Supercritical fluid extraction of bioactive compounds from Olea europaea pruning wastes

Importance of the topic

The valorization of olive (Olea europaea) pruning residues targets both environmental and economic objectives by converting abundant agricultural waste into sources of high-value bioactive molecules. Phenolic compounds, carotenoids and tocopherols extracted from olive leaves have applications in food, nutraceutical, cosmetic and pharmaceutical industries. Developing a single-step, scalable, and greener extraction workflow that recovers multiple chemical classes with diverse polarities supports circular-economy strategies and reduces solvent hazards and processing time compared to conventional multi-step approaches.

Objectives and study overview

This technical report describes optimization of a single-step supercritical fluid extraction (SFE) protocol to simultaneously recover three major classes of bioactives from olive leaves: phenolics (represented by oleuropein), carotenoids (lutein and ß-carotene) and tocopherols (α-tocopherol). The work compares SFE performance to validated ultrasound-assisted extraction (UAE) methods and aims to define operating parameters that maximize overall recovery while preserving the benefits of automation, reduced solvent use and faster throughput.

Methodology

Samples and preprocessing:

• Olive leaves were harvested, washed, air-dried to constant weight, milled and sieved to <0.3 mm particle size.

Comparative extractions:

• Three class-selective UAE protocols were used as reference methods: polar bio-EtOH/H2O for phenolics, acetone/methanol for carotenoids, and hexane for tocopherols; all applied repeatedly until exhaustion.

SFE design and optimization:

• SFE employed CO2 as the primary solvent and bio-ethanol as a polar modifier. A 0.2 mL extraction vessel loaded with 100 mg dried leaf powder was used for method development.
• All runs included an initial 2 min static fill with 100% CO2, followed by a dynamic stage of 5 min with 100% CO2 to preferentially extract very nonpolar compounds, then one or more dynamic steps with bio-ethanol modifiers (10–40%) to increase polarity and extract phenolics and polar pigments.
• Parameters explored: modifier fraction, division of dynamic steps, total dynamic times, flow rate (1–2 mL/min), extraction temperature (40–60 °C), and pressure (15–35 MPa).

Analytical characterization:

• HPLC-based quantitation was used with different detection modes per compound class: PDA and LC–MS (APCI for carotenoids, ESI for phenolics) and fluorescence detection for tocopherols.
• Chromatographic methods were optimized per class (C18 for phenols, C30 for carotenoids, silica for tocopherols) and are summarized in the report (gradient programs, detection wavelengths and injection conditions).

Instrumentation used

The SFE platform was a Shimadzu Nexera UC system comprising CBM-40 controller, SFE-30A module, LC-30ADSF CO2 pump, LC-40DXR modifier pump, LC-40D make-up pump, SFC-30A back pressure regulator, DGU-405 degasser and FRC-40 fraction collector. Extract analyses used HPLC with PDA (SPD-M30A), LC–MS single quadrupole (LCMS-2020) equipped with APCI and ESI interfaces, and RF-20AXS fluorescence detector for tocopherols.

Main results and discussion

Optimization outcome:

• The best compromise method (MET.6) employed: 2 min static 100% CO2; 5 min dynamic 100% CO2; 5 min dynamic with 10% bio-EtOH; 25 min dynamic with 30% bio-EtOH. Operating conditions: 35 MPa, 40 °C, 1 mL/min flow.
• Under these conditions, recoveries (relative to exhaustive UAE references) were ~76% for oleuropein, 84% for α-tocopherol, 88% for lutein and 90% for β-carotene.

Key process insights:

• CO2 alone favors extraction of nonpolar compounds (tocopherols, ß-carotene). Incorporation of a polar modifier (bio-ethanol) is essential to recover polar phenolics such as oleuropein.
• Pressure increase from 25 to 35 MPa notably improved recoveries across classes; lower pressure (15 MPa) reduced extraction efficiency.
• Temperature effects were compound-specific: temperatures above 40 °C enhanced recovery of some analytes but reduced oleuropein extraction; hence 40 °C was chosen as a compromise.
• High modifier fractions (>30–40%) degrade supercritical CO2 properties and provide limited incremental gains; excessive flow rate (2 mL/min) shortened residence time and reduced recovery of polar analytes.

Quantitative composition of optimized extract:

• Table 3 data summarized the major species quantified in the SFE extract (mg/kg dry leaf): oleuropein dominant at ~64,519 mg/kg, hydroxyoleuropein ~3,675 mg/kg, verbascoside ~2,298 mg/kg, oleuropein glucoside ~2,109 mg/kg, lutein ~865 mg/kg, trans-β-carotene ~217 mg/kg, and α-tocopherol ~171 mg/kg. Several other phenolics, carotenoids and tocopherol homologs were present at lower levels.
• Chromatograms confirmed co-extraction of representative compounds from each class; recovery patterns matched expectations from polarity tuning.

Benefits and practical applications of the method

The single-step SFE offers multiple practical advantages:

• Operational efficiency: complete multi-class extraction in ~40 minutes vs ~3 hours for three separate UAE procedures.
• Green credentials: use of CO2 and bio-ethanol reduces reliance on hazardous organic solvents, improving operator safety and environmental impact.
• Scalability and automation: SFE is readily automatable and amenable to scale-up for continuous or batch processing, supporting industrial valorization of pruning residues.
• Product relevance: extracts enriched in phenolics, carotenoids and tocopherols can serve as natural antioxidants, functional food ingredients, nutraceuticals, or cosmetic actives.

Limitations and trade-offs:

• Single-step SFE produced slightly lower recoveries for some polar compounds compared with targeted UAE protocols optimized per class; this reflects the compromise inherent to multi-class extraction.
• Equipment and high-pressure operation entail capital costs and engineering controls for scale-up.

Future trends and applications

Potential developments and uses include:

• Process intensification: adoption of sequential SFE stages, continuous extraction modules, or integrated fractionation (on-line fraction collectors) to improve class-specific yields while retaining automation.
• Modifier optimization: exploration of co-solvent mixtures, stepwise polarity gradients or intermittent pulsing strategies to further increase recovery of highly polar constituents.
• Downstream fractionation and formulation: coupling SFE extracts to chromatography, membrane separation, or encapsulation to produce standardized ingredients for food, pharma and cosmetic formulations.
• Lifecycle and techno-economic analyses: detailed assessment of energy, solvent and cost balances to validate large-scale circular-economy deployment for olive biomass valorization.

Conclusion

The report demonstrates that a carefully tuned SFE method using CO2 and bio-ethanol can efficiently produce a single-step extract from olive pruning residues enriched in phenolics, carotenoids and tocopherols. The optimized protocol achieves substantial recoveries (approximately 76–90% for representative markers) while delivering faster processing, improved safety and better environmental compatibility than multi-step solvent extractions. The approach is promising for sustainable production of natural bioactives, though further refinements could close the remaining recovery gap with class-specific UAE methods.

References

1. L. Olmo-García et al., Molecules, 2018, 23, e2419
2. L. Dugo et al., Food Anal. Methods, 2020, 13, 1027–1041
3. T. L. Silva Coelho et al., Ultrason. Sonochem., 2022, 84, e105980