (KH2PO4), sodium nitroprusside dehydrate (SNP), fluorescein sodium salt, sulphanilamide, naphthyl ethylene diamine dihydrochloride, gallic acid, Folin-Ciocalteu reagent, quercetin, limonene (analytical grade), and all-trans-β-carotene (≥ 97 %) were obtained from Sigma-Aldrich (Madrid, Spain). Lipoxygenase from Glycine max, 4-(amino-sulfonyl)-7-fluoro-2,1,3-benzoxadiazole (ABD-F), galantamine hydrobromide, betaine (Bet), choline chloride (ChCl), lactic acid (Lac) and 2,2-azobis(2-aminodipropane) dihydrochloride (AAPH) were purchased from TCI Chemicals (Tokyo, Japan). Soy phosphatidylcholine from lecithin was provided by Thermo Scientific GmbH (Karlsruhe, Germany) and sunflower lecithin was provided by Moara (Madrid, Spain). All 96-well microplate assays were performed using a spectrophotometer and fluorescence microplate reader (Cytation 5 imaging reader with auto-disperser, BioTek Instruments, Winooski, VT, USA).
Victor M. Amador-Luna, Lidia Montero, Carlos Pajuelo, Clovis Antonio Balbinot Filho, Marcelo Lanza, Sandra Regina Salvador Ferreira, Elena Ibáñez, Miguel Herrero, New approaches for improving bioactivity and stability of natural extracts: CO2-assisted compressed NADES and microencapsulation, The Journal of Supercritical Fluids, Volume 230, 2026, 106845, ISSN 0896-8446, https://doi.org/10.1016/j.supflu.2025.106845.
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