Application of ionic liquids based enzyme-assisted extraction of chlorogenic acid from Eucommia ulmoides leaves

Highlights

• An ionic liquid based enzyme-assisted extraction method of natural product was explored.

• ILEAE utilizes enzymatic treatment to improve permeability of ionic liquids solution.

• Enzyme incubation and solvent extraction process were ongoing simultaneously.

• ILEAE process simplified operating process and suitable for more complete extraction.

Abstract

A new approach for ionic liquid based enzyme-assisted extraction (ILEAE) of chlorogenic acid (CGA) from Eucommia ulmoides is presented in which enzyme pretreatment was used in ionic liquids aqueous media to enhance extraction yield. For this purpose, the solubility of CGA and the activity of cellulase were investigated in eight 1-alkyl-3-methylimidazolium ionic liquids. Cellulase in 0.5 M [C6mim]Br aqueous solution was found to provide better performance in extraction. The factors of ILEAE procedures including extraction time, extraction phase pH, extraction temperatures and enzyme concentrations were investigated. Moreover, the novel developed approach offered advantages in term of yield and efficiency compared with other conventional extraction techniques. Scanning electronic microscopy of plant samples indicated that cellulase treated cell wall in ionic liquid solution was subjected to extract, which led to more efficient extraction by reducing mass transfer barrier. The proposed ILEAE method would develope a continuous process for enzyme-assisted extraction including enzyme incubation and solvent extraction process. In this research, we propose a novel view for enzyme-assisted extraction of plant active component, besides concentrating on enzyme facilitated cell wall degradation, focusing on improvement of bad permeability of ionic liquids solutions.

Introduction

Eucommia ulmoides, with a Chinese name Duzhong, is one of the oldest edible natural herbs used for different medicinal purpose in some asian countries including mainly China and Japan. It is known to be used for nourishing the kidney and liver, strengthening bone and muscle and preventing abortion. A recent study showed [1] that the leaf of Eucommia ulmodies has the analogous chemical ingredients as the bark, leading to same therapeutic action. As one of most important active compounds in E. ulmodies leaves, chlorogenic acid (CGA, a kind of phenylpropanoids) shows anti-inflammatory, anti-microbial, anti-oxidation and other bioactivities [2], [3]. In conventional techniques, the extraction of CGA is achieved by using boiling, refluxing or heating. One shortcoming of these methods is the losing of CGA on account of oxidation, ionization and hydrolysis during extraction process [4], [5]. The other shortcoming is the use of the large amount of volatile organic solvent and the expense of long extraction time [6], [7].

Current extraction techniques require E. ulmodies leaves to be crushed into powder before the extraction intended for improving permeability of plant material matrix and the dissolution of target ingredients. However, gutta percha, a high content component in E. ulmodies leaves, has distinct properties such as: high flexibility, viscosity and anti-impact strength [8]. These properties of gutta percha result in that E. ulmodies leaves samples are not easy to be powdered into a smaller size, which might bring about low extraction yield of target ingredients. To make target analytes susceptible to extract, a pretreatment process is needed to increase contact area of plant cell with extraction solvent, conventional mechanical pulverizing method is not suitable for the E. ulmodies leaves. Therefore, an efficient pretreatment strategy is still one of the key barrier to target ingredients extraction.

Enzyme-assisted extraction of natural ingredients from plants is widely investigated in recent studies for its advantages in simple operation, high efficiency, and environment friendship [9]. Most of the studies in this field adopt cellulase, pectinase and β-glucosidase to hydrolyze and degrade plant cell wall constituents to improve the release of intracellular ingredients [10], [11], [12]. Common enzymatic extraction method includes two intermittent processes: A) enzyme incubation process in water (or buffer solution); B) organic solvent extraction process. When target ingredients are moderate polarity or low polarity components, there need a filter operation between A and B process [12], [13], [14], in order to separate plant materials from water, in which would lose part of target ingredients more or less. After filter operation, there may need a freeze-drying process for plant materials, or else extraction will consume more organic solvent.

Ionic liquids, which are composed of bulky organic cations and inorganic or organic anions, are liquid near room temperature. They have recently excited much research interest for various kinds applications thanks to their remarkable properties: negligible vapor pressure, chemical and thermal stabilities, designable structures, ease of recycling and manipulation, miscibility with water and organic solvents, as well as good solubility and extractability for various organic compounds [15], [16]. There is an increasing awareness of the need for environmental protection, and ionic liquids are considered to be promising alternatives to traditional solvents in the preparation of various active ingredients from plant samples such as alkaloids [17], lignans [18], and polyphenolic compounds [19], [20]. However, existence of ionic liquid in water brings about its worse permeability for solid plant matrix. Many researches resolve permeability problem by soaking samples [16], [18], [21], [22] for a long time, but stability of these natural compounds in ionic liquid solution are needed to be investigated.

We come up with an idea to utilize the enzymatic extraction in ionic liquids aqueous solution, to improve permeability of the solvent in the extraction process, while utilizing the enzyme activity in cell wall hydrolysis at the same time. In the proposed ionic liquids based enzyme-assisted extraction (ILEAE) method, enzyme incubation process and solvent extraction process were completed simultaneously in one system, without filter operation to minimize the loss of target ingredients. And regardless of low polarity or high polarity compounds, extraction method is suitable for more complete extraction. Besides that, treatment after enzymatic hydrolysis of plant cell wall would improve bad permeability for low smash degrees of E. ulmodies materials.

In this work, we aim to make it clear whether the used enzyme has activity in ionic liquids solutions. This is the first report of ionic liquids based enzyme-assisted extraction method. The objective of this current work was to investigate the feasibility of employing ILEAE as an efficient technique to recover CGA from E. ulmodies leaves. The optimization of ILEAE method was carried out, and the mechanism of the enhanced extraction by ILEAE was discussed by observing cell destruction of plant material by scanning electron microscopy.