Comparative Study of Microwave-Assisted Extraction and Ultrasound-Assisted Extraction Techniques (MAE vs. UAE) for the Optimized Production of Enriched Extracts in Phenolic Compounds of Camellia japonica var Eugenia de Montijo ^†

Abstract

Camellia japonica is an underexplored medicinal plant with associated bioactivities. Innovative approaches are proposed in regard to the large-scale application of C. japonica, being one of the main routes for the extraction of phenolic compounds. The optimum conditions for the extraction of phenolic compounds from the flowers of C. japonica var. Eugenia de Montijo were determined using the response surface methodology (RSM). A five-level experimental design was carried out and analyzed via RSM using, as variables, temperature (T), time (t) and solvent (S), in the case of microwave-assisted extraction (MAE), and power (P), t and S in the case of ultrasound-assisted extraction (UAE). The compounds were identified using HPLC–MS–MS. Two responses were studied: the extraction yield and concentration of phenolic compounds. The results showed that the maximum yields (80%) were obtained at high temperatures and low times (180 °C, 5 min) when using MAE. Lower yields (56%) were obtained using UAE (optimal conditions 62% amplitude, 8 min, 39% acidified ethanol). The main family of phenolic compounds were flavonols. Moreover, the present study contributes to the valorization of underused flower species commonly present in the North-West region of Spain, by obtaining extracts rich in phenolic compounds that can be potentially applied as ingredients in different industrial fields.

1. Introduction

Camellia spp. is a member of the Theaceae family, a group of perennial evergreen flowering plants that are widely commerzialized, which underlines their economic relevance [1]. This genus includes more than 250 plants with an intricate taxonomy, with Camellia sinensis L., Camellia oleifera Abel. and Camellia japonica L. being the most outstanding species. All these species have been traditionally used for multiple applications, including tea, the production of essential oils and ornamental purposes [2]. C. japonica and its hybrids are recognized as ornamentals since they have flowers of several colors and forms, long and varied blossoming seasons and different growth habitats [3]. According to many studies, C. japonica flowers possess several bioactive molecules that confer them several properties, such as anti-oxidant, antimicrobial, anti-inflammatory, and anti-cancer effects, among others [1,4,5,6]. These bioactivities are attributable to the occurrence of phenolic compounds (e.g., anthocyanins, flavan-3-ols, flavonols), polysaccharides, polyunsaturated fatty acids and pigments [1,7]. Despite these health-promoting activities, C. japonica flowers are still considered an underexploited resource at an industrial level [8]. However, in recent decades, several studies have disclosed the antioxidant activity, total phenolic contents, and specific phenolic acids of C. japonica flowers [9,10,11].

In recent decades, phenolic compounds are some of the compounds that have attracted the most attention due to the numerous beneficial properties attributed to them [12]. The extraction of phenolic compounds from plant matrices is complex and challenging since it includes a huge variety of molecules comprising very variable chemical properties [13]. So far, their extraction has been mainly performed using conventional methods; however, they require the use of large volumes of extraction solvents, extensive hands-on time and cannot be extracted using automated methods, so they are generally considered labour-intensive techniques [14]. These experimental drawbacks, along with the negative environmental impact they have, have led to the development of new extraction methods that are considered green technologies [15]. Among them, microwave-assisted extraction and ultrasound-assisted extraction techniques stand out for several reasons.

Microwave-assisted extraction (MAE) improves the extraction efficiency in comparison to conventional techniques due to the interaction among microwaves and the polar molecules in the extraction media, with an increase in the internal pressure of the solid material [13]. Moreover, in contrast to the conventional techniques, MAE reduces the thermal gradients and instant heating of the biomass, enhances the extraction yield, shortens extraction times and decreases solvent quantities [16]. MAE has been previously and succesfully applied to hassk or fruits from Camellia spp. [17,18,19].

Ultrasound-assisted extraction (UAE) improves the extraction efficiency in comparison to conventional techniques due to the implementation of high-frequency ultrasonic waves. These waves have the capacity to disrupt the plant cell walls, which facilitates the penetration of the solvent into the cells and therefore the extraction of molecules [20]. Several works have been recently published on the use of UAE applied to the extraction of phenolic compounds from Camellia spp. leaves [21,22].

To date, to the best of our knowledge, there is no study on the extraction of phenolic compounds from C. japonica flowers by using MAE or UAE. Hence, the aim of this study was to determine the extraction conditions required to best obtain extracts rich in phenolic compounds from C. japonica flowers by using these extraction methods.

2. Material and Methods

2.1. Chemicals and Reagents

Phenolic compound standards (cyanidin-3-glucoside, luteolin, quercetin, gallic acid, p-coumaric acid and resveratrol) were bought from Sigma (Saint Louis, MO, USA). Ethanol was bought from VWR (Radnor, PA, USA). All organic solvents used for the extraction and chromatographic analysis were HPLC-grade. High-purity water was obtained using Direct-Q 5UV Millipore equipment (Merck, Rahway, NJ, USA). Nylon syringe filters (0.22 µm pore size, 25 mm diameter) were acquired from Filter-Lab (Barcelona, Spain).

2.2. Sample Collection

C. japonica flowers (var. Eugenia de Montijo) were collected in Galicia (NW Spain) in the winter season of 2020. Samples were lyophilized (LyoAlfa10/15, Telstar, Thermo Fisher Scientific, Waltham, MA, USA), pulverized into a fine powder by a blender, and stored at −20 °C until extraction.

2.3. Sample Extraction Method

The process used to obtain the bioactive compounds was carried out via MAE and UAE. For MAE, the extraction was performed using the Multiwave 3000 (Anton Paar, Graz, Austria). The variables studied were temperature (T), time (t) and solvent (S), which were regarded as critical extraction parameters. Specifically, T varied between 50 and 180 °C, the t range was 5–25 min and the concentration of acidified ethanol was switched from 0 to 100% v/v. For UAE, the extraction was performed using the CY-500 (Optic Ivymen Systems). The variables studied were processing time (t, 5–45 min), power (P, 30–80%) and solvent (0–100% acidified ethanol), which were regarded as critical extraction parameters. Once the extractions were completed, the samples were centrifuged at 9000 rpm for 15 min and the supernatant was filtered. Extracts were stored in a freezer at −80 °C until their analysis.

The optimum conditions for the extraction of phenolic compounds from the flowers of C. japonica var. Eugenia de Montijo were determined using the response surface methodology (RSM) and using circumscribed central composite design (CCCD). As previously reported, this model allows one to identify the operating conditions that maximize the following responses: yield and phenolic compounds [23].

In addition, the extraction performance was calculated as follows (Equation (1)):

Y₁ (%) = (P₂ − P₁)/P₀ × 100

(1)

where P₀ is the mass of lyophilized flower prior to extraction (mg), P₁ is the mass of the empty crucible (mg), and P₂ is the mass of the dry extract in the crucible (mg).

2.4. Determination of Bioactive Compounds

The identification and quantification of phenolic compounds were carried out via HPLC–MS–MS (Thermo Scientific TSQ Quantis). Analytical separations were performed using a ThermoFisher C18 column (150 × 3.9 mm, 4 µm particle). The column was thermo-stated at 35 °C. The mobile phases used for the optimized analytical method were as follows: (A) milli-Q water acidified with 0.1% of formic acid; (B) acetonitrile acidified with 0.1% of formic acid. The flow rate was fixed at 0.350 mL/min. Briefly, 350 µL was pumped to the injection module in a C18 pre-concentration cartridge and further eluted by a loading pump following gradient conditions from 100% to 0% A. Tandem mass analysis was performed after optimizing the tube lens and the collision energy for each compound separately. Likewise, the ESI conditions were automatically adjusted to the set flow. Data acquisition and HPLC–MS–MS analysis interpretation were conducted by means of Xcalibur software (Version 4.3, Thermo Fisher Scientific, Waltham, MA, USA).

3. Results and Conclusions

The process was optimized by the RSM using a five-level central composite design, combining different independent variables. The RSM was performed in order to optimize the different responses associated with polyphenol production: the extraction yield and concentration of phenolic compounds. Theoretical models were fitted to experimental data, statistically validated, and used in the prediction and optimization steps.

Regarding the extraction efficiency of the two evaluated techniques, MAE provided better outcomes than UAE. For MAE, the results showed that the maximum yields (80%) were obtained using a combination of a high temperature and short time (180 °C, 5 min). Other approaches using the same temperature (180 °C) but longer incubation times (25 min) dropped the efficiency to 59%. Similarly, when using a combination of lower temperatures (50 °C) and short times (5 min), the extraction results were 52%. When applying longer times (25 min), recovery was around 50%. For UAE, the yields achieved under the optimal conditions (62% amplitude, 8 min, 39% acidified ethanol) showed a lower extractive efficiency, with values around 56%. Temperature does not have a significant impact on the results in UAE extraction. On the other hand, higher amplitudes provided yield efficiencies of up to 58%.

With regards to the phenolic composition of the extracts, the main family of phenolic compounds identified were flavonols, the major compounds of this family being quercetin-3-o-arabinose and kaempferol 3-o-acetyl-glucoside. These molecules possess a few associated biological activities, such as anti-oxidant, anti-microbial, anti-inflammatory and anti-viral effects [24,25,26].

Therefore, the present study underlines that MAE is a sustainable and effective technique that can be used to recover phenolic compounds from C. japonica flowers. The application of the optimized conditions would contribute to the valorization of underused flower species, common in the North-West region of Spain, via the obtainment of extracts rich in phenolic compounds that could potentially be applied as ingredients in different industrial fields.