3.2.3. Effect of the Concentration of [Na2S2O3]

The study of gold and silver extraction contained in a sedimentary ore, using the Na2S2O3-Air system, was done to establish the effect of the [Na2S2O3] concentration, temperature, and pH.

Figure 6A shows the leached fraction of Ag that was analyzed by the core model for diffusive control [41–43], which was [1 − 2/3XAg − (1 − XAg) 2/3]. Straight lines were obtained, and their slopes represented the experimental rate constant (kexp). Thiosulfate concentration had no effect on the rate of reaction. Since all experiences were done at a high stirring rate, the oxygen input could be enough to maintain a stoichiometric excess during the progress of the reaction. This caused a limited effect on the leaching of silver with respect to thiosulfate concentration, getting a low order of the reaction, n = −0.61 (Figure 6B).

Figure 7A shows the leached fraction of Au, which was evaluated by the same core model for diffusive control [1 − 2/3XAu − (1 − XAu) 2/3]. Similar to silver leaching, thiosulfate concentration did not affect the leaching reaction rate. In this case, the presence of pyrite and Ag2S could promote the generation of *S***0** (*s*)) and/or *S*+**2** (*s*) , which would be deposited on the gold surface, passivating its dissolution. This also provided a low order of reaction, n = −0.09 (Figure 7B).

**Figure 6.** Kinetic study of silver leaching; effect of the thiosulfate concentration: (**A**) *kexp* and (**B**) order of reaction n = −0.61.

**Figure 7.** Kinetic study of gold leaching; effect of the thiosulfate concentration: (**A**) *kexp* and (**B**) order of reaction n = −0.09.

#### 3.2.4. Effect of the Temperature

The effect of temperature for silver leaching in thiosulfate solutions is shown in Figure 8A,B. For low and high temperatures (298–328 K), the values were quite similar, giving low reaction rates. In conclusion, the influence of temperature was limited.

The activation energy was calculated by plotting the natural logarithm of *kexp* against the reciprocal of temperature. The slope (m= −(Ea/R)) of the linear curve represented the activation energy (Ea) divided by the negative value of the universal gas constant (Figure 8B). For silver leaching, the calculated energy of activation was of 3.15 kJ/mol, which was representative of a diffusive control [41–43]. This was also observed by the fitting obtained with the diffusive control model. The obtained order of reaction was low, and the curve type "S" had no induction period, then the process was not dependent on both thiosulfate and temperature, and the slow diffusion of products from the particle's surfaces to the deep of solution controlled the overall process.

For gold leaching, the effect of temperature according to the diffusive control model is shown in Figure 9A. The rate constants were quite similar in the range of temperature from 298 to 313 K (0.0011–0.0014), but he slight increase at higher temperatures (318 and 323 K) could indicate that the process was carried out by a combination of both chemical and diffusive control. Besides, the activation energy that is shown in Figure 9B was 36.44 kJ/mol, which, according to the literature, is representative of mixed control [41–43].

**Figure 8.** Kinetic study of silver leaching; effect of the temperature: (**A**) *kexp* and (**B**) energy of activation, Ea = 3.15 kJ/mol.

**Figure 9.** Kinetic study of gold leaching; effect of the temperature: (**A**) *kexp* and (**B**) energy of activation, Ea = 36.44 kJ/mol.

According to the obtained results, the kinetic expressions for silver (Equation (7)) and gold (Equation (8)) leaching in *S*2*O*−2 3 medium were:

For silver, diffusive control

$$\frac{r\_0^2}{V\_M} \left[ 1 - \frac{2}{3} \left| X\_{A\mathfrak{g}} - \left( 1 - \left| X\_{A\mathfrak{g}} \right)^{\frac{2}{3}} \right) \right. \right] = \ 2D\_\varepsilon \left[ \mathcal{S}\_2 O\_3^{-2} \right] \times t \tag{8}$$

For gold, when control could be by chemical reaction

$$\frac{r\_0^2}{V\_M} \left[ 1 - \left( 1 - X\_{A\mu} \right)^{\frac{1}{3}} \right] = 3.736 \,\text{x} \,\text{10}^3 \,\text{exp}^{\frac{-36.440}{RT}} \left[ S\_2 O\_3^{-2} \right]^{-0.09} \times \text{t} \tag{9}$$

For gold, when control could be by diffusion of products through the product layer

$$\frac{r\_0}{V\_M} \left[ 1 - \frac{2}{3} \left( X\_{Au} - \left( 1 - X\_{Au} \right)^{\frac{2}{3}} \right) \right] = \ 2D\_\varepsilon \left[ S\_2 O\_3^{-2} \right] \times t \tag{10}$$

where *VM* =1.44×10−9 m3·mol−1 for silver, and *VM* =6.22×10−9 m3·mol−1 for gold, *R*=8.31 J·mol−1·K−1, *r*0 in m, *De* is the diffusion coefficient through the product layer, T in Kelvin, [*S*2*O*3 −2] is in mol·m−3, and *t* is in seconds.

#### 3.2.5. Effect of the pH

The effect of the pH for silver leaching is shown in Figure 10A,B. Figure 10A represents the treatment of results with the diffusive control model and the obtained experimental rates. The rates decreased with increasing the pH, which was faster at pH 7, having a poor effect at a higher pH. Figure 10B shows that the influence of pH over the overall reaction rate was significant, finding an order of reaction of n = 5.03.

**Figure 10.** Kinetic study of silver leaching; effect of the pH: (**A**) *kexp* and (**B**) order of reaction n = 5.03.

To conclude, for gold leaching, the pH effect is shown in Figure 11A,B. Unlike what happened with the leaching of silver, the experimental rate constants were similar for all the analyzed pH values. Consequently, a poor effect of this variable over the overall rate of the process was observed (Figure 11A). However, all experimental rate constants were low. This might be explained by the presence of small amounts of sulfur minerals since their formation is inevitable even in small quantities, promoting a partial degradation of thiosulfate. The deposition of this formed sulfur on the surface of the gold particles promoted the decrease in its dissolution rate. Finally, Figure 11B displays the effect of pH over the overall gold leaching reaction rate. The order of reaction was n = 0.94, concluding that the pH had no effect on gold leaching under the thiosulfate concentration and temperatures considered.

**Figure 11.** Kinetic study of gold leaching; effect of the pH: (**A**) *kexp* and (**B**) order of reaction n = 0.94.

#### **4. Discussion**

The mineral studied here presented significant amounts of Ag and Au, including light rare earths contents, which increased its commercial importance. However, this research aimed to analyze the leaching kinetics of gold and silver in thiosulfate solutions without adding Cu(II), which allowed verifying the ability of the leaching reagent according to the nature and composition of the ore. Although leaching using thiosulfate solutions is considered a viable alternative to cyanidation [16–18,39], nowadays, it is not used at a big scale due to the high reagent consumption and the difficulty in recovering metallic gold and silver [9]. Still, it works appropriately with metallic ores containing carbonaceous materials [35], like the ore here studied (having about a 2% C).

The use of thiosulfate as a leaching reagent needs more attention since some problems can arise, the reagent is unstable, and it can self-decomposed or reduced to *S*0, *S*−, and *S*−2 3 . These species may deposit on the surface of the metal, hindering its dissolution [25]. Additionally, the reaction is prolonged without the addition of Cu(II) and ammonia [44]. However, for this study, we decided to use sodium thiosulfate solely since the characteristics of mineral gave a chance to get promising results. Yen et al. [45] concluded that high concentrations of thiosulfate, high dissolved oxygen, and high temperatures increased the consumption of thiosulfate. Consequently, low metallic recoveries were obtained. In parallel, dilute concentrations of thiosulfate, low oxygen concentration, and low temperatures could reduce the rate of gold dissolution. However, the results of this study were encouraging, getting 80% of silver recovery using 500 mol·m3 of sodium thiosulfate, pH 7, the temperature of 298 K, stirring rate of 500 s−1, and using air in low concentration (just by incorporation during mechanical stirring). In the case of gold, the maximum dissolution was of 20% using 130 mol·m3 of sodium thiosulfate, pH 9, the temperature of 298 K, stirring rate of 500 s−1, and using air in low concentration (just by incorporation during mechanical stirring). As known, the experimental conditions constantly change during leaching, and it could be difficult to control each of them with adequate precision. Similarly, the use of relatively low leaching reagent concentrations with limited oxygen supply is an easy method to avoid high thiosulfate consumption during the leaching process [9].

Some authors [28,38] reported interesting results for the silver leaching with thiosulfate solutions, concluding that the diffusion of oxygen controls the process through the product layer. The order of the reaction was similar to that found here, which was n = −0.61 for thiosulfate concentrations 200–500 mol·m−3. Other cases displayed values like n = 0.074 for thiosulfate concentrations 100–500 mol·m−3, for silver leaching contained in mining burrows [38], n = 0.41 for thiosulfate concentration 25–200 mol·m−3 [28], and n = 0 for thiosulfate concentration 200–600 mol·m−3, for leaching of metallic silver [28]. Thiosulfate concentration does not affect the reaction rate for silver leaching that contrast works with different mineralogies [9]. For this reason, more in-depth researches are needed to disclose the behavior of thiosulfate solutions over silver leaching, according to the nature of the minerals and species involved.

For gold leaching, the literature reports that the thiosulfate concentration, joint with some contents of Cu(II) and ammonia, have a detrimental effect on the gold dissolution by the presence of minerals, such as pyrite and other sulfurs [25]. This promotes a self-decomposition of the thiosulfate and, as a consequence, higher consumption of this reactant. In this study, it was found that the thiosulfate concentration had no effect on the reaction rate getting reaction order of n = −0.09 (quite similar to that obtained for silver leaching). The latter means that the absence of important amounts of sulfur minerals might avoid the formation of *S*0 and *S*−2, which deposit on the gold surface, reducing the dissolution.

According to the evaluation of the effect of the temperature on the rate of silver leaching, the activation energy found here was Ea = 3.15 kJ/mol. This corresponded to a diffusive control, and it validated the model used for the treatment of data [41–43]. In this case, the overall reaction was controlled by oxygen diffusion through the product layer because the chemical reaction of the complexation of Ag is too fast. The above result was consistent with that obtained during the silver leaching contained in a mining waste [38], (even with the absence of Cu(II) where the apparent energy of activation was of Ea = 1.91 kJ/mol.

When evaluating the effect of temperature during the gold leaching, it was found that the apparent energy of activation was Ea = 36.44 kJ/mol. According to previous studies, this corresponded to a mixed control [41–43]. At low temperatures, the experimental rates were low (0.0011–0.0014 s−1), and diffusive control was dominant since thiosulfate was more stable. Then, at higher temperatures, the instability of thiosulfate could lead to its decomposition, generating *S*0 and *S*2− that might deposit on the gold surface [9]. This avoided a fast chemical reaction, being this step that controlled the process.

Finally, due to the concentration of *S*2*O*−2 3 could be affected by the pH (below 4 and above 12), where thiosulfate degradation or decomposition occurred, with the consequent formation of elemental sulfur (especially below a pH 4) [28,38]. Consequently, this work was executed between the valid range pH, where the thiosulfate could be more stable and was not pH-dependent. For the leaching of silver, the order of reaction was n = 5.03, indicating an apparent effect of this variable but only at pH 7. At this condition, the rate of reaction was higher than at pH 8–10. For gold dissolution, the order of reaction was n = 0.94, indicating that the pH did not alter the reaction rate, which indicated the stability of the reactant. This was because of the operational conditions used in this work and the absence of minerals like pyrite that could promote thiosulfate decomposition.

**Author Contributions:** The conceptualization was done by E.S.-R., J.H.-Á., and E.C.-S.; the methodology was executed by E.R.-C., E.S.-R., and V.R.-L.; validation was done by E.S.-R.; data curation, E.S.-R.; writing—original draft preparation, E.S.-R., E.R.-C., and J.H.-Á.; writing—review and editing, E.S.-R. and R.I.J.; supervision, E.S.-R. and V.R.-L.; funding acquisition, N.T. and R.I.J. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Acknowledgments:** Authors want to thank the CONACyT of the Mexican Government for its support given through the Ph.D. scholarship to the student Edmundo R. C. (CVU 430931). Thanks also go to the Autonomous University of the State of Hidalgo, Mexico, especially to the Academic Group of Advanced Materials from the AACTyM-ICBI; University of Antofagasta, Chile; Polytechnic University of Cartagena, Spain, and Northern Catholic University, Chile.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).


**1. Introduction**

The use of biologically active molecules such as peptides and antibodies continues to increase for both diagnosis and therapy [1–3]. Peptides are attractive platforms for diagnostics due to their ability to achieve high target binding affinity and in part due to their small size which results in short biological half-life and rapid clearance from nontarget tissues, producing good target-to-non-target contrast, low toxicity, and generally low or absent immunogenicity [1]. Synthetic advantages of peptides include simple preparation and easy, flexible functionalization or chemical modification to further improve affinity, stability, selectivity, and overall pharmacokinetic properties [1,4]. However, some of the properties that are desirable for a diagnostic agent can hamper the translation to a therapeutic, which relies on a prolonged circulation for high and persistent uptake in the targeted tissue. Too rapid clearance can render the therapeutic ineffective, and poor clearance from non-target tissue can lead to off-target toxicity. Thus, peptides typically

**Citation:** Davis, R.A.; Hausner, S.H.; Harris, R.; Sutcliffe, J.L. A Comparison of Evans Blue and 4-(*p*-Iodophenyl)butyryl Albumin Binding Moieties on an Integrin αvβ6 Binding Peptide. *Pharmaceutics* **2022**, *14*, 745. https://doi.org/ 10.3390/pharmaceutics14040745

Academic Editor: Katona Gábor

Received: 1 March 2022 Accepted: 25 March 2022 Published: 30 March 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

require fine-tuning for therapeutic applications to balance circulation time and provide high target accumulation with sufficient clearance from non-target tissues [5–7].

Chemical modifications of peptides offer a route to improving these pharmacokinetic properties; this includes incorporation of polyethylene glycol (PEG; PEGylation), glycosylation, or the formation of protein conjugates (e.g., with serum albumin) [4,8–11]. PEGylation is a convenient approach as PEGs are commercially available in a variety of molecular sizes, including mono-disperse PEGs with various functional groups for synthetic orthogonality [1,9]. PEGylation increases hydrophilicity (reducing kidney, lung, and liver accumulation) [12,13], provides increased stability (by protection from proteases), and reduces immunogenicity (by masking the peptide) [9,13]. The size and placement of the PEG on the peptides can significantly affect the pharmacokinetics and tumor accumulation [12–14]. Stability, circulation time, and tumor uptake of peptides can also be increased by chemical ligation ex vivo to serum albumin (taking advantage of albumin's size, long circulation time, and renal recycling) [8,15–17]. Alternatively, the same benefit can be achieved by direct attachment of a small albumin binding moiety (ABM) onto the peptide without substantially increasing the size. The ABM binds reversibly to albumin in the blood, thereby increasing circulation time and facilitating renal recycling, which, in turn, increases target tissue accumulation [8,15,16,18]. Several ABMs have been employed to modify pharmaceuticals currently used in the clinic, with some being used on their own, primarily for measuring plasma volume [16]; among the first ABMs used to modify pharmaceuticals were long-chain fatty acids, such as myristic and palmitic acid [5], and later other lipophilic molecules including benoxaprofen, phenytoin, ibuprofen, and naproxen [16].

More recently, two ABMs in particular, a fragment of Evans blue (EB) dye and the 4-(*p*-iodophenyl)butytryl (IP) group, have also been used to modify the pharmacokinetic profile of radiopharmaceuticals, in particular small molecules (folic acid and prostate specific membrane antigen (PSMA) agents) and peptides (octreotide, exendin-4, and cRGDfK) [10,11,16,18–20]. The EB-ABM was derived from Evans blue dye, a dye which has been used clinically for over 90 years to measure plasma volume and determine blood-brain barrier integrity [16,17,21]. The EB-ABM fragment was first used in 2004 as an MRI contrast agent for imaging blood vessels [22] and has since been used for a variety of applications, including determining blood volume, vascular permeability, and as a conjugate to enhance receptor targeting agents (small molecules and peptides) for both cancer imaging and therapy [9,17,23–26]. The IP-ABM has also been studied extensively to enhance radiopharmaceuticals (small molecules and peptides), where the group at the *para*-position of the aromatic ring of the IP-ABM can be tuned to adjust serum albumin affinity [15,27,28], and a neighboring aspartate residue (D) has been shown to provide a more sustained tumor retention [29]. Numerous preclinical studies have evaluated both ABMs and noted prolonged blood circulation, with an increase in tumor uptake that can also lead to a reduction of kidney accumulation [7,11,18].

The Sutcliffe laboratory has spent over a decade developing and optimizing an integrin αvβ6-binding peptide (αvβ6-BP) [30] to selectively target integrin αvβ6, an epithelium cell surface receptor that is absent or expressed in low levels in healthy adult epithelia, but is highly expressed in numerous challenging cancers, where it is associated with angiogenesis, proliferation, invasion, metastasis, and chemoresistance [31–41]. Thus, the integrin αvβ6 has been recognized as negative prognostic indicator with the expression levels correlating to poor prognosis and overall survival in many cancers [31–41]. During the optimization of the αvβ6-BP, the bi-terminal PEGylation with monodispersed PEG28 of the 20 amino acid A20FMDV2-peptide (NAVPNLRGDLQVLAQKVART) derived from the integrin αvβ6 targeting foot and mouth disease virus, showed greatly improved integrin αvβ6 affinity and selectivity, and improved on the peptide's stability and tumor accumulation and retention [14]. Since then, further modifications have been tested in numerous preclinical models with an advancement of the peptide to >10-fold increase in tumor accumulation and the successful translation of the 4-[18F]fluorobenzoyl labeled [18F]αvβ6-BP into the

clinic for PET imaging of a variety of cancers, including pancreatic adenocarcinoma [30]. Further optimization of αvβ6-BP continues towards an integrin αvβ6 targeted peptide receptor radionuclide therapy (PRRT).

Recently, Hausner et al. described the IP-ABM modified αvβ6-BP radiolabeled using 1,4,7-triazacyclo-nonane-*N*,*N*',*N*"-triacetic acid (NOTA) for aluminum [18F]fluoride chelation, with the goals of improving the biodistributions and simplifying the fluorine-18 radiochemistry [42]. The [18F]AlF NOTA-IP-ABM-αvβ6-BP had increased blood circulation and tumor accumulation that allowed for high-contrast PET imaging at 6 h post-injection (p.i.) [42], and >3.5-fold lower kidney retention than the very early generation [18F]AlF NOTA-A20FMDV2-peptide [43]. Building on these data and to extend the imaging window beyond that of fluorine-18 (t1/2 = 109.7 min), a copper-64 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetracetic acid (DOTA) IP-ABM-αvβ6-BP (t1/2 = 12.7 h) was prepared, which again resulted in an increased tumor accumulation that allowed PET imaging up to 72 h p.i. [44].

In the present study, we describe a head-to-head comparison of the αvβ6-BP modified with either EB-ABM or IP-ABM, with the goal to examine if fine tuning of the ABM could further increase tumor accumulation. Copper-64 radiolabeled [64Cu]Cu DOTA-EB-αvβ6-BP ([64Cu]**1**) and [64Cu]Cu DOTA-IP-αvβ6-BP ([64Cu]**2**), along with the non-αvβ6-targeting ABM controls [64Cu]Cu DOTA-EB ([64Cu]**3**) and [64Cu]Cu DOTA-IP ([64Cu]**4**; Figure 1) were synthesized. Peptides [64Cu]**1** and [64Cu]**2** were evaluated in vitro by competitive ELISA, serum stability, albumin binding assays, and cell binding and internalization assays with DX3puroβ6 (αvβ6+), DX3puro (αvβ6−), and BxPC-3 (αvβ6+) cells (against controls [ 64Cu]**3** and [64Cu]**4**), and in vivo by PET/CT imaging and biodistribution studies in mice bearing BxPC-3 xenograft tumors (4–72 h, p.i., against controls [64Cu]**3** and [64Cu]**4** at 4 h, p.i.).

**Figure 1.** (**A**) Chemical structures of 64Cu-radiolabeled-ABM-αvβ6 -BP: [64Cu]Cu DOTA-EB-αvβ6 -BP and [64Cu]Cu DOTA-IP-αvβ6 -BP ([64Cu]**1** and [64Cu]**2**). (**B**) Chemical structures of 64Cu-radiolabeled non-targeting-ABM controls: [64Cu]Cu DOTA-EB and [64Cu]Cu DOTA-IP ([64Cu]**3** and [64Cu]**4**). [αvβ6 -BP = PEG28-NAVPNLRGDLQVLAQRVART-PEG28-CONH2 ].

#### **2. Materials and Methods**

#### *2.1. Materials and General Information*

Amino acids *N*-terminally protected with a fluorenylmethyloxycarbonyl (Fmoc) protecting group and acid labile side chain protecting groups (trityl, Pbf, *tert*-butyl, or Boc) were purchased from Novabiochem (MA, USA) or GL Biochem (Shanghai, China). The orthogonally protected lysine with a 1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)-3-methylbutyl

(ivDde) sidechain protecting group and an *N*-terminal Fmoc protecting group, Fmoc-Lys(ivDde)-OH was purchased from ChemPep (Wellington, FL, USA) and the reverse ivDde-Lys(Fmoc)-OH was purchased from EMD (MA, USA). The Fmoc-NH-PEG28 carboxylic acid was purchased from Polypure (Oslo, Norway) and the chelator DOTA-tris(*tert*butyl ester) was purchased from CheMatech (Dijon, France) and Macrocyclics (Plano, TX, USA). The coupling reagent 1-[bis(dimethylamino)methylene]-1*H*-1,2,3-triazolo[4,5 b]pyridinium 3-oxid hexafluorophosphate (HATU) was purchased from GL Biochem, and benzotriazol-1-yl-oxytripyrrolidinophosphoniumhexafluorophosphate (PyBOP) was purchased from Novabiochem. Ethylenediaminetetraacetic acid (EDTA), manganese chloride (MnCl2), and Tris were purchased from Sigma-Aldrich (St. Louis, MO, USA). Tween 20 and sodium chloride (NaCl) were purchased from Fisher (Hampton, NH, USA). The non-fat dry milk powder was purchased from Raley's (West Sacramento, CA, USA). Anhydrous *N*,*N*diisopropylethylamine (DIPEA) and hydrazine were purchased from Sigma-Aldrich and used without additional purification. Solvents *N*,*N*-dimethylformamide (DMF), dimethylsulfoxide (DMSO), acetonitrile (ACN), methanol (MeOH), dichloromethane (DCM), ethyl acetate (EtOAc), *n*-hexanes, and pyridine were purchased from EMD or Acros (NJ, USA). Water used was purified with a Millipore Integral 5 Milli-Q water system at 18.2 MΩ/cm resistivity through a 0.22 µm filter. All solid phase couplings were carried out by rotation in a fritted polypropylene reactor. Thin-layered chromatography (TLC) plates (silica gel 60 with 254 nm fluorescent indicator) from EMD were visualized by UV lamp at 254 nm and/or iodine staining (for the synthesis of **6**). Purification of compound **6** was carried out by normal phase flash column chromatography with silica gel (40–63 µm; Silicycle, QC, Canada). Characterization, purity, and stability were assessed by analytical C12-reverse-phase (RP) high-pressure liquid chromatography (HPLC) column (Jupiter Proteo, 250 mm × 4.6 mm × 4 µm; Phenomenex, Torrance, CA, USA). A Semi-preparative C18-RP-column (Proteo-Jupiter, 250 mm × 10 mm × 10 µm; Phenomenex) was used for purification as described in the Supporting Information (Table S3). All RP-HPLC were carried out on a Dionex Ultimate 3000 HPLC system or a Beckman Coulter Gold HPLC with the latter being used for all radio-RP-HPLC analysis. RP-HPLC were monitored by a UV detector at a wavelength of 220 nm; a serially connected gamma detector was used to monitor radioactivity. [64Cu]CuCl2 was from the University of Wisconsin Medical Physics Department (WIMR Cyclotron Labs, Madison, WI, USA). Tissue culture and cellular assays used Dulbecco's Modified Eagle Medium (DMEM), Roswell Park Memorial Institute (RPMI) 1640 medium, fetal bovine serum (FBS), bovine serum albumin (BSA), penicillin-streptomycin-glutamine (PSG), puromycin, and phosphate buffered saline (PBS; all: Gibco/Thermo Fisher). The DX3puroβ6 and DX3puro cells were a gift from Dr. John Marshall. The DX3puroβ6 and DX3puro cell lines were maintained in DMEM medium, supplemented with 10% FBS, 1% penicillin-streptomycin-glutamine, and 2 mg/mL-puromycin. The BxPC-3 cells were purchased from American Type Culture collection (ATCC, Manassas, VA, USA) and maintained in RPMI 1640 medium supplemented with 10% FBS and 1% penicillin-streptomycin-glutamine. Cells were kept in a humidified incubator at 37 ◦C under a 5%-carbon dioxide atmosphere. A Wizard 1470 or Wizard2 2470 automatic γcounter (Perkin-Elmer, Waltham, MA, USA) was used to measure radioactivity samples. Mass spectrometry analysis was performed at the UC Davis Mass Spectrometry Facility using either a matrix assisted laser desorption ionization time of flight (MALDI- TOF) spectrometer (UltraFlextreme; Bruker, Billerica, MA, USA) in positive ionization mode with a sinapic acid matrix (Sigma-Aldrich), or with electrospray ionization (ESI) using a quadrupole ion-trap mass spectrometer (Orbitrap; ThermoFisher). Nuclear magnetic resonance (NMR) spectra were collected at the UC Davis NMR Facility on an 800 MHz Bruker instrument with the chemical shifts referenced to the residual solvent of deuterium oxide (D2O, HOD 4.79 ppm).

#### *2.2. Synthesis of EB-ABM 8*

The synthesis of the Evans blue fragment (EB-ABM **8**) was based on previously described methods [7,17,45] (Scheme 1). In brief, *o*-tolidine **5** (531 mg, 2.5 mmol; TCI America, Inc., OR, USA) was dissolved in anhydrous pyridine (1 mL) followed by the addition of succinic anhydride (300 mg, 3.0 mmol) in DMF (1 mL) and allowed to react overnight at room temperature. The crude reaction mixture was concentrated under vacuum and purified by silica-gel column chromatography using a four solvent gradient system beginning with EtOAc/n-hexanes (1/1, *v*/*v*) to remove unreacted *o*-tolidine (**5**, yellow band). The solvent was then changed to 100% EtOAc before switching to MeOH/DCM (1/9, *v*/*v*) and gradually ramping to 3/7 (*v*/*v*) to obtain **6** (648 mg, rt = 0.13, 1:1 hexanes:EtOAc) as a white solid in 83% yield. Compound 6 was analyzed by analytical RP-HPLC and ESI mass spectrometry (Figure S17).

**Scheme 1.** (**A**) Synthetic route to modified EB-ABM **8**. (**B**) Radiochemical Synthesis of [64Cu]**1**–**4**. a. Succinic anhydride, DMF, pyridine (1:1), b. NaNO2 , MeOH, HCl/H2O, 0 ◦C, c. 1-amino-8-napthol-2,4-disulfonic acid, NaHCO3 , H2O, 0 ◦C, d. [64Cu]CuCl2 , NH4OAc, 37 ◦C.

Compound **6** (300 mg, 0.96 mmol) was added to a 25 mL round bottom flask with stir bar containing MeOH (7 mL) and water (5 mL). The contents were cooled to 0 ◦C (ice/brine solution) and allowed to stir for 15 min prior to addition of concentrated hydrochloric acid 240 µL (HCl, 12.1 N; EMD). The diazonium formation of **7** was most successful when the addition of sodium nitrite was done in two portions; the first portion of sodium nitrite (NaNO2, 70 mg, 1.01 mmol; Sigma-Aldrich) was allowed to react for 5 min before the addition of the second portion (NaNO2, 70 mg, 1.01 mmol), after which the reaction was stirred an additional 30 min to generate **7** in situ, which was produced in better yields using the methanol co-solvent than water alone [46]. During in situ formation of **7**, sodium bicarbonate (350 mg, 4.17 mmol; EMD) was dissolved in water (4 mL) with 1-amino-8 napthol-2,4-disulfonic acid (377 mg, 1.18 mmol; TCI America, Inc.) in a separate 25 mL round bottom flask and the contents cooled in an ice/brine solution (~20 min). Next, the diazonium **7** reaction mixture (yellow) was cannulated into the 1-amino-8-napthol-2,4-disulfonic acid (brown-purple) solution by drop-wise addition over 20 min while maintaining both solutions at 0 ◦C. Upon complete addition of **7**, the reaction contents were allowed to stir for 3 h at 0 ◦C, and the crude reaction mixture was lyophilized and purified by semi-preparative RP-HPLC, and the collected material lyophilized. The EB-ABM **8** was

afforded as a fluffy purple solid (480 mg, 78%) and was analyzed by analytical RP-HPLC, ESI mass spectrometry, and NMR (Figures S18 and S19).

## *2.3. Synthesis of DOTA-ABM-αvβ6-BPs* **1** *and* **2**

The αvβ6-BP (PEG28-NAVPNLRGDLQVLAQRVART-PEG28) was synthesized on NovaSyn TGR resin (NovaBiochem) and PEGylation was done using monodisperse Fmoc-amino-PEG-propionic acid (Fmoc-PEG28-CO2H; FW = 1544.8 g/mol) as previously described [30] using standard Fmoc-chemistry. After each coupling or deprotection the resin was rinsed with DMF (3×), MeOH (3×), and DMF (3×). The αvβ6-BP-resin was split in equal portions (100 mg, 0.0088 mmol) and further modified at the *N*-terminus for the synthesis of peptides **1** and **2**. The DOTA-EB-αvβ6-BP **1** was generated by first removing the *N*-terminal Fmoc of the αvβ6-BP with 20% piperidine (Sigma-Aldrich) in DMF (2 × 10 min) followed by the addition of Fmoc-Lys(ivDde)-OH (50.6 mg, 0.088 mmol) using HATU (32.3 mg, 0.085 mmol) and DI-PEA (30 µL, 0.172 mmol) in DMF (1 mL) for 2 h. The Fmoc was subsequently removed with 20% piperidine in DMF (2 × 10 min) and DOTA-tris(*tert*-butyl ester) (50.3 mg, 0.088 mmol) was coupled for 2 h to the *N*-terminus with HATU (32.3 mg, 0.085 mmol) and DIPEA (30 µL, 0.172 mmol) in DMF (1 mL). The removal of the ivDde lysine-sidechain protecting group was done with hydrazine (50 µL) in DMF (1 mL, 2 × 30 min) and the resin dried under vacuum. The EB-ABM **8** (60 mg, 0.093 mmol) was then coupled to the ε-amine of the sidechain of the DOTA-lysine on the αvβ6-BP-resin using PyBOP (125 mg, 0.24 mmol) and DIPEA (50 µL, 0.287 mmol) for 6 h to yield DOTA-EB-αvβ6-BP-resin **1** (Figure S4). The DOTA-EB-αvβ6-BP **1** was cleaved off the resin with concomitant removal of the protecting groups using trifluoroacetic acid (TFA, 2 mL; EMD), triisopropylsilane (TIPS, 50 µL; Alfa Aesar, Haverhill, MA, USA) and water (50 µL), concentrated, purified, and characterized by analytical RP-HPLC and MALDI-TOF (Figure S5). The IP-ABM containing-αvβ6-BP **2** was prepared as previously described [42,44] were upon removal the *N*-terminal Fmoc of the αvβ6-BP-resin, a ivDde-Lys(Fmoc)-OH was coupled. Completion of DOTA-IP-αvβ6-BP **2** was done by sequential coupling/deFmocing of (1) Fmoc-Asp(OtBu)-OH, (2) *N*-γ-Fmoc-γ-aminobutyric acid, and (3) 4-(*p*-iodophenyl)butyric acid using HATU and DIPEA for each coupling. Completion of DOTA-IP-αvβ6-BP **2** was achieved by removal of the *N*-terminal ivDde protecting group with 5%-hydrazine in DMF followed by attachment of DOTA-tris(*tert*-butyl ester) [44]. The completed DOTA-IP-αvβ6-BP **2** was cleaved, purified, and analyzed as described above for the DOTA-EB-αvβ6-BP **1** (Figure S8) [44].

#### *2.4. Synthesis of Non-Targeting ABMs* **3** *and* **4**

Using Fmoc-chemistry with Rink AM resin (200 mg, 0.114 mmol; GL Biochem), DOTA-ABM non-targeting compounds **3** and **4** were synthesized by first coupling Fmoc-Lys(ivDde)-OH (196.6 mg, 0.342 mmol) using HATU (123.5 mg, 0.325 mmol) and DIPEA (100 µL, 0.574 mmol) in DMF (1 mL). The Fmoc was removed with 20% (*v*/*v*) piperidine in DMF (1 mL, 2 × 10 min) and DOTA-tris(*tert*-butyl ester) (80 mg, 0.140 mmol) was coupled for 2 h with HATU (50 mg, 0.132 mmol) and DIPEA (50 µL, 0.287 mmol). Following the ivDde protecting group removal with hydrazine (50 µL) in DMF (1 mL, 2 × 30 min), the resin was dried under vacuum and split into equal portions for synthesis of **3** and **4**. For **3**, the EB-ABM **8** (165 mg, 0.256 mmol) was coupled using PyBOP (166.5 mg, 0.32 mmol) and DIPEA (100 µL, 0.574 mmol) for 6 h. The EB-ABM **3** was cleaved off the resin, purified, and characterized by analytical RP-HPLC and MALDI-TOF (Figure S11). IP-ABM **4** was prepared by sequential coupling/deFmocing of (1) Fmoc-Asp(OtBu)-OH (90 mg, 0.219 mmol), (2) *N*-γ-Fmoc-γ-aminobutyric acid (70 mg, 0.215 mmol), and (3) 4-(*p*-iodophenyl)butyric acid (65 mg, 0.224 mmol) using HATU (78 mg, 0.205 mmol) and DIPEA (100 µL, 0.574 mmol) for each coupling. The IP-ABM **4** was then cleaved off the resin, purified, and characterized by analytical RP-HPLC and MALDI-TOF (Figure S14).


*Article* **Extraction of Phenolic and Flavonoid Compounds from Mung Bean (***Vigna radiata* **L.) Seed Coat by Pressurized Liquid Extraction**

**Benya Supasatyankul 1 , Maythee Saisriyoot 2 , Utai Klinkesorn 1 , Kittipong Rattanaporn 3 and Sudathip Sae-Tan 1,\***

**Abstract:** Mung bean seed coat (MBC) is a by-product of the mung bean processing industry. It contains a large number of phenolic compounds with therapeutic anti-inflammatory, anti-diabetic and antioxidant properties. This research aimed to investigate the optimum conditions for phenolic and flavonoid extraction from MBC by pressurized liquid extraction (PLE). Response surface methodology (RSM) was used to study the effects of temperature (80–160 ◦C), pressure (1200–1800 psi) and ethanol concentration (5–95%) on total phenolic content (TPC), total flavonoid content (TFC) and 2,20 azinobis(3-ethylbenzothiazoline-6-sulfonic acid) scavenging activity (ABTS). Scale-up extraction was also performed. The optimum conditions for extraction were 160 ◦C, 1300 psi and 50% ethanol. Under optimum conditions, the TPC was 55.27 ± 1.14 mg gallic acid equivalent (GAE)/g MBC, TFC was 34.04 ± 0.72 mg catechin equivalent (CE)/g MBC and ABTS scavenging activity was 195.05 ± 2.29 mg trolox equivalent (TE)/g MBC. The TFC and ABTS scavenging activity of the extracts obtained at the pilot scale (10 L) was not significantly different from the laboratory scale, while TPC was significantly increased. The freeze-dried MBC extract contained vitexin and isovitexin 130.53 ± 17.89, 21.21 ± 3.22 mg/g extract, respectively. In conclusion, PLE was able to extract phenolics, flavonoids with ABTS scavenging activity from MBC with the prospect for future scale-up for food industry.

**Keywords:** flavonoid extraction; mung bean; phenolic extraction; pressurized liquid extraction; response surface methodology

#### **1. Introduction**

Mung bean (*Vigna radiata* L.) is a pulse crop widely cultivated and consumed in Asia, India and the warmer part of Europe and America, with a short growth cycle around 2–3 months [1]. Mung bean is a rich source of proteins, essential amino acids, complex carbohydrates, vitamins and minerals and it is easy to be digested [2]. Mung bean is also well known for a large number of bioactive compounds including protein, phenolic and flavonoid compounds with various health benefits [3]. The majority of by-products from the mung bean industry is mung bean seed coat and it is usually discarded. While phenolic and flavonoid compounds were reported to be abundant in mung bean seed coat for 84.2% and 83.9%, respectively [4]. Many biological activities of mung bean seed coat extract were reported. A recent study showed that mung bean seed coat water extract exerted antiinflammatory effects by inhibiting NF-κB activation via inhibition of TAK1 phosphorylation and IκBα degradation [5]. Mung bean seed coat water extract also showed antioxidant activity [6]. While ethanolic extract of mung bean seed coat inhibited α-glucosidase activity

**Citation:** Supasatyankul, B.; Saisriyoot, M.; Klinkesorn, U.; Rattanaporn, K.; Sae-Tan, S. Extraction of Phenolic and Flavonoid Compounds from Mung Bean (*Vigna radiata* L.) Seed Coat by Pressurized Liquid Extraction. *Molecules* **2022**, *27*, 2085. https://doi.org/10.3390/ molecules27072085

Academic Editor: Smaoui Slim

Received: 27 February 2022 Accepted: 22 March 2022 Published: 24 March 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

and exerted anti-diabetic activity in *db*/*db* mice [7]. The proposed bioactive compounds in mung bean seed coat were vitexin and isovitexin [5]. Total phenolic content in mung bean extract is ranging from 2.03 to 79.65 mg GAE/g extract, depending on the method of extraction [8–10]. Therefore, the study on the extraction method and optimum conditions is necessary.

Conventional extraction methods for polyphenols, phenolics and flavonoids from plant materials are maceration, hydro-distillation, agitated solvent extraction and Soxhlet extraction [11]. Recently, many innovative extraction methods have been introduced to increase the efficacy of extraction including ultrasound-assisted extraction (UAE), microwaveassisted extraction (MAE) and pressurized liquid extraction (PLE). Santos et al. compared the efficacy of α-bisabolol extraction between Soxhlet extraction and PLE [12]. The results showed that the amount of α-bisabolol from both methods was not significantly different. However, PLE used only 20 min, while Soxhlet extraction used 360 min for extraction. PLE was compared with UAE in extracting phenolic compounds from the residues of *Rubus fruticosus*, *Vaccinium myrtillus* and *Eugenia brasiliensis* [13]. The results showed that PLE gave a higher yield of phenolic compounds compared to UAE. PLE was also more efficient in the extraction of terpenes, fatty acids and vitamin E from *Piper gaudichaudianum* Kunth in terms of yield and time [14]. The efficacy of PLE and MAE in extracting phenolic compounds from *Moringa oleifera* leaves was compared by Barriada-Pereira et al. [15]. The results showed that TPC and TFC from PLE were higher than those from MAE. This indicated that PLE was a potential extraction method to increase the efficacy of phenolic compounds from mung bean seed coat.

Many factors play a role in extraction. The efficiency of PLE depends on the extraction temperature, pressure, time, solvent and the ratio between extraction solvent and sample [16]. The present study was designated to investigate the effects of extraction temperature, pressure and concentration of ethanol on TPC, TFC and ABTS of mung bean seed coat. The main goal of the present study was to optimize, validate and scale up the extraction of mung bean seed coat using PLE.

#### **2. Results**

#### *2.1. Comparison of Soxhlet and PLE Extraction*

Considering the high number of bioactive compounds and therapeutic properties of MBC, the extraction of phenolic and flavonoid compounds from MBC was interesting. The total content of bioactive compounds depends on many factors including the extraction method [17]. Here, MBC was extracted with Soxhlet and PLE extraction methods. The TPC, TFC and ABTS of the MBC extract using two extraction methods were shown and compared in Figure 1. The results showed no significant difference between TPC, TFC and ABTS of the extracted solutions from Soxhlet and PLE. About the extraction time, the PLE method spent only 10 min for extraction, while the Soxhlet method spent 6 h for extraction. Both extraction methods were carried out using 95% ethanol at 80 ◦C, however, the pressure of 1500 psi was applied in PLE. This indicated that pressure application did not have any effect on TPC, TFC and ABTS extraction, however pressure did reduce the extraction time. The present results agreed with the previous study showed that the PLE method reduced the extraction time in TPC extraction from passion fruit rinds by 12 times [18]. The PLE method also reduced the extraction time and the solvent use in polycyclic aromatic hydrocarbon (PAH) extraction from soil (18 and 10 times lower than Soxhlet extraction method, respectively) [19]. The extraction of phenolic compounds from *Mentha piperita*, *Origanum vulgare*, *Rosmarinus officinalis* L. and *Thymusvulgaris* L. by PLE method also gave a higher yield of phenolic and volatile compounds as well as the higher antioxidant activity compared to Soxhlet extraction method [20]. This did indicate the potential of PLE on MBC extraction.

**Figure 1.** Total phenolic content (**A**), total flavonoid content (**B**) and ABTS (**C**) of MBC extract obtained by Soxhlet extraction (80 °C and 95% ethanol) and PLE (80 °C, 1500 psi and 95% ethanol). **Figure 1.** Total phenolic content (**A**), total flavonoid content (**B**) and ABTS (**C**) of MBC extract obtained by Soxhlet extraction (80 ◦C and 95% ethanol) and PLE (80 ◦C, 1500 psi and 95% ethanol). There was no significant difference between methods on those response variables.

#### There was no significant difference between methods on those response variables. *2.2. Model Fitting and Analysis of Variance*

*2.2. Model Fitting and Analysis of Variance*  Optimization of extraction conditions was performed in 45 randomized runs to study the effect of different variables on the TPC, TFC and ABTS of the MBC extract. The coded values, uncoded values and the results of dependent variables were shown in Table 1. Phenolic compounds were extracted from MBC ranged from 3.34 ± 0.32 to 52.88 ± 2.48 mg GAE/g MBC (dry weight, d.w.). Extracted flavonoids ranged from 1.27 ± 0.26 to 17.07 ± 0.53 mg CE/g MBC d.w. and ABTS ranged from 3.34 ± 0.80 to 202.71 ± 6.26 mg TE/g MBC d.w. These results showed that TPC, TFC and ABTS considerably depended on the extraction condition, which indicated that the optimization of the extraction condition was im-Optimization of extraction conditions was performed in 45 randomized runs to study the effect of different variables on the TPC, TFC and ABTS of the MBC extract. The coded values, uncoded values and the results of dependent variables were shown in Table 1. Phenolic compounds were extracted from MBC ranged from 3.34 ± 0.32 to 52.88 ± 2.48 mg GAE/g MBC (dry weight, d.w.). Extracted flavonoids ranged from 1.27 ± 0.26 to 17.07 ± 0.53 mg CE/g MBC d.w. and ABTS ranged from 3.34 ± 0.80 to 202.71 ± 6.26 mg TE/g MBC d.w. These results showed that TPC, TFC and ABTS considerably depended on the extraction condition, which indicated that the optimization of the extraction condition was important. The optimization of the extraction process was performed by applying second-order polynomial equations. The regression coefficients for dependent variables along with the corresponding coefficient of determination value (R2 ) and lack of fit test of TPC, TFC and ABTS were shown in Table 2.

portant. The optimization of the extraction process was performed by applying secondorder polynomial equations. The regression coefficients for dependent variables along with the corresponding coefficient of determination value (R2) and lack of fit test of TPC, **Table 1.** Coded and uncoded levels of independent variables in experimental design, TPC, TFC and ABTS from the MBC extract.

4 +1 160 0 1500 +1 95 6.73 ± 0.73 e 6.73 ± 0.51 e,f 36.70 ± 2.64 f Means values with the different letters in the same column are significantly different (*p* ≤ 0.05).

5 −1 80 −1 1200 0 50 28.06 ± 2.11 c,d 7.22 ± 0.67 e 123.59 ± 5.90 c,d 6 −1 80 +1 1800 0 50 30.78 ± 1.94 c 6.80 ± 0.62 e,f 133.74 ± 2.01 c 7 +1 160 −1 1200 0 50 52.88 ± 2.48 a 17.07 ± 0.53 a 197.51 ± 7.99 a 8 0 120 0 1500 0 50 36.91 ± 1.63 b 14.13 ± 0.43 b 155.29 ± 9.98 b

10 0 120 0 1500 0 50 37.97 ± 3.74 b 13.90 ± 0.98 b 169.08 ± 8.90 b 11 +1 160 +1 1800 0 50 50.94 ± 0.53 a 13.13 ± 0.45 b,c 202.71 ± 6.26 a 12 −1 80 0 1500 −1 5 23.47 ± 0.44 d 7.26 ± 0.45 e 87.58 ± 0.62 e 13 0 120 −1 1200 −1 5 29.85 ± 2.72 c 9.76 ± 0.34 d 110.07 ± 2.07 d 14 0 120 +1 1800 −1 5 36.32 ± 1.22 b 11.81 ± 0.44 c,d 118.15 ± 1.29 c,d

**Table 2.** Regression coefficients, coefficient of determination and *p*-value of the predicted secondorder polynomial models for TPC, TFC and ABTS.

\* The *p*-value more than 0.05 is not significantly different at the 5% level.

The ANOVA results indicated that three independent variables on the model were able to explain the experimental variation for TPC, TFC and ABTS as shown by the significant *p*-value for each model (Table 2). The corresponding coefficients of determination (R2 ) of the model were 0.9426, 0.9507 and 0.9809 for TPC, TFC and ABTS, respectively. These values implied that more than 94.26% of the response variables variation could be explained by the regression models. Although the lack of fit was significant, the correlation between predicted values and actual values of all runs was high (R2 = 0.9740, 0.8486 and 0.9877) for TPC, TFC and ABTS respectively (data was not shown). The predicted second-order polynomial regression equations with enter elimination method were shown in Table 3 for TPC, TFC and ABTS (Equations (1)–(3), respectively).

#### *2.3. Effect of the Extraction Variables on TPC, TFC and ABTS*

The model showed a high significant value with the experimental data. ANOVA showed a positive linear effect of temperature (X1) and ethanol concentration (X3) was significant for TPC. The linear effect of ethanol concentration (X3) was significant for ABTS, while all dependent variables, temperature (X1), pressure (X2) and ethanol concentration (X3) were significant for TFC. The quadratic effect of temperature (X1), pressure (X2) and ethanol concentration (X3) were found to be significant for TFC, while the quadratic effect of only ethanol concentration (X3) was found to be significant for TPC and ABTS. The interaction effect of temperature (X1) and ethanol concentration (X3) was found to be significant for TPC, while the interaction effect of temperature (X1) and pressure (X2) was found to be significant for TFC. There was no interaction effect of dependent variables on ABTS.

The response surface plot of TPC using different combinations of temperature, pressure and ethanol concentration was shown in Figure 2. The TPC was higher at higher temperatures and vice versa, while pressure did not affect TPC. As shown in Table 2, TPC was significantly influenced by the interaction effect of temperature and ethanol concentration. Figure 2B showed that TPC increased with ethanol concentration increased, but this increase was up to 50% ethanol and then gradually decreased as ethanol concentration increased.

on ABTS.

on ABTS.

tion increased.

tion increased.

*Molecules* **2022**, *27*, x FOR PEER REVIEW 5 of 15

**Figure 2.** Response surface plots showing the interaction effects of extraction variables on TPC of MBC extract. (**A**) pressure and ethanol concentration at constant temperature (120 °C), (**B**) temperature and ethanol concentration at constant pressure 1500 psi and (**C**) temperature and pressure at constant ethanol concentration (50%). **Figure 2.** Response surface plots showing the interaction effects of extraction variables on TPC of MBC extract. (**A**) pressure and ethanol concentration at constant temperature (120 ◦C), (**B**) temperature and ethanol concentration at constant pressure 1500 psi and (**C**) temperature and pressure at constant ethanol concentration (50%). ature and ethanol concentration at constant pressure 1500 psi and (**C**) temperature and pressure at constant ethanol concentration (50%). The response surface plot of TFC using different combinations of temperature, pres-

sure and ethanol concentration was shown in Figure 3. The TFC was higher at higher

MBC extract. (**A**) pressure and ethanol concentration at constant temperature (120 °C), (**B**) temper-

to be significant for TPC, while the interaction effect of temperature (X1) and pressure (X2) was found to be significant for TFC. There was no interaction effect of dependent variables

The response surface plot of TPC using different combinations of temperature, pressure and ethanol concentration was shown in Figure 2. The TPC was higher at higher temperatures and vice versa, while pressure did not affect TPC. As shown in Table 2, TPC was significantly influenced by the interaction effect of temperature and ethanol concentration. Figure 2B showed that TPC increased with ethanol concentration increased, but this increase was up to 50% ethanol and then gradually decreased as ethanol concentra-

to be significant for TPC, while the interaction effect of temperature (X1) and pressure (X2) was found to be significant for TFC. There was no interaction effect of dependent variables

The response surface plot of TPC using different combinations of temperature, pressure and ethanol concentration was shown in Figure 2. The TPC was higher at higher temperatures and vice versa, while pressure did not affect TPC. As shown in Table 2, TPC was significantly influenced by the interaction effect of temperature and ethanol concentration. Figure 2B showed that TPC increased with ethanol concentration increased, but this increase was up to 50% ethanol and then gradually decreased as ethanol concentra-

The response surface plot of TFC using different combinations of temperature, pressure and ethanol concentration was shown in Figure 3. The TFC was higher at higher temperatures, pressure and vice versa. As shown in Table 2, TFC was significantly influenced by the interaction effect of temperature and pressure. The effect of temperature on TFC extraction seen in Figure 3C was positive but clearly lower than the effect of pressure. The response surface plot of TFC using different combinations of temperature, pressure and ethanol concentration was shown in Figure 3. The TFC was higher at higher temperatures, pressure and vice versa. As shown in Table 2, TFC was significantly influenced by the interaction effect of temperature and pressure. The effect of temperature on TFC extraction seen in Figure 3C was positive but clearly lower than the effect of pressure. The effect of ethanol concentration on TFC was similar to TPC but less potent. temperatures, pressure and vice versa. As shown in Table 2, TFC was significantly influenced by the interaction effect of temperature and pressure. The effect of temperature on TFC extraction seen in Figure 3C was positive but clearly lower than the effect of pressure. The effect of ethanol concentration on TFC was similar to TPC but less potent.

**Figure 3.** Response surface plots showing the interaction effects of extraction variables on TFC of MBC extract. (**A**) pressure and ethanol concentration at constant temperature (120 °C), (**B**) temperature and ethanol concentration at constant pressure 1500 psi and (**C**) temperature and pressure at constant ethanol concentration (50%). **Figure 3.** Response surface plots showing the interaction effects of extraction variables on TFC of MBC extract. (**A**) pressure and ethanol concentration at constant temperature (120 ◦C), (**B**) temperature and ethanol concentration at constant pressure 1500 psi and (**C**) temperature and pressure at constant ethanol concentration (50%).

MBC extract. (**A**) pressure and ethanol concentration at constant temperature (120 °C), (**B**) temperature and ethanol concentration at constant pressure 1500 psi and (**C**) temperature and pressure at constant ethanol concentration (50%). The response surface plot of ABTS using different combinations of temperature, pressure and ethanol concentration was shown in Figure 4. Figure 4A,B showed that ABTS increased with ethanol concentration increased, but this increase was up to 50% ethanol and then gradually decreased as ethanol concentration increased (>50%).

**Figure 3.** Response surface plots showing the interaction effects of extraction variables on TFC of

The present results were consistent with the previous studies showing that higher extraction temperature promoted higher phenolic compounds extracted from rosemary [21], jabuticaba skins [22], cocoa bean shell [23] and rice grains [16] using PLE. The use of high temperature promotes the recovery of phenolic and flavonoid compounds because it increases the molecular motion, which in turn causes a decrease of solvent viscosity, increased diffusivity and increased solubility of target compounds. High temperature also assists in making the cell walls more permeable, enabling the target compounds to

leach out into the solvent. In addition, high temperature assists in breaking down target compound-matrix interaction and promote the diffusion of the target compound to the matrix surface and mass transfer to solvent [16,24]. increased with ethanol concentration increased, but this increase was up to 50% ethanol and then gradually decreased as ethanol concentration increased (>50%).

The response surface plot of ABTS using different combinations of temperature, pressure and ethanol concentration was shown in Figure 4. Figure 4A,B showed that ABTS

*Molecules* **2022**, *27*, x FOR PEER REVIEW 6 of 15

**Figure 4.** Response surface plots showing the interaction effects of extraction variables on ABTS of MBC extract. (**A**) pressure and ethanol concentration at constant temperature (120 °C), (**B**) temperature and ethanol concentration at constant pressure 1500 psi and (**C**) temperature and pressure at constant ethanol concentration (50%). **Figure 4.** Response surface plots showing the interaction effects of extraction variables on ABTS of MBC extract. (**A**) pressure and ethanol concentration at constant temperature (120 ◦C), (**B**) temperature and ethanol concentration at constant pressure 1500 psi and (**C**) temperature and pressure at constant ethanol concentration (50%).

The present results were consistent with the previous studies showing that higher extraction temperature promoted higher phenolic compounds extracted from rosemary [21], jabuticaba skins [22], cocoa bean shell [23] and rice grains [16] using PLE. The use of high temperature promotes the recovery of phenolic and flavonoid compounds because it increases the molecular motion, which in turn causes a decrease of solvent viscosity, increased diffusivity and increased solubility of target compounds. High temperature also assists in making the cell walls more permeable, enabling the target compounds to leach out into the solvent. In addition, high temperature assists in breaking down target com-Although the use of elevated temperature promotes higher phenolic compounds recovery, the use of excessive extraction temperature may degrade phenolic compounds. The use of elevated temperature in potato peel extraction also increased phenolic compounds recovery, but further temperature to 190 ◦C resulted in a decrease in phenolic compounds yield [25]. Another study reported that the increase of temperature to a certain level (190 ◦C) did not affect the level of phenolic compounds [16]. The positive effect of temperature on antioxidant activity was also found in the papaya seed extraction [26]. A similar trend of TPC and ABTS in the present study was due to the highly correlated between the content of phenolic compounds and antioxidant activity as shown in other study [27].

pound-matrix interaction and promote the diffusion of the target compound to the matrix surface and mass transfer to solvent [16,24]. Although the use of elevated temperature promotes higher phenolic compounds recovery, the use of excessive extraction temperature may degrade phenolic compounds. The use of elevated temperature in potato peel extraction also increased phenolic compounds recovery, but further temperature to 190 °C resulted in a decrease in phenolic compounds yield [25]. Another study reported that the increase of temperature to a certain level (190 °C) did not affect the level of phenolic compounds [16]. The positive effect of temperature on antioxidant activity was also found in the papaya seed extraction [26]. A similar trend of TPC and ABTS in the present study was due to the highly correlated between the content of phenolic compounds and antioxidant activity as shown in other study [27]. Pressure plays an important role in PLE extraction because at high temperatures, the solvent becomes a gas state. High pressure in PLE allows the solvent to maintain a liquid state, which increases the penetration of the solvent in the sample matrix [24]. The present results indicated that pressure did not significantly affect TPC and ABTS in MBC extraction. This may be due to the lowest pressure in the present study (1200 psi) being enough to maintain the ethanol mixture in a liquid state. Therefore, the increase of pressure did not have any effect on extraction. The insignificant effect of pressure in the present study was consistent with a previous extraction study in the potato peel [25]. However, pressure showed a significant effect on TFC in the present study. Application of pressure in PLE alone without high temperature increases the viscosity of the solvent and solvents with high viscosity result in slow mass transfer, lower diffusivity and lower extraction efficacy [28]. Therefore, both high temperature and pressure need to be applied together in PLE.

Pressure plays an important role in PLE extraction because at high temperatures, the solvent becomes a gas state. High pressure in PLE allows the solvent to maintain a liquid state, which increases the penetration of the solvent in the sample matrix [24]. The present results indicated that pressure did not significantly affect TPC and ABTS in MBC extraction. This may be due to the lowest pressure in the present study (1200 psi) being enough to maintain the ethanol mixture in a liquid state. Therefore, the increase of pressure did not have any effect on extraction. The insignificant effect of pressure in the present study was consistent with a previous extraction study in the potato peel [25]. However, pressure showed a significant effect on TFC in the present study. Application of pressure in PLE alone without high temperature increases the viscosity of the solvent and solvents with Ethanol was used in the present study since it is relatively safer or less toxic. The concentration of ethanol also showed an important effect on the TPC, TFC and ABTS. The present study found that the optimum ethanol concentration for MBC extraction was 50% ethanol. The lower and higher ethanol concentration than 50% showed the lower responses, especially TPC and ABTS. The optimum ethanol concentration in the present study was consistent with previous studies. Zafari and Sharifi reported that the mixture of alcohol with 50% water gave the highest flavonoids including vitexin from *Prosopis farcta* [29]. Sixty percent of ethanol was the best concentration to extract phenolic compounds from brewers' spent grain compared to 20, 40 and 100% ethanol [30]. The vitexin and orientin yield from *Trollius chinesis* flowers extraction increased with the increase of ethanol concentration in the range of 0-60%. The highest yield was given at 60% ethanol extraction [31]. How-

ever, the yield sharply decreased when the ethanol concentration increased further. The present study and previous studies confirmed that ethanol concentration greatly impacted the phenolics and flavonoids extraction. Phenolic and flavonoid compounds are diverse with a wide range of solubilities in a single component [30]. Solvents with high polarity showed a higher ability to extract compounds with a wider polarity. Moreover, this allowed non-phenolic polar compounds such as protein and carbohydrates to dissolve during the extraction process, leading to the increased extraction yield [32]. This phenomenon was also explained by the fact that the addition of ethanol reduced the dielectric constant of extraction solvent, which facilitates the solubility and diffusion of phenolic compounds. Too high ethanol concentration, however, results in cell dehydration and denaturation of cell wall proteins, which inhibits the diffusion of phenolic compounds from plant material to extraction solvent [31]. Therefore, the mixture of ethanol and water was suitable for phenolic and flavonoid compounds extraction.

#### *2.4. Optimization and Validation of the Extraction Condition*

Multiple numerical optimizations were carried out to achieve the overall optimum PLE extraction conditions [33]. The goal was to set the optimum level of independent variables resulting in the highest TPC, TFC and ABTS. The predicted optimum extraction conditions from Minitab were: temperature of 160 ◦C, pressure of 1300 psi and 35% ethanol. Under recommended optimum extraction conditions, the predicted and experimental values provided residual standard error (RSE) of more than 10% (data was not shown). This indicated that the recommended optimum extraction conditions were not the actual optimum, which was indicated by the significant lack of fit of the models. Therefore, the steepest ascent method was carried out to find the actual optimum extraction condition. The procedure of the steepest ascend method is moving sequentially in the direction of the maximum response and based on the previous experiment [34]. According to Table 1, the conditions of temperature of 160 ◦C, pressure of 1200 psi and 50% ethanol provided the highest TPC, TFC and ABTS compared to other treatments. Therefore, 50% ethanol was selected to be a new optimum ethanol concentration. The new optimum conditions were temperature of 160 ◦C, pressure of 1300 psi and 50% ethanol. Under the new optimum extraction conditions, the predicted TPC, TFC and ABTS were 53.28 GAE/g MBC d.w., 32.88 mg CE/g MBS d.w. and 192.20 mg TE/g MBC d.w., respectively (Table 4). MBC was extracted under the new optimum conditions to verify the validity. The TPC, TFC and ABTS of the extract were then determined to verify the reliability of the new optimum conditions. The validation study showed the TPC, TFC and ABTS of the new MBC extracted solution were 55.267 ± 1.14 mg GAE/g MBC d.w., 34.041 ± 0.72 mg CE/g MBS d.w. and 195.046 ± 2.29 mg TE/g MBC d.w., respectively (Table 4). Under the new optimum extraction conditions, the experimental values showed that the models were in good agreement with the predicted values with RSE values of less than 4%. The present result confirmed the reliability of the new optimum extraction conditions.

**Table 4.** Predicted and experimental response values at the optimum extraction conditions.

#### *2.5. Scale-Up Study*

One of the main goals of this study was to scale up the extraction process of MBC. Many factors influence the upscaling extraction process such as process parameters, the kinetics of the whole process, the ratio between material and solvent [22]. The pilot-scale PLE (10 L extraction cell) was 25 times bigger than the laboratory-scale PLE (400 mL extraction cell). The optimum extraction conditions were used in the pilot-scale extraction with the same solid to solvent ratio (1:5 *w*/*v*; 2 kg MBC in 10 L ethanol). The extraction

time was constant with laboratory-scale PLE for 10 min. Comparing TPC, TFC and ABTS of the laboratory-scale PLE and pilot-scale PLE (Figure 5), the results showed that TFC and ABTS from pilot-scale PLE were not significantly different from TFC and ABTS from laboratory-scale PLE. Interestingly, TPC from pilot-scale PLE was significantly higher than that from laboratory-scale PLE. The increase of TPC might be due to the change of geometric factors of laboratory-scale PLE and pilot-scale PLE. Previous study showed that geometry and dynamic factors influenced the scale-up study [35]. However, this increase should be more investigated for the explanation. These results indicated that MBC extraction with pilot-scale PLE was efficiently comparable to laboratory-scale PLE. than that from laboratory-scale PLE. The increase of TPC might be due to the change of geometric factors of laboratory-scale PLE and pilot-scale PLE. Previous study showed that geometry and dynamic factors influenced the scale-up study [35]. However, this increase should be more investigated for the explanation. These results indicated that MBC extraction with pilot-scale PLE was efficiently comparable to laboratory-scale PLE.

**Table 4.** Predicted and experimental response values at the optimum extraction conditions.

One of the main goals of this study was to scale up the extraction process of MBC. Many factors influence the upscaling extraction process such as process parameters, the kinetics of the whole process, the ratio between material and solvent [22]. The pilot-scale PLE (10 L extraction cell) was 25 times bigger than the laboratory-scale PLE (400 mL extraction cell). The optimum extraction conditions were used in the pilot-scale extraction with the same solid to solvent ratio (1:5 *w*/*v*; 2 kg MBC in 10 L ethanol). The extraction time was constant with laboratory-scale PLE for 10 min. Comparing TPC, TFC and ABTS of the laboratory-scale PLE and pilot-scale PLE (Figure 5), the results showed that TFC and ABTS from pilot-scale PLE were not significantly different from TFC and ABTS from laboratory-scale PLE. Interestingly, TPC from pilot-scale PLE was significantly higher

*Molecules* **2022**, *27*, x FOR PEER REVIEW 8 of 15

**Response Variables Predicted Values Experimental Values RSE (%)**

TPC (mg GAE/g MBC d.w.) 53.28 55.27 ± 1.14 ns 3.73 TFC (mg CE/g MBC d.w.) 32.88 34.04 ± 0.72 ns 3.54 ABTS (mg TE/g MBC d.w.) 192.20 195.05 ± 2.29 ns 1.48

*2.5. Scale-Up Study*

**Figure 5.** Total phenolic content (**A**), total flavonoid content (**B**) and ABTS (**C**) of MBC extract obtained by laboratory-scale PLE and pilot-scale PLE. There was no significant difference between laboratory-scale PLE and pilot-scale PLE. The different alphabet indicated significant difference at **Figure 5.** Total phenolic content (**A**), total flavonoid content (**B**) and ABTS (**C**) of MBC extract obtained by laboratory-scale PLE and pilot-scale PLE. There was no significant difference between laboratory-scale PLE and pilot-scale PLE. The different alphabet indicated significant difference at *p* < 0.05.

#### *p* < 0.05. *2.6. Identification of Vitexin and Isovitexin in the Extract*

*2.6. Identification of Vitexin and Isovitexin in the Extract*  Vitexin and isovitexin are major flavonoids found in mung beans especially in seed coat [4]. Our previous study reported that mung bean seed coat extracted using boiling water contained vitexin and isovitexin 38.56 and 28.96 mg/g extract [5]. The present study Vitexin and isovitexin are major flavonoids found in mung beans especially in seed coat [4]. Our previous study reported that mung bean seed coat extracted using boiling water contained vitexin and isovitexin 38.56 and 28.96 mg/g extract [5]. The present study showed that mung bean seed coat extracted using PLE contain both vitexin and isovitexin at the concentration of 130.53 ± 17.89 and 21.21 ± 3.22 mg/g freeze-dried extract (Figure 6).

showed that mung bean seed coat extracted using PLE contain both vitexin and isovitexin at the concentration of 130.53 ± 17.89 and 21.21 ± 3.22 mg/g freeze-dried extract (Figure 6). In the present study, TPC and TFC of MBC extracted with the optimum conditions using scale-up PLE were 81.88 mg GAE/g MBC d.w. and 38.90 mg CE/g MBC d.w., respectively. TPC and TFC from the present study were higher than TPC and TFC of MBC extract obtained from maceration extraction method (29.58 mg GAE/g MBC and 22.08 mg CE/g MBC, respectively) [4]. UAE was also applied to increase TPC and TFC extraction. However, TPC and TFC from MBC extract obtained from UAE were only 42.22 mg GAE/g MBC and 1.13 mg CE/g MBC, respectively [9]. This indicated that PLE was a potential extraction method to extract phenolic and flavonoid compounds from MBC with high efficiency.

ficiency.

In the present study, TPC and TFC of MBC extracted with the optimum conditions using scale-up PLE were 81.88 mg GAE/g MBC d.w. and 38.90 mg CE/g MBC d.w., respectively. TPC and TFC from the present study were higher than TPC and TFC of MBC extract obtained from maceration extraction method (29.58 mg GAE/g MBC and 22.08 mg CE/ g MBC, respectively) [4]. UAE was also applied to increase TPC and TFC extraction. However, TPC and TFC from MBC extract obtained from UAE were only 42.22 mg GAE/g MBC and 1.13 mg CE/g MBC, respectively [9]. This indicated that PLE was a potential extraction method to extract phenolic and flavonoid compounds from MBC with high ef-

**Figure 6.** Representative high performance liquid chromatography-diode array detector chromato-**Figure 6.** Representative high performance liquid chromatography-diode array detector chromatographic profile (λ = 337 nm) of (**A**) mung bean seed coat extract from laboratory scale; (**B**) mung bean seed coat freeze-dried extract from pilot scale; (**C**) standard vitexin; (**D**) standard isovitexin.

#### graphic profile (λ = 337 nm) of (**A**) mung bean seed coat extract from laboratory scale; (**B**) mung bean seed coat freeze-dried extract from pilot scale; (**C**) standard vitexin; (**D**) standard isovitexin. **3. Materials and Methods**

#### *3.1. Materials and Chemicals*

**3. Materials and Methods**  *3.1. Materials and Chemicals*  Mung bean seed coat (MBC), a co-product of the mung bean dehulled process, was received from Kittitat Co., Ltd. (Bang Khun Thian, Bangkok, Thailand) in May 2019. After foreign matters removal, MBC was kept in sealed plastic bags at 4 °C until extraction. Mung bean seed coat (MBC), a co-product of the mung bean dehulled process, was received from Kittitat Co., Ltd. (Bang Khun Thian, Bangkok, Thailand) in May 2019. After foreign matters removal, MBC was kept in sealed plastic bags at 4 ◦C until extraction. Ethanol commercial grade (Mallinckrodt Baker Inc., Phillipsburg, NI, USA) was used for extraction. ABTS, vitexin and isovitexin were purchased from Sigma-Aldrich (St. Louis, MO, USA). All other chemicals were of analytical grade and obtained from reputable suppliers.

#### Ethanol commercial grade (Mallinckrodt Baker Inc., Phillipsburg, NI, USA) was used for *3.2. Extraction Procedures*

#### extraction. ABTS, vitexin and isovitexin were purchased from Sigma-Aldrich (St. Louis, 3.2.1. Soxhlet Extraction

MBC was extracted by Soxhlet extraction according to Weggler et al. [36]. Briefly, 8 g of MBC was placed in an extraction thimble. The thimble was then placed in a Soxhlet extractor and 400 mL of 95% ethanol was used as a solvent. The extraction was carried out for 6 h at 80 ◦C. The extract was collected in a Duran bottle and kept in a dark place at 4 ◦C. The extract was centrifuged at 7000 rpm, room temperature for 10 min. The supernatant was collected in a Duran bottle and kept in a dark place at 4 ◦C until further analysis.

pliers.

*3.2. Extraction Procedures*  3.2.1. Soxhlet Extraction

#### 3.2.2. Pressurized Liquid Extraction (PLE) at Laboratory Scale 3.2.2. Pressurized Liquid Extraction (PLE) at Laboratory Scale

*Molecules* **2022**, *27*, x FOR PEER REVIEW 10 of 15

PLE unit was assembled in the Chemical Engineering Laboratory, Department of Chemical Engineering, Faculty of Engineering, Kasetsart University, Bangkok, Thailand (Figure 7). The solvent was pumped by a preparative pump (Fluid Management System, Kalamazoo, MI, USA) into the extraction cell. The extraction cell was placed between an electrical heating system at the desired temperature until the required pressure was obtained. All connections within the system were made using stainless steel tubes. PLE unit was assembled in the Chemical Engineering Laboratory, Department of Chemical Engineering, Faculty of Engineering, Kasetsart University, Bangkok, Thailand (Figure 7). The solvent was pumped by a preparative pump (Fluid Management System, Kalamazoo, MI, USA) into the extraction cell. The extraction cell was placed between an electrical heating system at the desired temperature until the required pressure was obtained. All connections within the system were made using stainless steel tubes.

MO, USA). All other chemicals were of analytical grade and obtained from reputable sup-

MBC was extracted by Soxhlet extraction according to Weggler et al. [36]. Briefly, 8 g of MBC was placed in an extraction thimble. The thimble was then placed in a Soxhlet extractor and 400 mL of 95% ethanol was used as a solvent. The extraction was carried out for 6 h at 80 °C. The extract was collected in a Duran bottle and kept in a dark place at 4 °C. The extract was centrifuged at 7000 rpm, room temperature for 10 min. The supernatant was collected in a Duran bottle and kept in a dark place at 4 °C until further analysis.

**Figure 7.** Pressurized liquid extraction set-up. **Figure 7.** Pressurized liquid extraction set-up.

MBC was placed in the 400 mL extraction cell (OD 60.33 mm, length 28 mm, stainless steel 316) containing a metal filter at the top and bottom of the extraction cell. The cell containing the sample was filled with extraction solvent, heated and then pressurized. The sample was placed in the heating system for 10 min at desired temperature and pressure. After 10 min, the relief valve was carefully opened to lower pressure to atmospheric pressure. After temperature was below 70 °C, the cell was then exhaustively purged with 350 mL 50% ethanol to ensure that no residual extract solution was left in the extraction cell. The MBC extract was collected in a Duran bottle and kept in a dark place at 4 °C. The MBC extract was centrifuged at 7000 rpm, room temperature for 10 min. The supernatant MBC was placed in the 400 mL extraction cell (OD 60.33 mm, length 28 mm, stainless steel 316) containing a metal filter at the top and bottom of the extraction cell. The cell containing the sample was filled with extraction solvent, heated and then pressurized. The sample was placed in the heating system for 10 min at desired temperature and pressure. After 10 min, the relief valve was carefully opened to lower pressure to atmospheric pressure. After temperature was below 70 ◦C, the cell was then exhaustively purged with 350 mL 50% ethanol to ensure that no residual extract solution was left in the extraction cell. The MBC extract was collected in a Duran bottle and kept in a dark place at 4 ◦C. The MBC extract was centrifuged at 7000 rpm, room temperature for 10 min. The supernatant was collected in a Duran bottle and kept in a dark place at 4 ◦C until further analysis.

was collected in a Duran bottle and kept in a dark place at 4 °C until further analysis.

#### 3.2.3. Pressurized Liquid Extraction (PLE) at Pilot Scale

In order to scale up the extraction process, the pilot-scale PLE was used. This PLE was equipped with a 10L extraction cell (OD 143.2 mm, length 1200 mm, stainless steel 316). The optimum conditions for extraction were repeated at the pilot scale. The difference was that 2 kg of MBC was used in the extraction cell. After 10 min extraction, the MBC extract was collected in a plastic bottle and kept in a dark place at 4 ◦C. The MBC extract was centrifuged at 7000 rpm, room temperature for 10 min. The supernatant was collected and evaporated to remove the solvent at 60 ◦C, then the sample was freeze-dried and kept in an aluminum foil bag at 4 ◦C for further analysis.

#### *3.3. Extract Characterization*

#### 3.3.1. Determination of Total Phenolic Content (TPC)

TPC was analyzed according to Herald et al. [37]. Briefly, 75 µL of distilled water was added to each well of the 96-well plate, followed by 25 µL of sample and 25 µL of

folin ciocalteu reagent (diluted 1:1 (*v*/*v*) with distilled water). After the solutions had been mixed and left for 6 min, then 100 µL of 75 g/L Na2CO3 was added to each well. The solutions were mixed again, and the plates were covered and left at room temperature in a dark place for 90 min. After shaking for 60 sec, the absorbance was measured at 765 nm using a microplate reader (TECAN, Infinite 200 Pro, Manndenorf, Switzerland). Gallic acid was used as a reference standard and the results were expressed as mg GAE/g MBC (d.w.).

#### 3.3.2. Determination of Total Flavonoid Content (TFC)

TFC was analyzed according to Herald et al. [37]. Briefly, 100 µL of distilled water was added to each well of the 96-well plate, followed by 10 µL of 50 g/L NaNO2 and 25 µL of sample solution. After 5 min incubation, 15 µL of 100 g/L AlCl3 was added to the mixture. Six min later, 50 µL of 1 M NaOH and 50 µL of distilled water were added. The plate was shaken for 30 sec in the plate reader before measuring absorbance at 510 nm using a microplate reader (TECAN, Infinite 200 Pro, Manndenorf, Switzerland). Catechin was used as a reference standard and the results were expressed as mg CE/g MBC (d.w.).

#### 3.3.3. Determination of ABTS Radical Scavenging Activity (ABTS)

The 2,20 -azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical scavenging activity was determined according to Indracanti et al. [38]. Briefly, 10 µL of sample were mixed with 190 µL of ABTS solution (2.45 mM potassium persulfate solution and 7 mM ABTS). The mixture was incubated at room temperature for 6 min in a dark place, then the absorbance was measured at 734 nm using a microplate reader (TECAN, Infinite 200 Pro, Manndenorf, Switzerland). Trolox was used as a reference standard and the results were expressed as mg TE/g MBC (d.w.).

#### *3.4. Identification of Major Compounds in the Extract*

The major compounds in the extract were determined using high-performance liquid chromatography (HPLC) according to our previous method [39]. The extract was dissolved with deionized water and subjected to HPLC equipped with a diode array detector (Waters 600, Milford, MA, USA). An analytical column (C18) (4.6 × 250 mm, Inertsil ODS-3, 5 µm, GL Sciences, Tokyo, Japan) was used and kept at 30 ◦C while using. The spectra from 210–600 nm were recorded and UV absorbance at 337 nm was used to monitor flavonoids. The injection volume was 10 µL at a flow rate of 1 mL/min. Elution was done using two solvent gradients: solvent A (1% acetic acid in deionized water) and solvent B (1% acetic acid in methanol). The gradient program started with 10–35% B (10 min), 35–42% B (15 min), 42–75% B (10 min), 75% B (5 min), 75–10% B (5 min) and 10% B (5 min). Vitexin and isovitexin at the concentrations 20, 40, 60, 80 and 100 mg/kg were used as external standards to make standard curves and to determine the concentration of vitexin and isovitexin in the extract.

#### *3.5. Experimental Design and Statistical Analysis*

The effect of three independent variables: temperature (X1; 80–160 ◦C), pressure (X2; 1200–1800 psi) and ethanol concentration (X3; 5–95%) on the response variables: TPC (Y1), TFC (Y2) and ABTS (Y3) was evaluated using a three-factor-three-level Box-Behnken design (BBD). The three coded levels of temperature, pressure and ethanol concentration were incorporated into the design and were analyzed in 15 combinations (Table 1). The central point of the design was repeated three times to estimate the repeatability of the method. For each combination, three dependent variables were determined. A second-order polynomial model was used for fitting data and predicting the responses as Equation (4):

$$Y = b\_0 + b\_1 X\_1 + b\_2 X\_2 + b\_3 X\_3 + b\_{11} X\_1^2 + b\_{22} X\_2^2 + b\_{33} X\_3^2 + b\_{12} X\_1 X\_2 + b\_{13} X\_1 X\_3 + b\_{23} X\_2 X\_3 \tag{4}$$

where, *Y* is the response variable; *b*0, *b*1, *b*2, *b*3, *b*11, *b*22, . . . are regression coefficients and *X*1, *X*2 and *X*3 are uncoded values for temperature, pressure and ethanol concentration, respectively. An analysis of variance (ANOVA) was performed at a 95% confidence level to evaluate the predicted model on the response variables and assess the effect of each

factor. In addition, the regression coefficient (R2 ), the *p*-value of the regression model, the *p*-value of the lack of fit were used to determine the fitness of the regression model. The graphical and numerical optimization procedures were used to determine the optimum PLE condition. Three-dimensional (3D) response surface plots were applied for the interactions. Steepest ascend approach was also applied to determine the optimum PLE conditions to provide the highest TPC, TFC and ABTS. In order to assess the adequacy of the constructed model, the actual values were compared with the predicted values and the percentage of the residual standard error (RSE) was calculated for each response [40]. All of the treatments were carried out in triplicates. The experimental design matrix, data analysis, regression coefficients and numerical optimization were analyzed using Minitab statistical software (Trial version 16.1 Minitab Inc., State College, PA, USA) and 3D graphs were provided using Statistica 10.0 (StatSoft Inc., Tulsa, OK, USA). Differences between groups of data were assessed by student t-test. Results were expressed as mean ± standard deviation.

#### **4. Conclusions**

The present study showed the possibility of obtaining phenolics, flavonoids with antioxidant activity from MBC using PLE. The effects of temperature, pressure and ethanol concentration were studied to maximize TPC, TFC and ABTS of MBC extract. The results showed that TPC, TFC and ABTS were most affected by ethanol concentration. TPC and TFC were also affected by temperature, while pressure did not affect TPC and ABTS.  The optimum conditions obtained from numerical optimization and steepest ascend approach, were temperature of 160 ◦C, pressure of 1300 psi and 50% ethanol. The optimum conditions were performed in laboratory-scale and pilot-scale PLE. No significant decrease was observed in pilot-scale PLE. The profile of MBC extract from laboratory-scale and pilot-scale PLE also remained. This illustrated that the optimum conditions can be transferred to an industrial scale. However, industrial costs such as energy and materials are needed to be investigated. In conclusion, the PLE process appeared to be a potential technique in extracting phenolics, flavonoids with antioxidant activity from mung bean seed coat.

**Author Contributions:** Conceptualization, M.S., U.K., K.R., S.S.-T.; Data curation, S.S.-T.; Formal analysis, B.S. and S.S.-T.; Funding acquisition, S.S.-T.; Investigation, B.S.; Methodology, M.S., U.K., K.R. and S.S.-T.; Project administration, S.S.-T.; Resources, M.S.; Validation, M.S., U.K., K.R. and S.S.-T.; Visualization, B.S., M.S., U.K., K.R. and S.S.-T.; Writing—original draft, B.S.; Writing—review & editing, S.S.-T. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work is financially supported by Office of National Higher Education Science Research and Innovation Policy Council through Program Management Unit for Competitiveness (Grant number C10F630076).

**Data Availability Statement:** The dataset generated for this research are available on request to the corresponding author.

**Acknowledgments:** The authors acknowledge Department of Food Science and Technology, the Faculty of Agro-Industry and Department of Chemical Engineering, Faculty of Engineering, Kasetsart University for providing laboratory facilities and research equipment. We also thank Ravipim Chaveesuk from the Department of Agro-Industrial Technology, Faculty of Agro-Industry, Kasetsart University for giving suggestions about the optimization method.

**Conflicts of Interest:** The authors declare no conflict of interest.

**Sample Availability:** Samples of the compounds are not available from the authors.


**1. Introduction**

The motor is widely used in ship, municipal, electric power, port handling, and other fields, with broad development prospects and considerable market capacity. In recent years, with the upgrading of power system requirements, the traditional motor with low efficiency and low power to weight ratio has been unable to meet the market demand, which has prompted the need for the research and development of new oil-cooled motors with high efficiency, high power density, low vibration and noise, and strong overload capacity [1,2]. During operation, the motor will produce immense heat, which will reduce the operating efficiency of the motor. In order to ensure the efficient operation of the motor, a heat exchanger is often used to enhance heat transfer and cool the motor. Fluid mediums in heat exchangers are diverse, and the oil phase is widely used because of its superior heat exchange effect, low cost, and long service life [3]. However, when the motor is compact, there will still be areas with a high temperature in the motor after the oil phase heat exchange [4,5]. For instance, in an oil-cooled motor, the temperature at the

end of the coil outlet is higher than that of the iron core section, so further improving the heat transfer effect at the end of the coil outlet can enhance the performance of the whole motor [6–8]. Three ways are acknowledged to enhance heat transfer: increasing heat transfer area, increasing average temperature difference, and increasing the heat transfer coefficient [9]. According to the characteristics of coil oil cooling, the feasible heat transfer enhancement method of the coil end can be analyzed, and the vortex generator can be used to increase the heat transfer coefficient [10].

As a passive heat transfer enhancement technology, the vortex generator can produce vortexes to effectively improve the heat transfer rate of the heat transfer system. Because of its economy and convenience, it has attracted extensive attention in recent years. When it was initially proposed, it was mainly used in the field of aerodynamics [11]. Later, Johnson and Joubert [12] studied the heat transfer enhancement effect of the delta wing vortex generator on the air of the heat exchanger, which initiated the application of the vortex generator in the field of heat exchangers. Chai et al. [13] investigated the improvement of heat exchanger performance via the installation of vortex generators, based on the mechanism of the longitudinal vortex destroying the growth of the boundary layer, increasing the turbulence intensity, and producing secondary fluid flow on the heat transfer surface. There are various structures of vortex generators, such as rectangular wing, triangular wing, trapezoidal wing, cylindrical trapezoidal wing, cylindrical triangular wing, cylindrical rectangular wing, and so on [14–17]. Promvonge et al. [18] studied the influence of the vortex generator, combined with a fin and airfoil, on the heat transfer and drag characteristics of the flow passage under the condition of uniform heat flow boundary. The results showed that the heat transfer efficiency and friction loss of the fluid with the fin and airfoil vortex generator were higher than those with a smooth channel. Chen et al. [19] optimized the aspect ratio of the fluid channel and the height of the vortex generator. The results showed that, in a fluid channel with a large aspect ratio, the heat transfer performance could be enhanced while reducing the pressure loss. So far, many scholars have done a lot of research on the size and attack angle of vortex generators [20–25]. Wijayanta et al. [26] used the *k* − *ε* turbulence model to explore the heat transfer and pressure drop characteristics of vortex generators with various attack angles, and found that the maximum increases in Nusselt number and friction coefficient are 269% and 10.1 times higher than those of smooth tubes, respectively. Zhang et al. [27,28] explored the best combination of length, width, and longitudinal distance of the vortex generator, and its total efficiency was 7.2% higher than that without the vortex generator. Ebrahimi et al. [29,30] studied the heat transfer and fluid characteristics in the laminar flow channel installed with the vortex generator. It was noted that the channel of the vortex generator had higher efficiency, the friction coefficient increased by 2–25%, and the Nusselt number increased by 4–30%.

So far, the application of the vortex generator to enhance heat transfer has mainly been used in the heat exchanger, and air is primarily used as the fluid medium. In addition, the current research on the heat transfer of the vortex generator mainly focuses on the attack angle, size, and shape. However, only a few studies roughly explore the influence of the longitudinal distribution mode of the vortex generator on heat transfer, while there is a lack of detailed and orderly analysis and research on the specific longitudinal and transverse distribution mode of the vortex generator. Therefore, it is of great significance to study the arrangement of the vortex generator on the coil in the motor with oil as the fluid medium. With the help of computational fluid dynamics (CFD) software, the effect of different distribution types of the vortex generator on the heat transfer effect can be simulated, and the temperature change at the end of the coil and the pressure loss before and after the installation of the vortex generator can be analyzed. At the same time, the best distribution type can be obtained, and the mechanism of heat transfer enhancement by turbulence at the end of the coil can be revealed. Thus, through these explorations, this paper can provide a feasible idea for the design and application of the motor in industrial manufacturing.


*6.1.1 Intravenous hydration*

Adequate hydration for patients performing CM-enhanced imaging studies was suggested approximately 40years ago [30]. The beneficial effects of hydration were initially reported in the early 1980s by studies that compared outcomes of hydrated patients with historical controls [60, 61]. These reports were supported by the first RCT in 1994, concluding that patients with chronic renal impairment benefit better from intravenous (0.45%) saline administration (for 12h before and 12h after angiography) in comparison with saline plus mannitol or furosemide [62]. Since then, multiple RCTs have assured the benefit of intravenous normal saline (0.9%) hydration that is started 12h preceding to 12h after CM injection [63–65] in the prevention against CIN over 0.45% saline [65] or a fluid bolus (300mL) during CM administration only [66]. The rate of infusion was reported as 1mL/kg/h [67]. CM safety committee endorse a regime of intravenous infusion of 1.0–1.5mL/kg/h for at least 6h before and after CM administration [68].