1. Introduction
Extracts from vitis vinifera (red vine leaves) are used in herbal medicine and can help to relieve symptoms related to chronic venous insufficiency, such as swollen legs (edema), varicose veins, a feeling of heaviness, pain, tiredness, itching, and tension [
1,
2]. Red vine leaf extract primarily consists of secondary plant substances with polyphenols as the most important ones, e.g., flavonols, anthocyanins, and resveratrol [
3]. Polyphenols, which have the greatest potential as pharmaceutical drugs, are recovered from red vine leaves by leaching using appropriate solvents [
2], like acidified water and methanol due to polarity and stability reasons [
4]. In general, the solid–liquid extraction process is limited by the low yield of polyphenols and slow extraction kinetics, which is caused by the morphology of the plant material [
5]. For example, 1 kg red vine leaves yield about 70 g polyphenols depending on the strain of vitis vinifera, climate and location where the strain is grown as well as the timing of the harvest.
The cell morphology of red vine leaves mainly influences the thermodynamic partition equilibrium in the solid–liquid extraction process as the localization of polyphenols is in the vacuoles surrounded by robust and stable cell membranes [
5,
6]. The diffusion and mass transfer of polyphenols can be enhanced and accelerated by alternative methods of natural plant extraction and process intensification that promote cell membrane disruption. The energy to disrupt plant cell membranes is provided by ultrasonic waves (US) [
7,
8,
9], microwaves (MW) [
10,
11,
12], or by the use of pulsed electric fields (PEF) [
13,
14,
15,
16,
17]. US-assisted extraction generates turbulences and thermal effects promoting extraction, as well as production and growth of bubbles inside liquids causing cavitation leading to structural attacks [
18]. Cavitation bubbles can implode near a solid surface as a microjet [
19,
20] breaking up plant cell membranes [
21,
22,
23]. An alternative to improve efficiency of natural plant extraction processes is by applying MW [
24,
25,
26]. The MW radiation penetrates the target plant material and interacts with polar molecules through ionic conduction and dipole rotation [
27] to generate heat. Adsorption and penetration depth, which are dependent on the dielectric constant and the dielectric loss of the material [
28], are determined by the frequency of the MW [
29]. The MW radiation increases the local temperature leading to an increase of the internal pressure of plant cells. The plant cells are primarily comprised of a vacuole filled with intracellular water and secondary metabolites that consequently rupture under pressure [
30] and promote kinetics [
31]. The PEF-assisted technique is based on the electroporation phenomenon of the cell membranes, when a potential difference arises across a membrane [
32,
33]. During electroporation, molecular orientation takes place where the polar molecules align themselves with the electric field and migrate to the membrane induced by the electric field [
34]. The electrocompression starts to rupture the membrane and creates pores [
35]. This can result in a temporary (reversible) or permanent (irreversible) loss of membrane permeability [
36,
37]. The extent of the loss in permeability and the pore formation depends on the induction of a critical electric field strength and cell size in a range of 1–2 kV/cm for a plant cell size of 40 to 200 µm [
38].
In general, a typical extraction setup consists of a batch stirred vessel with temperature control and has been widely applied in the industry [
39]. Even though the set-up is ubiquitous, the optimum extraction conditions which maximizes solid-liquid extraction with minimal energy input and costs has not identified [
40,
41]. For identifying optimal conditions of a solid–liquid extraction, a laboratory robot provides a systematic and highly reproducible process development [
42,
43,
44]. Temperature, pH value, and solvent composition influence not only extraction kinetics and pseudostationary equilibrium but also solubility and stability of the extracted secondary metabolites [
45,
46]. A robot workstation allows high-throughput experiments and saves time by permitting unattended overnight operation [
47]. Additionally, solid–liquid extraction processing plants require an appropriate design reflecting the unique characteristics of any plant material, as the solute can be in root, leaf, fruit, etc. Thus, effective diffusivity is an important transport property to consider when designing mass transfer equipment and increasing the scale of the process [
48]. The most widely accepted models used to describe the extraction kinetics are: Fick’s law of diffusion [
49,
50,
51,
52], the modified chemical kinetic equations [
53,
54,
55], and the two-parametric empirical equations [
56,
57].
In this study, a custom-built laboratory robot is used to screen for the optimal conditions of a natural plant extraction process as temperature, pH value, and solvent composition are varied. For comparison, a standard stirred vessel experiment is used with alternative techniques, such as ultrasonification, microwaves or pulsed electric fields.
2. Materials and Methods
2.1. Preparation of Red Vine Leaves and Chemicals
Red vine leaves (
Vitis vinifera, DAKAPO GN7225-8 Deckrot x Portugieser Börner) were collected on 3 October 2014 in Geisenheim (RP), Germany, and dried at 75 °C for 48 h (UT6120, Heraeus Holding GmbH, Hanau, Germany). After drying, the red vine leaves were manually ground in a mortar using a pestle. The bruised red vine leaf powder was sieved into 3 fractions by riddle screens (Analysette 3 PRO, Frisch GmbH, Idar-Oberstein, Germany) with 200 µm, 450 µm, 2000 µm, and 4000 µm mesh sizes. Furthermore, the bulk densities
were determined by filling a 10 mL measuring cylinder (Brand GmbH & Co KG, Wertheim, Germany) with 5 mL the red vine leaves and the filled measuring cylinder was weighed using an analytical balance (1702, Sartorius AG, Göttingen, Germany). The particle size distribution is displayed in
Figure 1 and further details are given in
Table 1. For measurements with undried red vine leaves a part of the collection was stored in a freezer (GS26DN11, Siemens AG, München, Germany) at a temperature of −18 °C. After defrosting, the red vine leaves were cut into 2 to 4 mm pieces by a scalpel. To maintain comparability with the dried red vine leaves the moisture content was calculated by weighing the undried red vine leaves and reweighing these red vine leaves after drying. The loss of water averaged 72.7 ± 3.7% in the course of 5 experiments.
The solvents were deionized water (0.01 µS/cm) mixed with hydrochloric acid (35–38%, CHEMSOLUTE®, Th. Geyer GmbH & Co. KG, Renningen, Germany) at pH values of 1.21, 1.53, 2.00, 2.50, and 3.00 and pure methanol (≥99.9%, Sigma-Aldrich, St. Louis, MO, USA). The pH value was measured with a pH meter (pH 526, WTW, Weilheim, Germany).
2.2. Folin–Ciocalteu Assay
Concentration measurement was done by UV/Vis spectrometry (UV-mini 1240, Shimadzu Corporation, Kyōto, Japan). The Folin–Ciocalteu assay was performed as described in detail in [
58] using Folin–Ciocalteu reagent (Merck KGaA, Darmstadt, Germany) and Na
2CO
3 (Bernd Kraft GmbH, Duisburg, Germany).
2.3. Extraction Apparatus
Each extraction measurement was repeated 3 to 5 times and the ratio of red vine leaves to extractant was set to 40 g/L. In detail, the weighed portions
and the volumes of the solvents
are given in
Table 1. In order to determine the optimal extraction conditions and partition equilibria, a custom-built laboratory robot (Lissy 4G200, Zinsser Analytic GmbH, Eschborn, Germany) and red vine leaves with particle size of 200 to 450 µm were used. In 8 vials red vine leave powder is suspended and shaken in time intervals of t = 1, 5, 10, 15, 30, 60, 90, and 120 min. The regulated thermostat temperatures of 25 °C, 35 °C, 45 °C, 55 °C, 60 °C, and 65 °C yielded temperatures in the extraction vials of 23.0 °C, 34.0 °C, 43.0 °C, 51.0 °C, 56.0 °C, and 60.5 °C, respectively. After agitation with a shaking rate of
samples were taken and filtered using a mesh size of 1 µm (7700-9905, Whatman plc, Little Chalfont, UK). Details of the laboratory robot and its handling is described in detail in [
42].
For comparison an 1 L jacketed tank held at 50 °C was used. The 1 L jacketed tank is equipped with a propeller mixer adjusted to and a metal mesh cage that retains the dried red vine leaves (2000 to 4000 µm) when using a solvent volume of 250 mL.
2.4. Alternative Extraction Techniques
For the alternative extraction techniques, red vine leaves with particle sizes of 450 to 2000 µm (size small, SS) and 2000 to 4000 µm (size large, SL) were used. Additionally, the temperature of the extraction slurry was measured with a PT100 probe when a sample was removed for the UV/Vis analysis (UV-mini 1240, Shimadzu Corporation, Kyōto, Japan).
The microwave assisted extraction (MW) was performed in a microwave oven (MW 4000, Landgraf Laborsysteme HLL GmbH, Langenhagen, Germany) using a 50 mL vessel containing a stirring bar and an immersed PT100 probe and operates at (100%). For temperature control of the extraction batch vessel (50 °C or 60 °C) the immersed PT100 controller is connected to a two-level controller, which regulates the power of the microwave.
The ultrasonic-assisted extraction (US) was executed using an ultrasonic probe (Bioblock Scientific Vibra Cell VC 750, Standard Probe ½”, Thermo Fisher Scientific Inc., Waltham, MA, USA) at a frequency of 20 kHz. The ultrasonic probe was dipped in a stirred 150 mL jacketed tank and the maximum amplitude (114 µm) was reduced to 30% (34.2 µm) or 40% (45.2 µm). During 120 min of application the US probe generates and of energy at 30% and at 40% of the maximal amplitude, respectively. With the energy input is correlated to an effective power of , respectively .
For the pulsed electric field assisted extraction (PEF) the setup consists of a high voltage generator (610C, Trek Inc., Lockport, NY, USA), an impulse generator (8035, Hameg Instruments GmbH, Mainhausen, Germany), a Schmitt trigger circuit, a high voltage switch, an oscilloscope (D1010, Siemens AG, München, Germany), and 2 plate electrodes (1.4301). Plate electrodes with a separation distance of 0.42 cm and a surface of 6 cm2 were located in a 20 mL mixed glass beaker. The pulsed electric field setup generates monopolar exponential pulses for a duration of with intervals between pulses. The voltage was set to or and using the setup generates an electric field strength of 3.33 kV/cm or 1.67 kV/cm, respectively. The electrical power is given by where the current is calculated by Ohm’s law and the electric resistance is defined by . The conductivity was measured after 120 min application time with a conductivity electrode (Seven2GoTM S3, Mettler-Toledo, Columbus, OH, USA) giving for , resp. for . The resulting effective power is , respectively . Using undried red vine leaves the PEF assisted extraction process was executed with a voltage of , a conductivity of and an electric power of .
2.5. Mass Transfer
Fick’s law is used to describe the mass transfer and several simplifications have been made to enable the comparison of different techniques. The diffusion of the polyphenols is not hindered by other components and there is only one pseudo-solute (gallic acid [
59,
60]) diffusing. It is assumed that the dispersed solid material is an assembly of spherical particles of the same size with radius r and bulk density
[
61]. The volume
of the red vine leaves is then related to their surface area
and to their total mass
:
A decrease in the thickness of the diffusion layer, which surrounds each particle, as stirring increases, is neglected. Thus, the flux
is equal to the amount of polyphenols
entering the bulk solution
in unit time
. The mass transfer from the beginning until equilibrium is analyzed from experimental data. Thus, the flux
is given by
where
is the diffusion coefficient,
is the volume of the bulk, and
is the difference of the polyphenol concentrations at the center and at the periphery of a given particle, considering the
to
ratio:
where
is the equilibrium concentration and
the mass of the bulk liquid resp. of
ms of the solid. With Equation (3), when knowing both masses and the equilibrium concentration, the initial (pseudo)polyphenol content in the particle can be calculated. For calculating the effective diffusion coefficient
according to Equation (2), all data are given in
Table 1. The mass of the bulk
is given by
where
and
are the mass percentage of water and methanol, neglecting the amount of HCl. The amounts of water
and methanol
are defined by the density
[
62] and the volume
.
4. Conclusions
The influence of temperature and solvent composition on extraction kinetics and saturation and degradation limits is screened using a fully automated laboratory robot for the optimization of solid–liquid extraction when leaching polyphenols from red vine leaves. Gallic acid was considered to be the representative pseudo-solute. The results generated by the laboratory robot show that varying the acidity of extracting agent does not influence the polyphenols yield and extraction kinetics. However, an increasing temperature markedly enhances the extraction yield and saturation concentration, but does not significantly improve extraction kinetics. An upper limit is given as polyphenols are thermally sensitive and the extraction efficiency is reduced at temperatures higher than 60 °C. When investigating different amounts of methanol as a modifier at an extraction temperature of 56 °C, a mixture of methanol:water 50:50 (v/v) independent of the pH value gives fast kinetics and the highest yield. However, methanol/water solutions give nearly results as acidified methanol/water solutions, and best results were when using only acidified water. In conclusion, the laboratory robot allows systematic and highly reproducible screenings of process conditions. Furthermore, the use of the laboratory robot allows massive time savings during screening with parallel and unattended overnight work.
With nonconventional processing techniques, like microwave, ultrasonic, and pulsed electric field, smaller particle size positively influences the extraction process due to a shorter diffusion path and higher surface area per volume. As a result, an appropriate sample preparation and combination is recommended with respect to industrial application with either filtering limits after maceration or a limiting pressure loss with percolation.
Generally, the microwave-assisted extraction process followed by the ultrasonic-assisted extraction process gives the highest yield of polyphenols at approximately 50 °C. PEF are less effective than MW or US assisted extraction in comparison to conventional batch extraction. Interestingly, US when combined with undried plant material presents a promising technique for benign extraction of thermal sensitive solutes. Finally, the best industrial extraction procedure for leaching polyphenols from red vine leaves uses a batch reactor with implemented magnetrons to generate microwaves and quickly heat suspended plant material.