Extraction of oleuropein and luteolin-7-O-glucoside from olive leaves: Optimization of technique and operating conditions

Highlights

• Oleuropein and luteolin-7-O-glucoside have been extracted from olive leaves.

• Response surface methodology has been used to optimize extraction.

• Extraction using pressurized liquids is more effective than dynamic maceration.

• Optimal extraction conditions are reached after 5 min of operation.

• It is advisable to use pressurized liquids to extract phenols at the industrial level.

Abstract

Olive leaves have become a promising source of phenolic compounds and flavonoids with high added value. Phenolic compounds and flavonoids are important sources of antioxidants and bioactives, and one of the processes used to effectively produce them is extraction via solvents, using aqueous ethanol solutions. To obtain the highest extraction yield per kg of biomass, olive leaves were extracted using a conventional technique (dynamic maceration) and an emerging technology, such as pressurized liquid extraction. Studies of the factors that influence these processes were performed: temperature, leaf moisture content, solvent/solid, and aqueous ethanol concentration were optimized using the central composite and Box-Behnken experiment designs. Pressurized liquid extraction resulted in more efficient oleuropein and luteolin-7-O-glucoside extraction than dynamic maceration. The operational conditions for maximizing the recovery of phenolic compounds and flavonoids and antioxidant capacity were determined to be 190 °C, leaf moisture content of 5%, and aqueous ethanol concentration of 80%.

Introduction

Olive leaves, an agricultural waste obtained during harvesting or processing of olive fruits, are found in large quantities in the olive oil and olive table industries, where they are separated from olives using pneumatic separation systems and create a residue without industrial interest. However, from an economic point of view, as industrial residue from vegetable materials, olive leaves are an excellent source of phytochemicals. Olive leaves contain significant amounts of valuable compounds such as phenolic and flavonoid compounds, which have attracted considerable interest due to their potential use as food additives and/or nutraceuticals in both the food and pharmaceutical industries (Şahin et al., 2018). The amount of olive leaves that accumulate annually from these industries may exceed 1 million tons. Therefore, this olive industry residue can present interest in a biorefinery context. Moreover, it is worth recovering high added-value compounds from this material, as these compounds can present great interest for the pharmaceutical, food, and cosmetic sectors, due to the trend of using natural products instead of synthetic ones.

Since olive leaf extracts are rich in phenolic and flavonoid compounds, they present great potential as natural antioxidants for food preservation, bioactive food, etc. Oleuropein is the most abundant phenolic compound in olive leaves and the main compound responsible for the antioxidant and bioactive properties of hydroalcoholic extracts (Fakhraei et al., 2014). There has been increasing evidence that helped establish a scientific basis for the use of oleuropein-rich extracts as functional foods. The antioxidant and anti-inflammatory effects of oleuropein and the ability to treat pro-oxidant and inflammatory-related diseases (i.e., cancer, cardiovascular disease, hepatic disorders, obesity, diabetes, etc.) are its main biological activities, and these have been confirmed by previously published papers (Hassen, Casabianca, & Hosni, 2015). Qualitative and quantitative compositional analysis using high-performance liquid chromatography (HPLC) also revealed that luteolin-7-O-glucoside, a flavonoid, is another major polyphenolic compound present in olive leaf extracts (Guinda et al., 2015, Kiritsakis et al., 2018). This phytocompound can be a potent drug against colon carcinogenesis (Baskar, Ignacimuthu, Michael, & Al Numair, 2011) that also demonstrated gastroprotective effects when studied on different ulcer models using rats (Antonisamy et al., 2016).

Several methods have been used for the extraction of phenolic and flavonoid compounds from olive leaves and other by-products which originate from the olive oil industry (Romero-García et al., 2016). A traditional solvent extraction via maceration is a favorable process since heat sensitive compounds can be recovered at low temperatures. However, the extraction rate is limited by the dissolution, mass transfer to the solution by diffusion, and osmotic pressure of soluble compounds. Since slow extractions require large equipment to increase the yield, and long exposure to solvent may cause degradation, researchers have been seeking to provide alternative physical extraction methods using emerging technologies to replace the conventional processes (Cruz, Brito, Smirniotis, Nikolaidou, & Vieira, 2017).

Pressurized liquid extraction (PLE) uses solvents to carry out extraction at high pressure and temperature, always below their critical points, thus maintaining the liquid state of the solvents during the entire extraction process. When applying such conditions, extraction processes can occur faster, and typically, higher extraction yields are obtained using low volumes of organic solvents (e.g., 20 min and 10–50 mL solvent for PLE can be compared with a traditional extraction step where 10–48 h and up to 200 mL solvent are required), therefore decreasing the dilution of the samples. These characteristics are mainly due to the improvement in mass-transfer kinetics achieved at high temperature and pressure. Using high temperatures increases the solubility of the analytes in the solvent and decreases the viscosity and surface tension of the solvent, thus allowing for a better penetration of the solvent into the matrix (Herrero, Castro-Puyana, Mendiola, & Ibañez, 2013). PLE is broadly recognized as a green, eco-friendly, efficient, and cost effective extraction approach. As previously mentioned, it is mainly due to its low solvent consumption. Although the majority of PLE applications developed so far are aimed at the extraction of contaminants, herbicides and pesticides from different natural, food and environmental samples (Martínez, Gonzalo, Cruz, Álvarez, & Méndez, 2007), this technique has also demonstrated its usefulness for the extraction of bioactive compounds from natural matrices thus considering as a promising innovative extraction technology for recovering polyphenols from olive leaves. PLE has been used successfully in polyphenols extraction from various plant matrices and it can be implemented at industrial-scale (Putnik et al., 2017).

In addition, the extraction of bioactive compounds from plant materials requires several preliminary steps, such as drying, size reduction, or carrying out studies on their solvent retention capacities and extraction kinetics, to obtain higher extraction yields. These require labor-intensive and time-consuming preparation procedures. The recovery of phenols from olive leaves is clearly influenced by a number of factors, and the effect of drying plays a significant role in the amount of phenols recovered and their antioxidant capacities. Drying is the most common commercial technique employed before the extraction of high added-value compounds from plant materials. Using this technique helps to reduce the water content of the plants and prevents their microbial spoilage as enzymatic degradation (Ahmad-Qasem et al., 2016). Investigation results demonstrated that the oleuropein content (OC) extracted from fresh olive leaves was low, thus indicating that drying the leaves would be required for high oleuropein recovery (Afaneh et al., 2015, Kamran et al., 2015, Şahin et al., 2018). However, no research has been conducted on the influence of the moisture content (MC) of leaves on the extraction process. To optimize the recovery of bioactive compounds, this parameter should be investigated.

In this study, we used the response surface methodology (RSM) to optimize the extraction of phenolic compounds and flavonoids from olive leaves, with focus on oleuropein and luteolin-7-O-glucoside. The application of the RSM allows studying the influence of several process factors on one or more responses of the design. Experimental designs are able to determine interactions between parameters and obtain and predict in a quick and reliable way optimum extraction conditions using a minimum number of experimental runs (Arabi et al., 2016, Ostovan et al., 2018). Experimental design methodology has recently used in the optimized extraction of biologically active constituents and several biological indicators in different samples (Arabi et al., 2017, Ostovan et al., 2018, Bagheri et al., 2019). We used dynamic maceration (DM) and PLE as extraction methods, which allowed us to compare our results with those obtained using conventional extraction methods. The aim of this work was to obtain antioxidant rich-oleuropein and luteolin-7-O-glucoside extracts featuring high antioxidant capacities. The effects of treatment temperature, MC of leaves, solvent/solid ratio, and ethanol concentration on the contents of oleuropein and luteolin-7-O-glucoside biophenols in the extracts and on their antioxidant capacities were studied using both techniques, and corresponding mathematical models were developed for determining the optimal operational conditions which would favor a better valorization of this plant by-product.