Extraction of Natural Pigments from Gardenia Jasminoides J.Ellis Fruit Pulp Using CO2-Expanded Liquids and Direct Sonication

separations

Article Extraction of Natural Pigments from Gardenia Jasminoides J.Ellis Fruit Pulp Using CO2-Expanded Liquids and Direct Sonication

Hiroki Sakai 1, Kento Ono 1, Shinichi Tokunaga 1, Tanjina Sharmin 1,2, Taku Michael Aida 1,2 and Kenji Mishima 1,2,*

1 Department of Chemical Engineering, Faculty of Engineering, Fukuoka University, 8-19-1 Nanakuma Jonan-ku, Fukuoka 814-0180, Japan; [email protected] (H.S.); [email protected] (K.O.); [email protected] (S.T.); [email protected] (T.S.); [email protected] (T.M.A.) 2 Research Institute of Composite Materials, Fukuoka University, 8-19-1, Nanakuma Jonan-ku, Fukuoka 814-0180, Japan * Correspondence: [email protected]; Tel.: +81-92-871-6631 (ext. 6428); Fax: +81-92-865-6031

Abstract: In this work, a carbon dioxide-expanded liquid (CXL) extraction system was used with or without direct sonication for the extraction of highly polar natural pigments (crocin-1 and crocin-2) from Gardenia jasminoides Ellis fruit pulp. The effects of different parameters, including modifiers (ethanol, water, aqueous ethanol), temperature (5–25 ◦C), pressure (8–14 MPa), and sonication time (0–200 s) on extraction concentrations were examined using the CXL system. Aqueous ethanol (50% or 80%, v/v) was selected for the CXL system as a modifier due to its efficiency. The best conditions for extraction were found at 25 ◦C and 10 MPa. The CXE 80% extraction system with direct sonication extracted a significantly higher amount of crocin-1 and crocin-2, 13.63 ± 0.5 and 0.51 ± 0.05 µg/mL, respectively, compared to conventional solid–liquid methanol extraction (10.43 ± 0.3 and 0.37 ± 0.02 µg/mL, respectively). Under these conditions, a water-rich phase, an ethanol-rich phase, and a CO2-rich gas phase coexisted in the high-pressure cell in the CXE 80% Citation: Sakai, H.; Ono, K.; extraction system, which was vigorously disrupted by the addition of sonication, resulting in a Tokunaga, S.; Sharmin, T.; Aida, T.M.; compressed aqueous ethanol phase and an aqueous ethanol-modified CO2-rich phase, and may have Mishima, K. Extraction of Natural a positive influence on extraction. Pigments from Gardenia Jasminoides J.Ellis Fruit Pulp Using Keywords: CO2-expanded liquid; crocin; sonication; natural pigments; gardenia fruits CO2-Expanded Liquids and Direct Sonication. Separations 2021, 8, 1. https://dx.doi.org/10.3390/ separations8010001 1. Introduction

Received: 4 December 2020 Natural pigments extracted from natural resources (plants, minerals, insects, Accepted: 21 December 2020 microorganisms such as algae, cyanobacteria, fungi, etc.) are less toxic, less polluting, Published: 24 December 2020 and noncarcinogenic and tend to have potential positive health effects [1–4]. Natural pigments are therefore now a market demand for use in foods and cosmetics over synthesized Publisher’s Note: MDPI stays neu- natural-identical pigments due to the rising awareness of harmful and undesirable risks tral with regard to jurisdictional claims related to chemically modiﬁed synthetic pigments. in published maps and institutional Crocins are among the plant-based, water-soluble, natural yellow pigments, derived from afﬁliations. C–20 mono or di-glycosyl esters of crocetin (crocin-2 and crocin-1, respectively). Crocins, mostly present in the stigmas of saffron (Crocus sativus L) and the fruits of gardenia (Gardenia jasminoides J.Ellis), are responsible for their color [5]. At present, the global extrac-

Copyright: © 2020 by the authors. Li- tion of crocins is mostly dependent on saffron, which is the most expensive spice, and the censee MDPI, Basel, Switzerland. This production is labor-intensive and time-consuming. Alternatively, gardenia, an evergreen article is an open access article distributed shrub belonging to the family Rubiaceae, commonly grows wild or horticulturally and is under the terms and conditions of the native to many Asian countries such as Vietnam, southern China, Korea, Japan, Myanmar, Creative Commons Attribution (CC BY) India, and Bangladesh. Therefore, it is more attractive economically for the extraction of license (https://creativecommons.org/ crocin. Crocins from gardenia fruits have long been used as natural yellow dyes in food licenses/by/4.0/). coloring, especially in Japan and China, receiving government approval in both countries

Separations 2021, 8, 1. https://dx.doi.org/10.3390/separations8010001 https://www.mdpi.com/journal/separations Separations 2021, 8, 1 2 of 14

for use as a food additive [6,7]. Due to their powerful antioxidant ability and capacity to absorb free radicals [8], crocins are also widely used in the treatment of inflammatory [9,10] and cardiovascular [11] diseases, suppression of growth of cancer cells [12], diabetes treatment [13], and in the prevention of Alzheimer’s disease [14]. However, with so much potential, very little research has been conducted to find an effective extraction method. The common methods of extracting crocin from gardenia fruits are heat extraction [15] and solid–liquid extraction (SLE) [8]. In the heat extraction method, diffusivity increases with increasing temperature, giving faster mass transfer; however, decomposition at higher extraction temperatures limits its application for thermally unstable compounds. On the other hand, SLE can be performed at room temperature using organic solvents such as methanol, diethyl ether, or acetone; however, the diffusion rate is slow, and it takes a long time for completion. Solutions have been reported to speed up the diffusion rate by using a homogenator [15] or ultrasound-assisted extraction [16]; however, the amount of organic solvent used in these extraction processes is quite significant, and proper safety protocols should be followed because of their negative effects on the quality of the extracted compounds as well as on human health and the environment. Therefore, it is necessary to develop an environmentally friendly extraction technology using generally recognized as safe (GRAS) substances. Carbon dioxide (CO2), an alternative to organic solvents, is unique because its sol- vency power can be changed by changing its physical condition to liquid or supercritical simply by adjusting the temperature and pressure during extraction. Supercritical carbon dioxide (ScCO2) typically replaces nonpolar solvents such as hexane or toluene in terms of solubility, but has a gas-like viscosity, liquid-like density, and around a hundred times faster diffusivity than organic solvents, reducing the amount of solvent used. However, it operates at a much higher temperature and is not suitable for thermally unstable com- ◦ pounds such as crocins (which degrade at 60 C and above) [17,18]. Instead, liquid CO2 can be an interesting substitute for ScCO2 and operates similarly to ScCO2 extraction, except its diffusivity is greater [19] and, operating at lower temperature, it can maintain most thermally unstable compounds in plant materials. Furthermore, the affinity of non- polar liquid CO2 can be diverted to polar components such as crocins by adding a small amount of entrainer (co-solvent or modifier), such as water, ethanol, or aqueous ethanol. In this case, instead of adding an entrainer to the CO2 upstream (as co-solvent), compressed liquid CO2 is added to the entrainer (as modifier), which is well known as CO2-expanded liquid (CXL), used for various applications, including extraction, reaction, and separation [19–23]. Unlike ScCO2, a gas phase may exist in the CXL system in addition to the CO2-expanded liquid phase; therefore, density change with pressure is negligible. As a result, excess solvent power sensitivity with the change of operation pressure can be avoided. Additionally, the application of direct sonication to the high-pressure CO2 system has been well documented for the extraction [24] or production of high-yielding liposomes [25]. Direct sonication of the water/CO2 two-phase system caused rapid physical mixing between the water and CO2 phases, including micro-phase separation and cavitation, which may speed up the mass transfer at lower frequencies, typically 20 kHz [24,25]. Therefore, the objective of this work was to develop a green extraction method based on the use of CO2-expanded liquids (CXLs) with or without direct sonication to enhance the concentrations of crocin-1 and crocin-2 from gardenia fruit pulp powder. To do so, the effects of different solvents (ethanol and water, and methanol as a control), the modifier concentration (aqueous ethanol 30, 50, or 80% v/v), and the extraction time (10–120 min) on extraction were examined. Finally, parameters concerning CXL extraction included modifiers (ethanol, water, and aqueous ethanol 50 or 80% v/v), temperature (5–25 ◦C), and pressure (8–14 MPa) with or without direct sonication and the effect of sonication time (25–200 s) were evaluated. The efficacy of direct sonication was evaluated using scanning electron microscopy (SEM). The influence of each factor was analyzed by a single factor experiment. Separations 2021, 8, 1 3 of 14

2. Materials and Methods 2.1. Materials and Chemicals Dried gardenia fruits (native to China) were purchased from S & B Foods Co., Ltd., Tokyo, Japan. The skins were peeled off and the fruit pulp was powdered (average particle size, 75.4 µm) with a 6750 Freezer/Mill (SPEX CertiPrep Co. Ltd., Metuchen, NJ, USA). Crocin-1 and crocin-2 (both >98%; ChemFaces Biochemical Co., Ltd., Wuhan, China) were used as references. HPLC-grade methanol (>99.7 wt%) and analytical-grade ethanol (>99.5 wt%) were purchased from Wako Pure Chemical Industries Ltd., Osaka, Japan, and used as received. High-purity CO2 (>99%) was supplied by Fukuoka Sanso Co. Ltd., Fukuoka, Japan.

2.2. Conventional Solid–Liquid Extraction (SLE) To compare the effects of different solvents on the extraction concentration, conventional SLE was performed using the solvents methanol, ethanol (99.5%), and water as described elsewhere [26]. In brief, gardenia fruit pulp milled powder (10 mg) and solvent (10 mL) were loaded into a screw bottle (30 mL) and sealed. The extraction was carried out by stirring with a magnetic stirrer for 1 h at 25 ◦C and atmospheric pressure. The extract was filtered using a polytetrafluoroethylene (PTFE) membrane filter (0.45 µm) and analyzed by HPLC-UV for quantitative determination of crocin-1 and crocin-2. The effects of different concentrations of ethanol at 0−100% (v/v) were also evaluated with or without sonication to extract crocins from gardenia fruit pulp powder using the conventional SLE method at 25 ◦C and atmospheric pressure. In the extraction experiments with sonication, irradiation was performed for 125 s at a constant amplitude of 20% using a titanium ultrasound horn driven by electrical signals from a 20 kHz ultrasound processor (VC-505, Sonic and Materials, Inc., Suffolk, UK) with a maximum power capability of 500 W. Sampling was performed after 1 h and analyzed by HPLC-UV. To characterize the solvent extraction behavior at different time points of extraction of crocin-1 and crocin-2 from the fruit pulp of gardenia, a time-dependent extraction assay was performed using 80% aqueous ethanol solution (80:20 v/v ethanol:water) as the solvent at 25 ◦C and atmospheric pressure. Sampling was performed at 10, 20, 30, 60, and 120 min after starting the extraction and analyzed by HPLC-UV.

2.3. CO2-Expanded Liquids (CXLs) Extraction with or without Direct Sonication

Liquid CO2 extraction apparatus (Super-200, JASCO Co., Ltd., Tokyo, Japan) described previously was used for the CXL extraction system [24,25]. The equipment setup for system is shown in Figure1. Gardenia fruit pulp milled powder (10 mg) and 10 mL of the modiﬁer, ethanol or water, or 50 to 80% of aqueous ethanol solution were placed in the high-pressure extractor (150 cc, 34 mm id 165 mm long; Toyo Koatsu Co., Ltd., Hiroshima, Japan) equipped with a titanium ultrasound horn driven by electrical signals from a 20 kHz ultrasound processor (VC-505, Sonic and Materials, Inc., Suffolk, UK) with a maximum power capability of 500 W. The loaded extractor was then immersed in a water bath to maintain the desired extraction temperature (5, 20, and 25 ◦C) controlled within ±0.1 ◦C. The temperature of the extraction cell was measured from a thermocouple located inside the cell. Liquid CO2 was pumped into the extraction cell using an HPLC pump equipped with a cooler (SCF-GET, JASCO, Tokyo, Japan) to reach the desired extraction pressure (8, 10, and 14 MPa) at a constant ﬂow rate of 10 mL/min. Under these conditions, the CO2-expanded liquid phase coexists with the gas phase in the high-pressure cell. A cooling unit was used to maintain the CO2 in its liquid state. A backpressure regulator (880-81, JASCO, Tokyo, Japan) was installed at the outlet of the extraction cell to control the desired pressure within ±0.3 MPa. In the extraction experiment with direct sonication, ultrasound irradiation was applied directly to the pressurized extractor for 25 to 200 s at a constant amplitude of 20% (max. 40%) after reaching the desired extraction pressure. Separations 2021, 8, x FOR PEER REVIEW 4 of 15

Separations 2021, 8, 1 the desired pressure within ± 0.3 MPa. In the extraction experiment with direct sonication,4 of 14 ultrasound irradiation was applied directly to the pressurized extractor for 25 to 200 s at a constant amplitude of 20% (max. 40%) after reaching the desired extraction pressure.

FigureFigure 1. 1.Schematic Schematic diagram diagram of of CO CO2-expanded2-expanded liquid liquid (CXL) (CXL) extraction extraction apparatus. apparatus. (1) (1) CO CO22cylinder; cylinder; (2)(2) cooling cooling unit;unit; (3) filter; ﬁlter; (4) (4) pump; pump; (5) (5) pressure pressure ga gauge;uge; (6) (6)safety safety valve; valve; (7) high-pressure (7) high-pressure cell; cell;(8) (8)quartz quartz glass glass window; window; (9) (9) thermometer; thermometer; (10) ultrasonicultrasonic processor; processor; (11) (11) ultrasonic ultrasonic horn; horn; (12) (12) water water bath; (13)bath; pressure (13) pressure gauge; gauge; (14) safety (14) safety valve; valve; (15) cold (15) traps; cold traps; (16) gas (16) ﬂow gas meter;flow meter; (V-1, V-5)(V-1, back V-5) pressureback regulators;pressure regulators; and (V-2, V-6)and stop(V-2, valves. V-6) stop valves.

UltrasonicUltrasonic irradiation irradiation during during the the extraction extraction experiment experiment was was performed performed at at a a constant constant amplitudeamplitude of of 12.2 12.2µ mμm (amplitude (amplitude control control was was set toset 20%). to 20%). Constant Constant amplitude amplitude was suppliedwas sup- byplied the by ultrasonic the ultrasonic processor processor by automatically by automatically adjusting adjusting the power the power supply. supply. The ultrasonic The ultra- processorsonic processor was capable was capable of conducting of conducting safe treatmentsafe treatment of temperature-sensitive of temperature-sensitive samples sam- atples high at intensityhigh intensity and provided and provided mixing mixing by repeatedly by repeatedly allowing allowing the sample the sample to settle to backsettle underback under the probe the probe after each after burst each usingburst anusing independent an independent on/off on/off pulsar pulsar (ON: 5(ON: s, OFF: 5 s, 10 OFF: s). After10 s). the After extraction the extraction process process was completed, was completed, the CO2 thewas CO vented2 was to vented the atmosphere to the atmosphere through athrough gas flow a metergas flow (Alexander meter (Alexander Wright and Wright Co. Ltd.,and Co. Thornaby, Ltd., Thornaby, UK), which UK), was which used was to verifyused to the verify amount the ofamount CO2 used of CO in2the used experiment. in the experiment. The contents The contents of the high-pressure of the high-pres- cell andsure trap cell (liquidand trap and (liquid solid) and were solid) collected were collected and filtered and filtered through through a PTFE a membrane PTFE membrane filter (0.45filterµ m)(0.45 to μ obtainm) to aobtain liquid extract.a liquid Theextract. resulting The liquidresulting extract liquid was extract analyzed was by analyzed HPLC-UV by forHPLC-UV quantitative for quantitative determination determination of crocin-1 of and crocin-1 crocin-2. and Thecrocin-2. structure The structure and morphology and mor- ofphology the gardenia of the fruitgardenia pulp fruit milled pulp powder milled and po thewder residues and the after residues extraction after wereextraction analyzed were usinganalyzed a scanning using a electron scanning microscope electron micros (SEM;cope JEOL (SEM; JSM6060, JEOL Tokyo, JSM6060, Japan). Tokyo, Japan).

2.4.2.4. ConventionalConventional Ultrasound-Assisted Ultrasound-Assisted Extraction Extraction (UAE) (UAE) ConventionalConventional ultrasound-assistedultrasound-assisted extractionextraction (UAE)(UAE) waswas performedperformed asas described described by by Wang et al. [16]. In brief, 10 mg of milled gardenia fruit pulp powder was mixed in 10 mL Wang et al. [16]. In brief, 10 mg of milled gardenia fruit pulp powder was mixed in 10 mL of solvent (distilled water or ethanol or methanol) and loaded into a screw bottle (30 mL). of solvent (distilled water or ethanol or methanol) and loaded into a screw bottle (30 mL). The experimental setup was placed in an ultrasonic washer (SU-3T, Sibata Scientific Tech- The experimental setup was placed in an ultrasonic washer (SU-3T, Sibata Scientific Tech- nology Ltd., Tokyo, Japan). Ultrasonication was conducted at 34 kHz and 40 W for 30 min. nology Ltd., Tokyo, Japan). Ultrasonication was conducted at 34 kHz and 40 W for 30 min. The temperature of water inside the ultrasonic washer was controlled to a target value The temperature of water inside the ultrasonic washer was controlled to a target value (40 (40 ◦C) by using a water circulation system (CCN-1000, GL Science Inc., Tokyo, Japan). °C) by using a water circulation system (CCN-1000, GL Science Inc., Tokyo, Japan). The The resulting liquid extract was filtered through a PTFE membrane filter (0.45 µm) and resulting liquid extract was filtered through a PTFE membrane filter (0.45 μm) and ana- analyzed by HPLC-UV for quantitative determination of crocin-1 and crocin-2. lyzed by HPLC-UV for quantitative determination of crocin-1 and crocin-2. 2.5. Analytical Procedures 2.5.1.2.5. Analytical High-Performance Procedures Liquid Chromatography (HPLC) Analysis 2.5.1.Qualitative High-Performance and quantitative Liquid analysesChromatography of the liquid (HPLC) extract Analysis were conducted by HPLC-UV using a method modified from [27]. HPLC-UV analysis was performed under the following conditions: TSK-GEL (Tosoh Co., Tokyo, Japan) ODS-80Ts column (250 mm × 4.6 mm), column oven set at constant 40 ◦C, UV detector (875-UV, JASCO, Tokyo, Japan) wavelength set at 438 nm. Methanol and 0.1% acetic acid were used as phases. Table1 shows the gradient profile of methanol. The total mobile phase flow rate was set at 1.0 mL/min.

Separations 2021, 8, 1 5 of 14

Table 1. Time course of methanol gradient for HPLC-UV analysis.

Time (min) Methanol (vol%) 0 5 25 40 40 60 50 90 60 90 65 5 75 5

2.5.2. Analytical Method Validation The method was validated according to the International Conference on Harmonisa- tion (ICH) guidelines in terms of linearity, limit of detection (LOD), limit of quantiﬁcation (LOQ), and precision. The LOD and LOQ for the tested compounds were determined by the signal-to-noise (S/N) ratio. The intra-day and inter-day precision were evaluated by repeated injection. The intra-day experiment was obtained by 3 replicates for a day, and the inter-day was determined by 3 injections for 3 days for the peak area. Precision was expressed as relative standard deviation (RSD, %) according to Equation (1). The results obtained by testing various parameters during the validation of the analytical method are given in Table2.

% RSD = Standard deviation of peak area/Average of peak area × 100 (1)

Table 2. Validation parameters. RSD, relative standard deviation.

Parameter Crocin-1 Crocin-2 Concentration range of crocin-1 standards (µg/mL) 2.25–36.0 0.125–2.0 Correlation coefﬁcient of linearity equation 0.9995 0.9997 Linearity Intercept of linearity equation 17439 −8999.8 Slope of linearity equation 2.0 × 108 4.0 × 108 Limit of detection (µg/mL) 0.34 0.01 Limit of quantiﬁcation (µg/mL) 1.13 0.04 Intra-day (RSD, % of peak area, n = 3) Day 1 2.74 1.84 Precision Day 2 2.75 0.53 Day 3 1.09 1.52 Inter-day (RSD, % of peak area n = 3) 3.47 1.05

2.5.3. Standard Solutions and Calibration Curve The standard stock solutions of crocin-1 and crocin-2 were prepared with 80% ethanol at a ﬁnal concentration of 300 µg/mL. Standard solutions of crocin-1 at concentrations of 2.25, 4.5, 9.0, 18.0, and 36.0 µg/mL and crocin-2 at concentrations of 0.125, 0.25, 0.5, 1.0, and 2.0 µg/mL were prepared by diluting the stock solutions with 80% ethanol. The calibration curve was prepared over a concentration range of 2.25–36.0 µg/mL for crocin-1 and 0.125–2.0 µg/mL for crocin-2 with 5 concentration levels. Linearity for crocins was plotted using linear regression of the peak area versus concentration. The coefﬁcient of correlation (R2) was used to judge linearity. The extraction concentrations of crocin-1 and crocin-2 were calculated according to Equation (2):

Extraction concentration = y/m − c (2) Separations 2021, 8, x FOR PEER REVIEW 6 of 15

and 0.125–2.0 μg/mL for crocin-2 with 5 concentration levels. Linearity for crocins was plotted using linear regression of the peak area versus concentration. The coefficient of correlation (R2) was used to judge linearity. The extraction concentrations of crocin-1 and crocin-2 were calculated according to Equation (2): Extraction concentration = y/m − c (2)

Separations 2021, 8, 1 where y is the peak area of crocin-1 and crocin-2 determined by HPLC-UV, 6m of is 14 the slope of the calibration curve, and c is the intercept of the regression line with the y-axis.

2.6. Statisticalwhere y is Analysis the peak area of crocin-1 and crocin-2 determined by HPLC-UV, m is the slope Allof the analyses calibration were curve, performed and c is the in intercept triplicates. of the The regression data were line with analyzed the y-axis. by one-way analysis of2.6. variance Statistical (ANOVA) Analysis accompanied by Tukey’s post-hoc test. The level of significance was set atAll p < analyses 0.05. were performed in triplicates. The data were analyzed by one-way analysis of variance (ANOVA) accompanied by Tukey’s post-hoc test. The level of signiﬁcance 3. Resultswas set and at p Discussion< 0.05.

3.1. HPLC-UV3. Results andProfile Discussion Chromatograms3.1. HPLC-UV Proﬁle of the extracts of gardenia fruit pulp and crocin-1 and crocin-2 standardsChromatograms were determined of the extractsusing ofHPLC-UV gardenia fruit analysis. pulp and The crocin-1 calibration and crocin-2 curve stan- was con- structeddards with were crocin determined concentration using HPLC-UV versus analysis. peak area The calibration for crocin-1 curve (y was = 2E constructed + 08x, R2 = 0.9995, with crocin concentration versus peak area for crocin-1 (y = 2E + 08x, R2 = 0.9995, linear 2 linearrange: range: 2.25–36 2.25–36µg/mL) μg/mL) and and crocin-2 crocin-2 (y = 4E (y += 08x,4E + R 208x,= 0.9997, R = 0.9997, linear range: linear 0.125–2 range: 0.125–2 μg/mL).µg/mL). Peaks Peaks were were identified identiﬁed by by comparing comparing their HPLC-UV HPLC-UV retention retention time time with with the the tar- getedtargeted compounds, compounds, as shown as shown in Figure in Figure 2.2.

Figure 2. HPLC-UVFigure 2. HPLC-UV chromatograms chromatograms of (a of) extracted (a) extracted substances substances fromfrom gardenia gardenia fruit fruit pulp pulp using using liquid COliquid2, 80% CO ethanol,2, 80% ethanol, and ultrasoundand ultrasound at 25 °C atand 25 ◦ 10C andMPa; 10 MPa;(b) standard (b) standard sample sample of of crocin-1; crocin-1; (c ()c standard) standard sample sample of crocin-2. of crocin-2.

A typical HPLC-UV chromatogram of CO2-expanded ethanol (CXE80%) with direct Asonication typical extractionHPLC-UV at 25 chromatogram◦C and 10 MPa from of CO gardenia2-expanded fruit pulp ethanol was recorded (CXE80%) at 438 nm with direct sonication(Figure extraction2a). Standard at crocin-125 °C and and 10 crocin-2 MPa chromayogramsfrom gardenia (Figure fruit 2pulpb,c, respectively) was recorded at 438 nm (Figureconﬁrmed 2a). the Standard peaks appearing crocin-1 at and 40.23 crocin and 43.20-2 chromayograms min, respectively (Figure (Figure2a). 2b,c, respectively) confirmed the peaks appearing at 40.23 and 43.20 min, respectively (Figure 2a). 3.2. Conventional Solid–Liquid Extraction (SLE)

For the selection of modiﬁers for the CO2-expanded liquid (CXL) extraction system 3.2. Conventionalfollowing SLE, Solid–Liquid such as the effectsExtraction of different (SLE) solvents (water, ethanol, and methanol),

Separations 2021, 8, x FOR PEER REVIEW 7 of 15

For the selection of modifiers for the CO2-expanded liquid (CXL) extraction system following SLE, such as the effects of different solvents (water, ethanol, and methanol), Separations 2021, 8, 1 ethanol concentration (aqueous ethanol 30, 50, or 80% v/v), and time (10–1207 of min) 14 on extraction, concentrations were pre-examined.

3.2.1. ethanolEffect of concentration Solvents on (aqueous Extraction ethanol Concentration 30, 50, or 80% v/v), and time (10–120 min) on extraction, concentrations were pre-examined. In this study, conventional solid–liquid extraction (SLE) was carried out using three types 3.2.1.of solvents Effect of (water, Solvents onethanol, Extraction and Concentration methanol as control) to extract the highest concentrations ofIn crocins this study, from conventional gardenia solid–liquid fruit pulp extraction powder. (SLE) was As carried shown out in using Figure three types3a, methanol of solvents (water, ethanol, and methanol as control) to extract the highest concentrations extraction was found to be the highest for crocin-1 and crocin-2 (10.43 ± 0.3 and 0.37 ± 0.02 of crocins from gardenia fruit pulp powder. As shown in Figure3a, methanol extraction μg/mL,was respectively) found to be the followed highest for by crocin-1 water and ex crocin-2traction (10.43 (6.93± ±0.3 0.3 and and 0.37 0.22± 0.02 ± 0.002µg/mL, μg/mL, respectively),respectively) whereas followed extraction by water with extraction pure ethanol (6.93 ±showed0.3 and the 0.22 lowest± 0.002 values.µg/mL, This may be becauserespectively), methanol whereas has a extractionhigher dielectric with pure ethanolconstant showed than the ethanol, lowest values. which This enabled may be us to extract morebecause polar methanol compounds. has a higher However, dielectric constant because than ethanol ethanol, is which safer enabled for human us to extract consumption, more polar compounds. However, because ethanol is safer for human consumption, it was it waschosen chosen for for subsequent subsequent experiments. experiments.

Figure 3. ExtractionFigure 3. Extraction concentrations concentrations of crocin-1 of crocin-1 and andcrocin-2 crocin-2 from from gardenia gardenia fruit pulppulp using using conventional conventional solid–liquid solid–liquid extraction (SLE):extraction (a) effect (SLE): of (a) different effect of different solvents solvents on extraction on extraction concentration; concentration; ( b()b effect) effect of ethanol of ethanol concentration concentration on extraction on extraction ◦ concentrationconcentration at 25 °C under at 25 C atmospheric under atmospheric pressure pressure with with (■) (or) without or without (■ () direct) direct sonication sonication (125(125 s, s, 20% 20% amplitude). amplitude). Error ± bars representError means bars represent ± SE (n means = 3). LettersSE (n = 3).on Letters top of on the top co of thelumns columns represent represent significant signiﬁcant differencesdifferences among among methods methods at at the the 0.05 level. (c) Effect of extraction time on extraction concentration of crocin-1 and crocin-2 in conventional SLE system 0.05 level. (c) Effect of extraction time on extraction concentration of crocin-1 and crocin-2 in conventional SLE system using 80% ethanol at 25 ◦C under atmospheric pressure. using 80% ethanol at 25 °C under atmospheric pressure.

3.2.2. 3.2.2.Effect Effect of Ethanol of Ethanol Conc Concentrationentration on on the the Extraction Extraction The composition of the mixture of ethanol/water and the solid–liquid ratio of plant Thematerial composition are important, of becausethe mixture the concentration of ethanol/ in thewater solvent and increases the solid–liquid until saturation ratio in of plant material are important, because the concentration in the solvent increases until saturation in the conventional SLE system. The results of the effect of different concentrations of ethanol at 0−100% (v/v) with or without sonication are presented in Figure 3b. The concentrations of crocin-1 and crocin-2 were dramatically increased by the addition of water to ethanol (30, 50, or 80% aqueous ethanol) (Figure 3b). However, the highest concentrations

Separations 2021, 8, 1 8 of 14

Separations 2021, 8, x FOR PEER REVIEW 8 of 15

the conventional SLE system. The results of the effect of different concentrations of ethanol at 0−100% (v/v) with or without sonication are presented in Figure3b. The concentrations wereof crocin-1 achieved and with crocin-2 50 and were 80% dramatically aqueous etha increasednol for bycrocin-1 the addition (9.76 ± of0.5 water and to9.49 ethanol ± 0.2 μ(30,g/mL, 50, respectively) or 80% aqueous and ethanol)crocin-2 (Figure(0.40 ± 0.023b). and However, 0.31 ± 0.01 the highestμg/mL, concentrationsrespectively). On were the otherachieved hand, with no significant 50 and 80% changes aqueous in ethanolconcentrations for crocin-1 were (9.76 found± with0.5 and sonication. 9.49 ± 0.2 Thisµg/mL, may berespectively) because of andthe short crocin-2 time (0.40 application± 0.02 and (125 0.31 s) ±of0.01 sonication,µg/mL, which respectively). is insufficient On the to other in- ducehand, a nosignificant significant response. changes Crocins in concentrations are generally were large found molecules with sonication. (molecular This weight may of be crocin-1because and of the crocin-2 shorttime is 977 application and 814.42 (125 g/mol, s) of respectively) sonication, which that combine is insufficient two polar to induce heads a ofsignificant gentiobiosyl response. esters with Crocins a non-polar are generally C20 carbon large molecules chain. Due (molecular to their amphiphilic weight of crocin-structure,1 and the crocin-2 addition is 977of water and 814.42to ethanol g/mol, increa respectively)ses the dielectric that combine constant two and polar the solubility heads of andgentiobiosyl enables greater esters withconcentrations a non-polar for C20 both carbon crocin-1 chain. and Due crocin-2. to their Aqueous amphiphilic ethanol structure, is also knownthe addition as a better of water solvent to ethanol than pure increases ethanol the for dielectric the extraction constant of green and the pigment solubility [23,28]. and Therefore,enables greater in the concentrationssubsequent expe forriment, both crocin-180% aqueous and crocin-2. ethanol (80:20 Aqueous v/v ethanol:water) ethanol is also wasknown chosen as a to better see the solvent effects than of extraction pure ethanol time for on theconcentration. extraction of green pigment [23,28]. Therefore, in the subsequent experiment, 80% aqueous ethanol (80:20 v/v ethanol:water) 3.2.3.was chosenEffect of to Extraction see the effects Time of on extraction the Concentration time on concentration. 3.2.3.To Effect obtain of Extractionmaximum Timeconcentrations on the Concentration of crocin-1 and crocin-2, a time-dependent ex- tractionTo experiment obtain maximum was performed concentrations using of a crocin-1conventional and crocin-2, SLE system a time-dependent with aqueous extrac- etha- noltion (80% experiment v/v) of gardenia was performed fruits at using 25 °C aunder conventional atmospheric SLE systempressure, with as shown aqueous in ethanolFigure 3c.(80% Thev/ massv) of of gardenia extracts fruits of crocin-1 at 25 ◦C and under crocin-2 atmospheric generally pressure, increased as over shown 0–60 in Figuremin and3c. wereThe massthen saturated of extracts to of concentrations crocin-1 and crocin-2 of approximately generally increased9.84 and 0.35 over μg/mL, 0–60 min respectively. and were Therefore,then saturated the appropriate to concentrations extraction of time approximately was set at 60 9.84 min andfor the 0.35 followingµg/mL, experiments. respectively. Therefore, the appropriate extraction time was set at 60 min for the following experiments. 3.3. CO2-Expanded Liquid (CXL) Extraction System 3.3.1.3.3. CO Effect2-Expanded of Modifiers Liquid on (CXL) Extraction Extraction Conc Systementration with or without Direct Sonication 3.3.1.The Effect effects of Modifiers of different on modifiers—pure Extraction Concentration ethanol (CXE) with oror withoutwater (CXW), Direct or Sonication 50 or 80% of aqueousThe effects ethanol of solution different (CXE50% modifiers—pure and CXE80%, ethanol respectively)—on (CXE) or water the (CXW), extraction or 50con- or centration80% of aqueous of crocin-1 ethanol and solution crocin-2 (CXE50% in the CXL and extraction CXE80%, system respectively)—on was tested theat 25 extraction °C and ◦ 10concentration MPa and compared of crocin-1 with and the crocin-2 conventional in the SLE CXL using extraction methanol, system as wasshown tested Figure at 25 4. AC comparisonand 10 MPa between and compared extraction with conditions the conventional and concentrations SLE using methanol, using different as shown methods Figure is4. summarizedA comparison inbetween Table 3. extractionCrocins are conditions polar compounds, and concentrations therefore liquid using differentCO2 sole methodsextraction is wassummarized not executed. in Table The3. Crocinsresults show are polar that compounds, the extraction therefore concentrations liquid CO were2 sole greatly extraction af- fectedwas not by executed. changing The the resultsmodifiers. show that the extraction concentrations were greatly affected by changing the modifiers.

Figure 4. Effect of different modiﬁers on extraction concentration of crocin-1 and crocin-2 in CO2-expanded liquid (CXL) ◦ Figureextraction 4. Effect system of withdifferent () or modifiers without (on) extraction direct sonication concentration (125 s, 20% of crocin-1 amplitude) and at crocin-2 25 C and in CO 10 MPa.2-expanded Error barsliquid represent (CXL) ■ extractionmeans ± SE system (n = 3).with Letters ( ) or on without top of ( the□) direct columns sonication represent (125 signiﬁcant s, 20% amplitude) differences at 25 among °C and methods 10 MPa. at Error the 0.05 bars level. represent means ± SE (n = 3). Letters on top of the columns represent significant differences among methods at the 0.05 level.

Separations 2021, 8, 1 9 of 14

Table 3. Extraction conditions and concentrations using different methods.

Extraction Conditions Concentration Extraction Methods T P t US ampl. Crocin-1 Crocin-2 Solvent Modiﬁer (◦C) (MPa) (min) (%) (µg/mL) (µg/mL) Methanol - 25 0.1 60 - b 10.43 ± 0.31 c 0.37 ± 0.02 Solid–liquid Ethanol - 25 0.1 60 - a 0.76 ± 0.17 a 0.08 ± 0.01 Water - 25 0.1 60 - b 6.93 ± 0.21 b 0.22 ± 0.002 Methanol - 40 0.1 40 - b 12.08 ± 0.35 a 0.46 ± 0.02 Con. UAE Ethanol - 40 0.1 40 - a 3.36 ± 0.31 c 0.17 ± 0.01 Water - 40 0.1 40 - b 10.22 ± 0.35 c 0.33 ± 0.01 a a Liquid CO2 Ethanol 25 10 60 - 1.64 ± 0.23 0.11 ± 0.03 b b,c Liquid CO2 Water 25 10 60 - 7.84 ± 0.32 0.25 ± 0.02 CXLs b,c c,d Liquid CO2 50% Ethanol 25 10 60 - 11.57 ± 0.12 0.42 ± 0.03 b,c c,d Liquid CO2 80% Ethanol 25 10 60 - 10.77 ± 0.29 0.41 ± 0.01 a a Liquid CO2 Ethanol 25 10 60 20% 2.63 ± 0.13 0.13 ± 0.01 b b Liquid CO2 Water 25 10 60 20% 8.25 ± 0.34 0.26 ± 0.02 CXLs + DS b,c,d d Liquid CO2 50% Ethanol 25 10 60 20% 12.91 ± 0.75 0.52 ± 0.03 d d,e Liquid CO2 80% Ethanol 25 10 60 20% 13.63 ± 0.52 0.51 ± 0.05 Mean values in the same column with different letters are signiﬁcantly different at p < 0.05. Con. UAE, conventional ultrasonic-assisted extraction; CXLs, carbon dioxide expanded liquids; DS, direct sonication; US ampl., ultrasonication amplitude.

The effect of the modifier on the extraction concentration followed the order: CXE80% > CXE50% > CXW > pure CXE (Figure4). The highest concentration was obtained using CO2-expanded aqueous ethanol solution CXE50% (11.57 ± 0.12 and 0.42 ± 0.03 µg/mL for crocin-1 and crocin-2, respectively) or CXE80% (10.77 ± 0.29 and 0.41 ± 0.01µg/mL for crocin-1 and crocin-2, respectively), whereas CO2-expanded pure ethanol (CXE) was the lowest (1.64 ± 0.23 and 0.11 ± 0.03 µg/mL for crocin-1 and crocin-2, respectively; p > 0.01). This result is in agreement with the extraction of chlorophyll, where mixtures of CO2 and aqueous ethanol were much more effective than mixtures containing only pure ethanol [28]. In the case of the conventional UAE system according to Wang et al. [16], using ethanol or methanol as solvent, no significant enhancement was observed. However, the concentrations of crocin-1 and crocin-2 were significantly increased with the addition of direct sonication (DS) in CXE50% + DS (12.91 ± 0.75 and 0.52 ± 0.03 µg/mL for crocin-1 and crocin-2, respectively; p < 0.05) or CXE 80% + DS (13.63 ± 0.52 and 0.51 ± 0.05 µg/mL for crocin-1 and crocin-2, respectively; p < 0.05) extraction system (Figure4, Table3).

3.3.2. Effects of Temperature and Pressure on Extraction Concentration in CXE80% System with or without Direct Sonication The effects of temperature on the concentration of crocin-1 and crocin-2 extracted at various temperatures (5, 20, and 25 ◦C) at constant pressure (10 MPa) in the CXE80% system with and without direct sonication are shown in Figure5a. As shown in Figure 5a, the extraction concentration of both crocin-1 and crocin-2 increased from 10.18 ± 0.1 and 0.41 ± 0.01 µg/mL to 10.78 ± 0.29 and 0.43 ± 0.01 µg/mL, respectively, by increasing temperature from 5 to 25 ◦C in the CXE80% extraction system without direct sonication. This may be attributed to the reduced viscosity of liquid CO2, and thereby increased solubility of the target components. It is well known that increasing temperature decreases the surface tension between the solvent, solute, and matrix, reduces viscosity, and facilitates better solvent penetration into the matrix, which together improves the extraction concentration [22]. Separations 2021, 8, x FOR PEER REVIEW 10 of 15

Mean values in the same column with different letters are significantly different at p < 0.05. Con. UAE, conventional ultrasonic-assisted extraction; CXLs, carbon dioxide expanded liquids; DS, direct sonication; US ampl., ultrasonication amplitude.

3.3.2. Effects of Temperature and Pressure on Extraction Concentration in CXE80% Sys- tem with or without Direct Sonication The effects of temperature on the concentration of crocin-1 and crocin-2 extracted at various temperatures (5, 20, and 25 °C) at constant pressure (10 MPa) in the CXE80% system with and without direct sonication are shown in Figure 5a. As shown in Figure 5a, the extraction concentration of both crocin-1 and crocin-2 increased from 10.18 ± 0.1 and 0.41 ± 0.01 μg/mL to 10.78 ± 0.29 and 0.43 ± 0.01 μg/mL, respectively, by increasing temperature from 5 to 25 °C in the CXE80% extraction system without direct sonication. This may be attributed to the reduced viscosity of liquid CO2, and thereby increased solubility of the target components. It is well known that increasing temperature decreases the surface tension between the solvent, solute, and matrix, reduces viscosity, and facilitates bet- Separationster2021 solvent, 8, 1 penetration into the matrix, which together improves the extraction concentra-10 of 14 tion [22].

Figure 5. Effects of (a) temperature and (b) pressure on extraction concentration of crocin-1 and crocin-2 from gardenia fruit ◦ pulpFigure in CXE80% 5. Effects extraction of (a system) temperature at 25 C and and 10 ( MPab) pressure obtained withon extraction () and without concentration (N) ultrasound of crocin-1 (amplitude and 20%, 125crocin-2 s). Error bars from represent gardenia means fruit± SE pulp (n = in 3). CXE80% extraction system at 25 °C and 10 MPa obtained with (■) and without (▲) ultrasound (amplitude 20%, 125 s). Error bars represent means ± SE (n = 3). The effect of pressure on the extraction concentration of crocin-1 and crocin-2 extracted at pressure ranging from 8 to 14 MPa at a constant temperature (25 ◦C) in the CXE80% The effect of systempressure with on and the without extraction direct sonication concentration is shown of in crocin-1 Figure5b. Asand shown crocin-2 in Figure ex-5b, tracted at pressureno ranging signiﬁcant from increases 8 to were 14 foundMPa inat the a extractionconstant concentrations temperature of crocin-1(25 °C) and in crocin-2the CXE80% system withwith increasingand without pressure. direct Generally, sonication increasing is shown pressure in Figure increases 5b. the As density shown of ain CXL Figure 5b, no significantextraction increases system, andwere it isfound more likelyin the to extraction be increased concentrations in solubility. However, of crocin-1 as previously observed [24,26], the density of liquid CO2 under these conditions is very similar and crocin-2 with increasing pressure. Generally, increasing◦ pressure increases3 the den- (density of CO2 at 8, 10, and 14 MPa at 25 C is 777, 818, and 867 kg/m , respectively), sity of a CXL extractionsuggesting system, that these and pressure it is more levels likely have littleto be effect increased on liquid in CO solubility.2 solubility. How- ever, as previously observedAs shown [24,26], in Figure the5a,b, density the extraction of liquid concentrations CO2 under ofcrocin-1 these conditions and crocin-2 wereis very similar (densitygreatly of affectedCO2 at by8, the10, additionand 14 ofMPa direct at ultrasound25 °C is 777, in the 818, CXE80% and system867 kg/m regardless3, re- of temperature and pressure. This is because adding direct sonication to the system facilitates spectively), suggesting that these pressure levels have little effect on liquid CO2 solubility. microstreaming and cavitation, breaking the cell membranes and allowing more solvent to penetrate, followed by better contact between the solvent and solute and improved mass transfer, resulting in a high extraction concentration [26,29]. Similar effects of sonication have been previously conﬁrmed [24].

3.3.3. Effect of Sonication Time on Extraction Concentration in CXE80% System The concentrations of crocin-1 and crocin-2 were gradually increased with increasing sonication time in the CXE80% system at 25 ◦C and 10 MPa (Figure6) and saturated at 125 s. As described above, increasing the sonication time of the system may facilitate more microstreaming and cavitation to break more cell membranes, resulting in better contact and interactions of solvents to wash out the plant materials [26,29]. From this result, therefore, it is considered that 125 s of direct sonication in CXL can produce a sufﬁcient Separations 2021, 8, x FOR PEER REVIEW 11 of 15

As shown in Figure 5a,b, the extraction concentrations of crocin-1 and crocin-2 were greatly affected by the addition of direct ultrasound in the CXE80% system regardless of temperature and pressure. This is because adding direct sonication to the system facilitates microstreaming and cavitation, breaking the cell membranes and allowing more solvent to penetrate, followed by better contact between the solvent and solute and improved mass transfer, resulting in a high extraction concentration [26,29]. Similar effects of sonication have been previously confirmed [24].

3.3.3. Effect of Sonication Time on Extraction Concentration in CXE80% System The concentrations of crocin-1 and crocin-2 were gradually increased with increasing sonication time in the CXE80% system at 25 °C and 10 MPa (Figure 6) and saturated at 125 s. As described above, increasing the sonication time of the system may facilitate more Separations 2021, 8, 1 11 of 14 microstreaming and cavitation to break more cell membranes, resulting in better contact and interactions of solvents to wash out the plant materials [26,29]. From this result, therefore, it is considered that 125 s of direct sonication in CXL can produce a sufficient extraction effect, withextraction the advantages effect, with of reduced the advantages extraction of time reduced and increased extraction extract time and concen- increased extract tration. concentration.

Figure 6. EffectFigure of sonication 6. Effect of time sonication on extraction time on concentration extraction conc of crocin-1entration and of crocin-2crocin-1 inand CXE80% crocin-2 extraction in CXE80% system with ◦ direct sonicationextraction (125 s, 20%system amplitude) with direct at 25sonicationC and 10(125 MPa. s, 20% Error amplitude) bars represent at 25 means°C and ±10SE MPa. (n = Error 3). bars represent means ± SE (n = 3). Figure7 shows a comparison of the cell morphology of gardenia pulp powder before Figure 7and shows after a comparison extraction with of the or withoutcell morphology sonication of ingardenia different pulp extraction powder methods. before There were and after extractionno significant with morphologicalor without sonicati changeson in shapedifferent and extraction structure observed methods. in There untreated material were no significant(Figure morphological7a) or material changes treated with in shape aqueous and ethanolstructure 80% observed (Figure in7b). untreated However, wrinkles material (Figureand 7a) swelling or material of cell treated walls werewith aqueous observed ethanol in the samples80% (Figure treated 7b). inHowever, the CXE80% system (Figure7c). Furthermore, significant morphological changes in shape and structure after wrinkles and swelling of cell walls were observed in the samples treated in the CXE80% the addition of direct sonication (125 s) to the CXE80% system caused cell rupture and system (Figure 7c). Furthermore, significant morphological changes in shape and struc- clearance of cell contents, leaving a smooth surface area (Figure7d). These structural ture after the addition of direct sonication (125 s) to the CXE80% system caused cell rup- changes confirm the extraction of cell contents and may facilitate the enhancement of ture and clearance of cell contents, leaving a smooth surface area (Figure 7d). These struc- diffusion, resulting in higher extraction concentrations in CXE80% with a direct sonication Separationstural 2021, changes8, x FOR PEER confirm REVIEW the extraction of cell contents and may facilitate the enhancement12 of 15 extraction system. of diffusion, resulting in higher extraction concentrations in CXE80% with a direct sonication extraction system.

Figure 7. SEM 1000 images of (a) raw gardenia fruit pulp powder; (b) sample after SLE using 80% ethanol; (c) sample after Figure 7. SEMCXE80% 1000 extraction; images of(d) ( asample) raw after gardenia CXE80% fruit extraction pulp with powder; direct (sonication.b) sample after SLE using 80% ethanol; (c) sample after CXE80% extraction; (d) sample after CXE80% extraction with direct sonication. 3.4. Proposed Mechanism of Extraction in CXL Extraction System With or Without Direct Sonication Although the mechanisms of extraction in the CXL extraction system using different modifiers and their effects on extraction efficiency are not completely understood, it is clear that the extraction concentrations in the system were greatly affected by the addition of CO2 to the modifiers, which may be related to different phase behavior and polarity. The visual observation of the phase behavior upon expanding CO2 in different modifiers is shown in Figure 8. As shown in Figure 8a, in the CXE extraction system, it was observed that ethanol expanded well with the addition of CO2. It is known that CO2 dissolves in pure ethanol and decreases the dielectric constant and polarity of ethanol [30], which may explain the cause of poor crocin concentration when using pure ethanol as a modifier (Fig- ure 4). In the CXW system, it was found that the water phase did not expand well with addition of CO2 (Figure 8b). This was expected, because CO2 is poorly soluble in water, and it resulted in a greater concentration of crocins than in CXE. CO2-saturated water may alter the polarity of the interphase region of CO2-water, therefore there was increased solubility of the target compounds and a higher concentration than with pure ethanol. This result also agreed with the study of Nerome et al., who showed that water with ScCO2 is superior to methanol as a modifier for the extraction of crocin [31]. Interestingly, as shown in Figure 8c, the addition of CO2 to the aqueous ethanol CXE80% system split the aqueous ethanol into distinct two liquid phases, which can be defined, according to Jessop et al. [30], as a water-rich and an ethanol-rich phase in addition to the CO2-rich gas phase. This may turn the process into a three-phase rather than a two-phase system and allow more CO2 to diffuse through the matrix and transport more target materials to the aqueous ethanol solution, which may be the reason for the enhanced concentration of crocins (Figures 4 and 7c). However, as shown in Figure 8d,e, adding direct sonication to the CXE80% system, three phases were disrupted with vigorous mixing, and interestingly, only two

Separations 2021, 8, 1 12 of 14

3.4. Proposed Mechanism of Extraction in CXL Extraction System with or without Direct Sonication Although the mechanisms of extraction in the CXL extraction system using different modifiers and their effects on extraction efficiency are not completely understood, it is clear that the extraction concentrations in the system were greatly affected by the addition of CO2 to the modifiers, which may be related to different phase behavior and polarity. The visual observation of the phase behavior upon expanding CO2 in different modifiers is shown in Figure8. As shown in Figure8a, in the CXE extraction system, it was observed that ethanol expanded well with the addition of CO2. It is known that CO2 dissolves in pure ethanol and decreases the dielectric constant and polarity of ethanol [30], which may explain the cause of poor crocin concentration when using pure ethanol as a modifier (Figure4). In the CXW system, it was found that the water phase did not expand well with addition of CO2 (Figure8b). This was expected, because CO 2 is poorly soluble in water, and it resulted in a greater concentration of crocins than in CXE. CO2-saturated water may alter the polarity of the interphase region of CO2-water, therefore there was increased solubility of the target compounds and a higher concentration than with pure ethanol. This result also agreed with the study of Nerome et al., who showed that water with ScCO2 is superior to methanol as a modifier for the extraction of crocin [31]. Interestingly, as shown in Figure8c, the addition of CO 2 to the aqueous ethanol CXE80% system split the aqueous ethanol into distinct two liquid phases, which can be defined, according to Jessop et al. [30], as a water-rich and an ethanol-rich phase in addition to the CO2-rich gas phase. This may turn the process into a three-phase rather than a two-phase system and allow more CO2 to diffuse through the matrix and transport more target materials to the aqueous ethanol solution, which may be the reason for the enhanced concentration of crocins (Figures4 and7c). However, as shown in Figure8d,e, adding direct sonication to the

Separations 2021, 8, x FOR PEER REVIEWCXE80% system, three phases were disrupted with vigorous mixing,13 of 15 and interestingly, only two phases could be observed, a compressed aqueous-ethanol and an aqueous ethanol- modiﬁed CO2-rich phase (Figure8e). We assume that vigorous mixing with direct sonica- phasestion may could cause be observed, micro-phase a compressed separation aqueous-ethanol [25], leading and an aqueous to the dispersionethanol-modi- of ethanol micelles fiedbetween CO2-rich aqueous phase (Figure and 8e). CO We2-rich assume phases, that vigorous with amixing resulting with direct increase sonication in surface area of the may cause micro-phase separation [25], leading to the dispersion of ethanol micelles be- extraction. Ethanol is more soluble in CO2 than in water, therefore we presume that more tween aqueous and CO2-rich phases, with a resulting increase in surface area of the ex- micelles of ethanol dissolved in the CO -rich phase than in the water-rich phase, resulting in traction. Ethanol is more soluble in CO2 than in2 water, therefore we presume that more micellestwo visible of ethanol phases, dissolved as shown in the CO in2-rich Figure phase8e. than Besides, in the water-rich sonication phase, produces resulting cavitation bubbles, inwhich two visible induce phases, shockwaves as shown in toFigure damage 8e. Besides, the cellsonication matrix. produces Therefore, cavitation altogether, bub- direct sonica- bles,tion which may enhanceinduce shockwaves the mass to transfer damage ofthe targetcell matrix. materials, Therefore, providing altogether, the direct highest concentration sonicationwith the may CXE80% enhance system the mass than transfer any of other target methodmaterials, (Figureproviding4 andthe highest Table con-3). centration with the CXE80% system than any other method (Figure 4 and Table 3).

◦ FigureFigure 8. Phase 8. Phase behavior behavior observed observed upon addition upon of CO addition2 (25 °C 10 of MPa) CO to2 different(25 C modifiers: 10 MPa) (a to) different modiﬁers: ethanol(a) ethanol in CXE in extraction CXE extraction system; (b system;) water in (CXWb) water extraction in CXW system; extraction (c) 80% ethanol system; in CXE80% (c) 80% ethanol in CXE80% extraction system; (d) CXE80% extraction system during ultrasonication assistance (UA); (e) CXE80%extraction extraction system; system (d) after CXE80% UA. Schematic extraction diagrams system above during visual ultrasonicationobservations depict assistance them (UA); (e) CXE80% asextraction EtOH-rich systemor water- afterand CO UA.2-rich Schematic conditions.diagrams above visual observations depict them as EtOH-rich

or water- and CO2-rich conditions. 4. Conclusions Natural pigment components (crocin-1 and crocin-2) from gardenia fruit pulp were extracted in a CO2 expanded liquid (CXL) extraction system using modifiers of ethanol, water, aqueous ethanol (CXE50% or 80%, v/v), and direct sonication. The parameters temperature, pressure, and sonication time were optimized using a combination of single- factor experiments. The optimal temperature and pressure were 25 °C and 10 MPa. The extraction concentrations of crocin-1 and crocin-2 were higher using direct sonication (125 s, 20% amplitude) in the liquid CO2-expanded aqueous ethanol (80%, v/v) system than without sonication. In summary, in order for the extraction system to be fast and efficient, the addition of direct sonication to CO2-expanded aqueous ethanol (80%) is the most influential factor in the extraction concentration of polar compounds.

Author Contributions: Conceptualization, T.S., T.M.A. and K.M.; data curation, H.S.; formal analysis, H.S.; funding acquisition, K.M.; investigation, H.S.; methodology, H.S.; project administration, K.M.; resources, H.S., K.O. and S.T.; software, H.S.; supervision, K.M.; validation, H.S., K.O. and S.T.; visualization, H.S.; writing—original draft, H.S.; writing—review and editing, T.S., T.M.A. and K.M. All authors have read and agreed to the published version of the manuscript. Funding: This work was funded by JSPS KAKENHI Grant Number 17K06899. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: No new data were created or analyzed in this study. Data sharing is not applicable to this article.

Separations 2021, 8, 1 13 of 14

4. Conclusions Natural pigment components (crocin-1 and crocin-2) from gardenia fruit pulp were extracted in a CO2 expanded liquid (CXL) extraction system using modifiers of ethanol, water, aqueous ethanol (CXE50% or 80%, v/v), and direct sonication. The parameters temperature, pressure, and sonication time were optimized using a combination of single- factor experiments. The optimal temperature and pressure were 25 ◦C and 10 MPa. The extraction concentrations of crocin-1 and crocin-2 were higher using direct sonication (125 s, 20% amplitude) in the liquid CO2-expanded aqueous ethanol (80%, v/v) system than without sonication. In summary, in order for the extraction system to be fast and efficient, the addition of direct sonication to CO2-expanded aqueous ethanol (80%) is the most influential factor in the extraction concentration of polar compounds.

Author Contributions: Conceptualization, T.S., T.M.A. and K.M.; data curation, H.S.; formal analysis, H.S.; funding acquisition, K.M.; investigation, H.S.; methodology, H.S.; project administration, K.M.; resources, H.S., K.O. and S.T.; software, H.S.; supervision, K.M.; validation, H.S., K.O. and S.T.; visualization, H.S.; writing—original draft, H.S.; writing—review and editing, T.S., T.M.A. and K.M. All authors have read and agreed to the published version of the manuscript. Funding: This work was funded by JSPS KAKENHI Grant Number 17K06899. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: No new data were created or analyzed in this study. Data sharing is not applicable to this article. Conﬂicts of Interest: The authors declare no conﬂict of interest.

References 1. Hong, I.K.; Jeon, H.; Lee, S.B. Extraction of natural dye from Gardenia and chromaticity analysis according to chi parameter. J. Ind. Eng. Chem. 2015, 24, 326–332. [CrossRef] 2. Yen, H.-W.; Yang, S.-C.; Chen, C.-H.; Jesisca; Chang, J.-S. Supercritical fluid extraction of valuable compounds from microalgal biomass. Bioresour. Technol. 2015, 184, 291–296. [CrossRef][PubMed] 3. Sinha, K.; Saha, P.D.; Datta, S. Extraction of natural dye from petals of flame of forest (Butea monosperma) flower: Process optimization using response surface methodology (RSM). Dye. Pigment. 2012, 94, 212–216. [CrossRef] 4. Ali, S.; Hussain, T.; Nawaz, R. Optimization of alkaline extraction of natural dye from Henna leaves and its dyeing on cotton by exhaust method. J. Clean. Prod. 2009, 17, 61–66. [CrossRef] 5. Carmona, M.; Zalacain, A.; Sánchez, A.M.; Novella, J.L.; Alonso, G.L. Crocetin esters, picrocrocin and its related compounds present in Crocus sativus stigmas and Gardenia jasminoides fruits. Tentative identification of seven new compounds by LC-ESI-MS. J. Agric. Food Chem. 2006, 54, 973–979. [CrossRef] 6. Yamada, S.; Oshima, H.; Saito, I.; Hayakawa, J. Adoption of crocetin as an indicator compound for detection of gardenia yellow in food products (Analysis of natural coloring matters in food V). J. Food Hyg. Soc. Jpn. 1996, 37, 372–377. [CrossRef] 7. Yang, W.; Wang, J.; Li, X.; Du, Z. A new method research for determination of natural pigment crocin yellow in foods by solid-phase extraction ultrahigh pressure liquid chromatography. J. Chromatogr. A 2011, 1218, 1423–1428. [CrossRef] 8. Chen, Y.; Zhang, H.; Tian, X.; Zhao, C.; Cai, L.; Liu, Y.; Jia, L.; Yin, H.-X.; Chen, C. Antioxidant potential of crocins and ethanol extracts of Gardenia jasminoides ELLIS and Crocus sativus L.: A relationship investigation between antioxidant activity and crocin contents. Food Chem. 2008, 109, 484–492. [CrossRef] 9. Nam, K.N.; Park, Y.-M.; Jung, H.-J.; Lee, J.Y.; Min, B.D.; Park, S.-U.; Jung, W.-S.; Cho, K.-H.; Park, J.-H.; Kang, I.; et al. Anti-inflammatory effects of crocin and crocetin in rat brain microglial cells. Eur. J. Pharmacol. 2010, 648, 110–116. [CrossRef] 10. Koo, H.-J.; Lim, K.-H.; Jung, H.-J.; Park, E.-H. Anti-inflammatory evaluation of gardenia extract, geniposide and genipin. J. Ethnopharmacol. 2006, 103, 496–500. [CrossRef] 11. He, S.-Y.; Qian, Z.-Y.; Tang, F.-T.; Wen, N.; Xu, G.-L.; Sheng, L. Effect of crocin on experimental atherosclerosis in quails and its mechanisms. Life Sci. 2005, 77, 907–921. [CrossRef][PubMed] 12. Aung, H.H.; Wang, C.Z.; Ni, M.; Fishbein, A.; Mehendale, S.R.; Xie, J.T.; Shoyama, A.Y.; Yuan, C.S. Crocin from crocus sativus possesses significant anti-proliferation effects on human colorectal cancer cells. Exp. Oncol. 2007, 29, 175–180. [PubMed] 13. Yorgun, M.A. Effects of crocin on diabetic maculopathy: A placebo-controlled randomized clinical trial. Am. J. Ophthalmol. 2019, 204, 141–142. [CrossRef][PubMed] 14. Ebrahim-Habibi, M.-B.; Amininasad, M.; Ebrahim-Habibi, A.; Sabbghian, M.; Nemat-Gorgani, M. Fibrillation of α-lactalbumin: Effect of crocin and safranal, two natural small molecules from crocus sativus. Biopolymers 2010, 93, 854–865. [CrossRef][PubMed] Separations 2021, 8, 1 14 of 14

15. Zhu, X.; Mang, Y.; Shen, F.; Xie, J.; Su, W. Homogenate extraction of gardenia yellow pigment from Gardenia Jasminoides ellis fruit using response surface methodology. J. Food Sci. Technol. 2014, 51, 1575–1581. [CrossRef][PubMed] 16. Wang, X.; Dong, S.; Chen, F. Ultrasonic-assisted extraction of gardenia yellow from fructus Gardeniae. Adv. Mat. Res. 2012, 396, 1075–1078. [CrossRef] 17. Budisa, N.; Schulze-Makuch, D. Supercritical carbon dioxide and its potential as a life-sustaining solvent in a planetary environment. Life 2014, 4, 331–340. [CrossRef][PubMed] 18. Karasu, S.; Bayram, Y.; Ozkan, K.; Sagdic, O. Extraction optimization crocin pigments of saffron (crocus sativus) using response surface methodology and determination stability of crocin microcapsules. J. Food Meas. Charact. 2019, 13, 1515–1523. [CrossRef] 19. del Pilar Sánchez-Camargoa, A.; Mendiolab, J.A.; Ibáñez, E. Gas expanded-liquids. In Supercritical and Other High-Pressure Solvent Systems: For Extraction, Reaction and Material Processing; Hunt, A.J., Attard, T.M., Eds.; Royal Society of Chemistry: London, UK, 2018; pp. 512–529. 20. Rodríguez-Pérez, C.; Mendiola, J.A.; Quirantes-Piné, R.; Ibáñez, E.; Segura-Carretero, A. Green downstream processing using supercritical carbon dioxide: CO2-expanded ethanol and pressurized hot water extractions for recovering bioactive compounds from Moringa oleifera leaves. J. Supercrit. Fluids 2016, 116, 90–100. [CrossRef] 21. Paudel, A.; Jessop, M.J.; Stubbins, S.H.; Champagne, P.; Jessop, P.G. Extraction of lipids from microalgae using CO2-expanded methanol and liquid CO2. Bioresour. Technol. 2015, 184, 286–290. [CrossRef] 22. Al-Hamimi, S.; Mayoral, A.A.; Cunico, L.P.; Charlotta Turner, C. Carbon dioxide expanded ethanol extraction: Solubility and extraction kinetics of α-pinene and cis-verbenol. Anal. Chem. 2016, 88, 4336–4345. [CrossRef][PubMed] 23. del-Valle, J.M.; Martín, Á.; Cocero, M.J.; de la Fuente, J.C.; de la Cruz-Quiroz, R. Supercritical CO2 extraction of solids using aqueous ethanol as static modiﬁer is a two-step mass transfer process. J. Supercrit. Fluids 2019, 143, 179–190. [CrossRef] 24. Kawamura, H.; Mishima, K.; Sharmin, T.; Ito, S.; Kawakami, R.; Kato, T.; Misumi, M.; Suetsugu, T.; Orii, H.; Kawano, H.; et al. Ultrasonically enhanced extraction of luteolin and apigenin from the leaves of Perilla frutescens (L.) Britt. using liquid carbon dioxide and ethanol. Ultrason. Sonochem. 2016, 29, 19–26. [CrossRef][PubMed] 25. Tokunaga, S.; Tashiro, H.; Ono, K.; Sharmin, T.; Kato, T.; Irie, K.; Mishima, K.; Satho, T.; Aida, T.M.; Mishima, K. Rapid production of liposomes using high pressure carbon dioxide and direct ultrasonication. J. Supercrit. Fluids 2020, 160, 104782. [CrossRef] 26. Mishima, K.; Kawakami, R.; Yokota, H.; Harada, T.; Kato, T.; Kawamura, H.; Matsuyama, K.; Mustofa, S.; Hasanah, F.; Siregar, Y.D.I.; et al. Extraction of Luteolin and Apigenin from leaves of Perilla frutescens (L.) Britt. with Liquid Carbon Dioxide. Solvent Extr. Res. Dev. Jpn. 2014, 21, 55–63. [CrossRef] 27. Cristea, D.; Bareau, I.; Vilarem, G. Identification and quantitative HPLC analysis of the main flavonoids present in weld (Reseda luteola L.). Dye. Pigm. 2003, 57, 267–272. [CrossRef] 28. Hojnik, M.; Škerget, M.; Knez, Ž. Isolation of chlorophylls from stinging nettle (Urticadioica L.). Sep. Purif. Technol. 2007, 57, 37–46. [CrossRef] 29. Chemat, F.; Zill-e-Huma; KamranKhan, M. Applications of ultrasound in food technology: Processing, preservation and extraction. Ultrason. Sonochem. 2011, 18, 813–835. [CrossRef] 30. Jessop, P.G.; Subramaniam, B. Gas-expanded liquids. Chem. Rev. 2007, 107, 2666–2694. [CrossRef] 31. Nerome, H.; Ito, M.; Machmudah, S.; Wahyudiono; Kanda, H.; Goto, M. Extraction of phytochemicals from saffron by supercritical carbondioxide with water and methanol as entrainer. J. Supercrit. Fluids 2016, 107, 377–383. [CrossRef]