Current Methods for the Extraction and Analysis of Isothiocyanates and Indoles in Cruciferous Vegetables

: Cruciferous vegetables are characterized by the presence of sulfur-containing secondary plant metabolites known as glucosinolates (GLS). The consumption of cruciferous vegetables such as broccoli, cabbage, rocket salad, and cauliﬂower has been related to the prevention of non-communicable diseases. Their beneﬁcial effects are attributed to the enzymatic degradation products of GLS, e.g., isothiocyanates and indoles. Owing to these properties, there has been a shift in the last few years towards the research of these compounds and a wide range of methods for their extraction and analytical determination have been developed. The aim of this review is to present the sample preparation and extraction procedures of isothiocyanates and indoles from cruciferous vegetables and the analytical methods for their determination. The majority of the references that have been reviewed are from the last decade. Although efforts towards the application of eco-friendly non-conventional extraction methods have been made, the use of conventional solvent extraction is mainly applied. The major analytical techniques employed for the qualitative and quantitative analysis of isothiocyanates and indoles are high-performance liquid chromatography and gas chromatography coupled with or without mass spectrometry detection. Nevertheless, the analytical determination of isothiocyanates presents several problems due to their instability and the absence of chromophores, making the simultaneous determination of isothiocyanates and indoles a challenging task.


Introduction
Cruciferous vegetables are members of the Brassicaceae (Cruciferae) family and include vegetables such as broccoli, cauliflower, Chinese cabbage, radish, arugula, cabbage, Brussels sprouts, turnip, watercress, kale, horseradish, garden cress, and wasabi. They are abundant in bioactive compounds, e.g., flavonoids, phenolic acids, carotenoids, terpenoids, phytosterols, and glucosinolates (GLS) [1]. Cruciferous vegetables can be considered functional foods, since their consumption can improve normal body function and can help prevent the development of cancer, cardiovascular disease, diabetes, and chronic inflammation [2][3][4][5][6]. The series of S-and N-secondary metabolites, known as GLS, underpin this beneficial effect. When GLS undergo enzymatic hydrolysis from the endogenous enzyme myrosinase, there is a release of aglucon products, which have a positive impact on human health [7]. Depending on the pH values, temperature, and other factors, the predominant aglucon products are isothiocyanates (ITCs) and indoles [8]. These compounds also contribute highly to the defense mechanism system of plants against herbivores and pathogens [9][10][11].
According to recent epidemiological research, certain types of cancer are less likely to develop when the dietary intake of cruciferous vegetables is high, providing support for the classification of these vegetables as functional foods. [48][49][50][51][52]. Consequently, the market has been inundated with dietary supplements that contain different extracts or compounds derived from cruciferous vegetables. GLS, and the products of their enzymatic hydrolysis are the primary source of the positive health effects of cruciferous vegetables [53], while polyphenols and triterpenes are beneficial to health as well [54,55]. The action against cancer, oxidants, inflammation, and cardiac disease that has been attributed to cruciferous vegetables can be also attributed to the synergistic activity of the above-mentioned phytochemicals [55][56][57].
During the chewing, cutting, or wounding of cruciferous vegetables, endogenous myrosinase is released and catalyzes the hydrolysis of GLS to glucose with an intermediate lack of stability (Figure 1), which is immediately transformed into different compounds such as isothiocyanates, thiocyanates, nitriles, epithionitriles, and oxazolidine-2-thiones [67]. The ITCs are usually formed when enzymatic hydrolysis occurs at a neutral pH. The generated ITCs are cyclized to oxazolidine-2-thiones if the C-2 of the GLS side chain contains a hydroxyl group. Nitriles are formed in vitro when the pH level is low and Fe 2+ ions are present [68]. The nitrile formation in vivo occurs when protein factors such as the epithiospecifier protein (ESP), thiocyanate-forming protein (TFP), and epithiospecifier modifier protein (EMP) are active [69,70]. It is likely that the epithiospecifier modifier (ESM1) interferes with ESP and directs the hydrolysis towards the production of ITCs [71]. Glucosinolates undergo enzymatic hydrolysis to epithionitriles when ESP and TFP are present. Thiocyanates are formed from the hydrolysis of benzyl-, allyl-, and 4-methylsulfinylbutyl GLS [72].
Unlike other GLS hydrolysis products, those with an indole ring are distinct because the ITCs formed at a neutral pH are unstable and ultimately undergo conversion to indol-methanols, ascorbic acid conjugates, and oligomeric mixtures [67,70,73]. When glucobrassicin is hydrolyzed (Figure 2), indole-3-acetonitrile and I3C are formed as a result of the fast reaction of the unstable isothiocyanate intermediate. The formation of indole-3-acetonitrile is promoted by the ESP protein in a way that is comparable to aliphatic GLS. Furthermore, the epithiospecifier modifier 1 gene (ESM1) [74,75] regulates indole-3-acetonitrile generation as well. Compared to indole-3-acetonitrile, I3C displays a greater chemical instability and yields products such as 3,3-diindindolylmethane, indole-3carboxaldehyde, and ascorbigen ( Figure 2) [14,73].
The content of these compounds is indicative as it is influenced by numerous factors including variety, geography, season, and environmental factors, e.g., the infestation of pathogenic microorganisms, soil fertility, and plant growth regulators [85]. In addition, another important factor to note is the sample preparation and extraction conditions. The enzymatic hydrolysis of GLS must be complete and under appropriate conditions conducive to the production of ITCs over the corresponding nitriles. This explains the discrepancies between studies regarding the SFN concentration in broccoli, which has been reported to be in the range of 0.14-370.3 mg/100 g fresh weight (Table 2). [84]

Conventional Extraction
The optimum extraction conditions must be chosen carefully, given that ITCs and indoles are the products of GLS enzymatic hydrolysis. Glucosinolates have to undergo complete hydrolysis to ITCs rather than other compounds (e.g., nitriles) that exhibit a potent genotoxic risk [86]. In conventional extraction (Table 3), fresh or lyophilized samples are subjected to homogenization in a blender, with the addition of water or a buffer solution [25,[87][88][89]. For Brassicaceae seeds, n-hexane is usually employed as the solvent for defatting purposes before the addition of water or a buffer. In this step, sonication for several minutes may be used [90]. To complete the hydrolysis of GLS, the mix is then allowed to autolyze at an ambient temperature or is subjected to incubation in a water bath at 35-45 • C for 1-4 h. A buffer solution is typically added instead of deionized water because it affords a constant neutral pH that is required to ensure that GLS hydrolysis yields ITCs [25,87,91,92]. Nevertheless, it has been argued that for SFN, water with a pH of 3-6 should be added to attain the maximum amount of this compound when glucoraphanin is hydrolyzed [19,93]. Once hydrolysis is completed, a suitable solvent is used for the extraction of ITCs and indoles. At this step, derivatization may be also applied [25,87,94]. Table 3 lists the organic solvents necessary for standard ITC and indole extraction, with the chlorinated solvents dichloromethane (CH 2 Cl 2 ) [42,43,78,93,[95][96][97] and chloroform (CHCl 3 ) [90] being preferred. Nevertheless, other solvents have been also proposed such as ethyl acetate (AcOEt) [98] acetone [96,99], and methyl t-butyl ether (MTBE) [19,96]. The general procedure is briefly described in Figure 3. In the case of analysis using gas chromatography with a flame ionization detector (GC-FID) or a mass spectrometer (GC-MS), CH 2 Cl 2 is mainly used for the extraction of the hydrolysis products of GLS (Table 4) [100][101][102][103][104][105][106]. Additionally, Clevenger hydrodistillation has been also employed to extract the essential oil of cruciferous vegetables in which a great variety of ITCs, nitriles, epithionitriles, and oxazolidine-2-thiones have been identified [107,108].         Glucosinolates can be subjected to enzymatic hydrolysis indirectly by an initial extraction of intact GLS. In this approach, the plant material is pulverized and the myrosinase enzyme is deactivated with liquid nitrogen or boiling water. Methanol or aqueous methanol is then employed to extract the intact GLS, which are desulfated on a diethylaminoethyl (DEAE) Sephadex column, with the addition of an artificial or natural myrosinase enzyme. This technique is advantageous primarily because of the possibility to adjust the column conditions, including pH (acidic, basic, or neutral) and temperature (low, room temperature, or high) depending on the type of hydrolysis product that is targeted to be obtained [59].

Non-Conventional Extraction
The conventional extraction methods are simple, especially for lab-scale processes; however, due to the large quantities of solvents and the long extraction times required, they may be replaced or enhanced by non-conventional extraction techniques such as High-Pressure Processing (HPP), High Voltage Electrical Discharges (HVED), Ultrasound-Assisted Extraction (UAE), Microwave-Assisted Extraction (MAE), Supercritical Fluid Extraction (SFE), and Pressurized Fluid Extraction (PFE) ( Table 5). Non-conventional extraction may be preferable, as it manages to reduce the extraction temperature and time, and therefore the consumption of the solvent, but at the same time it can achieve a higher efficiency and an improvement in the quality of the extracted compounds compared to the conventional methods. However, in the case of ITCs and indoles, the research for non-conventional extraction procedures is still ongoing and more studies are required for the establishment of these techniques as an alternative to conventional extraction.
The technique of HPP, (also known as High Hydrostatic Pressure (HHP)), is a nonthermal technology that utilizes liquids for a pressure transmission medium. Compared to thermal processing, HPP is more effective in the preservation of the flavor, texture, nutrients, and appearance of food products [109]. Applications of HPP in foods include the inactivation of microorganisms and enzymes and the increase in the shelf life of the products. Furthermore, the use of chemical preservatives is reduced [110]. After HPP on broccoli sprouts with 600 MPa, 85% of GLS were converted to ITCs [111] which suggested the inactivation of the ESP protein. Moreover, the glucosinolate-myrosinase system in broccoli sprouts was not negatively affected. Two studies [110,112] have reported that the application of HHP at 400 MPa in red cabbage and broccoli generated the highest amounts of ITCs. Moreover, it was found that the activity of myrosinase increased after processing, indicating that the increase in ITCs content is related to the stimulation of myrosinase activity [110].  [118] High voltage electrical discharges are a non-thermal technique based on the electrical breakdown in water, while the air bubbles in water may accelerate the process. The HVED process initiates from the avalanche of electrons that is caused by the intense electrical field and constitutes the starting point of streamer propagation. The application of HVED improves the extraction yield of compounds through the destruction of the cellular structure and the enhancement of the mass transfer [119,120]. Barba et al. [114] employed HVED technology for the extraction of protein, polyphenols, and ITCs from rapeseeds and rapeseed press-cake. Different energy inputs (0-400 kJ/kg) were tested, and the researchers concluded that the optimal treatment energy input was 80 or 240 kJ/kg, while when this was exceeded, the yield decreased. It is important to note that the nature of the sample influences the treatment efficiency to a great extent; thus, for each starting material, an optimization of the HVED process is required.
Ultrasound-assisted extraction (UAE) facilitates cell disruption and solvent penetration in the samples. This technique manages to enhance the extraction yield of compounds owing to the cavitation phenomena that are created by the ultrasound pressure waves in the solvent used for the extraction. Although the use of a solvent is required, the amount of solvent and energy consumption are reduced [121]. Besides, eco-friendly solvents such as natural deep eutectic solvents may be used with UAE to obtain high-quality extracts with increased yields [122]. Diatuo et al. [118] applied the UAE at a frequency of 24 kHz in order to obtain extracts from cauliflower by-products that are rich in ITCs. The researchers developed a powder from the plant material, which was subjected to UAE. From the three solvents (distilled water, 70% methanol, and 80% acetonitrile) that were tested under UAE, the distilled water was the most efficient for the extraction of ITCs. The MAE is an alternative extraction procedure that has the potential to reduce the extraction time and the amount of solvent consumption while simultaneously increasing the extraction yield. Microwaves are electromagnetic waves with a frequency of 300 MHz to 300 GHz. The process is based on the ionic conduction and dipole movement which are responsible for the warming of substances. The choice of the appropriate solvent in the MAE process is crucial. Solvents transparent to microwaves such as hexane do not heat up when subjected to the microwaves, while microwave absorbing solvents, such as ethanol or water, are more suitable for the MAE extraction process [123]. Tanongkankit et al. [115] studied the MAE of SFN from white cabbage in comparison to the conventional solvent extraction method. Different solvent types (CH 2 Cl 2 or water), microwave power, and extraction time were tested. MAE led to a higher extraction yield of SFN with a reduced extraction time compared to the classic solvent extraction method while both solvents, CH 2 Cl 2 , and water, provided similar yields.
In supercritical extraction, a supercritical fluid solvent, such as carbon dioxide, is used. The advantage of this technique is the avoidance of the use of organic solvents, which eventually reduces environmental concerns. The surface tension is not present in supercritical fluids and thus they have the potential to penetrate into small pores which are inaccessible to liquid solvents. This technique is very effective for the extraction of volatile compounds [124]. Li et al. [116] applied SFE for the extraction of allyl ITC from wasabi. The researchers tested different ranges of pressure (15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25) and temperature (35-55 • C) and found that the extraction yield was increased with the increase in pressure and the decrease in temperature. The highest extraction yield was obtained at 25 MPa and 35 • C, while the most significant parameters for the SFE process were pressure, temperature, and moisture content. Wu et al. [117] extracted allyl-ITC from horseradish using water extraction, hydrodistillation, and supercritical fluid extraction with carbon dioxide (SFE-CO 2 ). Hydrodistillation and SFE-CO 2 provided similar extraction yields, whereas the conventional water extraction yield was significantly lower. Rafińska et al. [118] used UAE, SFE, and Accelerated Solvent Extraction (ASE) for the extraction of bioactive compounds from garden cress. It was found that the SFE method applied in dried garden cress sprouts was more efficient in the extraction of ITCs compared to ASE and UAE. The main ITC was found to be benzyl-ITC.
Rodrigues et al. [83] reported the use of Pressurized Fluid Extraction (PFE) with supercritical CO 2 for the recovery of ITCs and phenolic compounds from watercress. Pressurized fluid extraction is also an advantageous alternative method to the traditional extraction processes as it requires less solvent and a shorter extraction time. The results showed that the application of the PFE process was highly selective for the isolation of ITCs from watercress and the main isothiocyanate that was recovered was phenethyl-ITC. Moreover, an alternative extraction method for ITCs has been proposed using lactic acid bacteria (LAB). Jaiswal et al. [125] performed a fermentation-assisted extraction of ITCs from York cabbage using LAB. The researchers concluded that the employment of LAB facilitated the hydrolysis of GLS to the formation of ITCs. The factors with the highest influence in obtaining a high yield of ITCs were the fermentation time, the solid-to-liquid ratio, and the agitation rate. Yuan et al. [126] developed an ultrasound-assisted dispersivefilter extraction technique based on poly(deep eutectic solvent)-graphene oxide (PDES-GO) adsorbent for the isolation of I3C from broccoli. For the preparation of the PDES-GO, the researchers used a mixture of choline chloride and methacrylic acid in a 1:2 molar ratio in order to modify the graphene oxide surface. The specific method proved to be rapid, accurate, and low-cost. The efficiency of aqueous micellar systems (AMSs) was explored by Coscueta et al. [127] for the extraction of phenylethyl-ITC from watercress by-products. For this purpose, the surfactants Genapol X-080 (Clariant, Louisville, KY, USA) and Tergitol 15-S-7 (Sigma-Aldrich, Burlington, MA, USA) were utilized as it is known that they are effective in interacting with the low polar molecules of plant tissues. Liquid-liquid extraction was also performed using n-hexane and MeCN/CHCl 3 . The highest content of phenylethyl-ITC was obtained using n-hexane; however, no statistically significant differences were observed between the extraction systems with Tergitol 15-S-7 (Sigma-Aldrich, Burlington, MA, USA) and MeCN/CHCl 3 compared to n-hexane, while the use of Genapol X-080 (Clariant, Louisville, KY, USA) afforded a slightly lower yield of phenylethyl-ITC.

Methods of Analysis
The analysis of ITCs and indoles has been performed using a great variety of chromatographic, spectrophotometric, and other techniques. However, the techniques that have been commonly employed are LC and GC, with or without the use of mass spectrometry detection (Tables 3 and 4). Furthermore, prior to the analysis of ITCs, derivatization may be applied owing to the high volatility and instability of these compounds and the absence of chromophore groups.

UV-Vis Spectrophotometry
Spectrophotometry is defined as the measurement of the absorption or reflectance properties of a substance as a function of the wavelength. The key advantages of the UV-Vis spectrophotometry methods are the low analysis time and the reduced labor consumption, while they simultaneously exhibit a high sensitivity and precision [128]. A UV-Vis spectrophotometric method for the determination of total ITCs content has been developed [44,129]. This method is indirect, and it is based on the quantitative reaction of ITCs with an excess of 1,2-benzenedithiol ( Figure 4). The product of the cyclocondensation reaction, 1,3-benzodithiole-2-thione, is determined spectrophotometrically at 365 nm. The reaction of 1,2-benzenedithiol with ITCs is highly selective; thus, other enzymatic hydrolysis products of GLS, such as thiocyanates, do not react. Although the sensitivity of the method is very low (1 nmol), it can be considered a time-consuming method of analysis due to the pre-required stage of derivatization. However, this cyclocondensation reaction has been utilized in several studies for the determination of total ITCs with High-Performance Liquid Chromatography (HPLC) [21,130,131] or GC-MS [132].

Fourier-Transform Infrared Spectroscopy (FT-IR)
Another method that has been reported in the literature for the determination of the total ITCs in cruciferous vegetables is the Attenuated Total Reflectance Fourier-Transform Infrared Spectroscopy (ATR-FTIR) method in combination with the partial least-squares (PLS) algorithm [45]. The sampling technique of ATR is advantageous as it enables the direct examination of samples without the requirement of a derivatization step [133]. The spectral range 2150-2020 cm −1 was used for the quantification of total ITCs in broccoli and the results showed that it is an equivalent method in terms of reproducibility and accuracy to the UV-Vis spectrophotometric method [44]. This method seems to be simple and rapid and constitutes a useful alternative for the determination of total ITCs in cruciferous vegetables.

Paper Chromatography (PC)
Paper chromatography is the oldest method used to analyze GLS hydrolysis products [134,135]. Ammonia reacts with ITCs and produces thiourea-type derivatives which are separated by PC. The components that are hydrophobic are analyzed using water saturated CHCl 3 , while the hydrophilic substances are analyzed with a mixture of water, butanol, and toluene as the developing solvent system. However, PC exhibits low reproducibility for the analysis of ITCs; thus, alternative chromatographic methods have been developed for their analysis [136].

Gas Chromatography (GC)
Gas chromatography is an excellent separation technique for the identification and determination of volatile organic substances. This technique in combination with a Flame Ionization Detector (FID) has been used for the determination of SFN [29,[138][139][140] and other ITCs [100][101][102][103]108] in a great variety of cruciferous vegetables. However, the thermal degradation of SFN to 3-butenyl ITC has been observed under split/splitless conditions [31]. Besides, the thermal degradation studies that have been performed for ITCs show that these compounds are thermolabile [32,141,142]. Therefore, LC methods are often preferred for their analysis.

High-Performance Liquid Chromatography (HPLC)
For the analysis of ITCs and certain indoles, HPLC coupled with a UV detector has been proposed by various researchers [19,25,78,88,92,93,[95][96][97]99]. For the determination of I3C, indole-3-acetonitrile, and 3,3 -diindolylmethane, HPLC with a Fluorescence Detector (FLD) is usually employed [87,99,143]. The determination is performed at excitation at 280-285 nm, and emission at 340 nm. In the case of SFN, the UV detection has been performed at 202 nm [95], 205 nm [93], and 196 nm [19]. However, the lack of strong UV chromophores in several ITCs, and especially SFN, creates analytical problems. For this reason, Nakagawa et al. [144] and Liang et al. [20] proposed the use of an Evaporative Light-Scattering Detector (ELSD) for analyzing SFN, achieving a higher sensitivity compared to a UV detector. Another problem that also arises in the HPLC analysis of ITCs is their precipitation when aqueous mobile phases are used. This impacts the accuracy of determinations and leads to operational problems [22].

High Speed Counter Current Chromatography (HSCCC)
High speed counter current chromatography is a simple and low-cost method which is based on the distribution of substances between two immiscible liquids. This technique enables the recovery and purification of samples; thus, it can be used for the isolation of natural products from plant extracts [145,146]. Liang et al., applied HSCCC in broccoli seed meal in order to purify and recover SFN [147]. The two-phase liquid system used in this study was n-hexane/ethyl acetate/methanol/water (1:5:1:5, v/v/v/v) which was selected based on the partition coefficient of SFN. The results of HSCCC were compared with the preparative HPLC method. Sulforaphane was obtained with a slightly higher purity (97%) via HSCCC compared to preparative HPLC (95%), while the recovery obtained from HSCCC was higher (98.5%) than the preparative HPLC method (87.4%). High speed counter current chromatography has been also employed for the purification of sulforaphene from radish seeds [148]. The two-phase solvent system used in this study was n-hexane-ethyl acetate-methanol-water (35:100:35:100, v/v/v/v). High purity sulforaphene was obtained (96.9%), while the recovery of the compound was 95.2%.

Supercritical Fluid Chromatography (SFC)
Supercritical fluid chromatography constitutes a variation of HPLC. The difference between HPLC and SFC lies in the fact that in SFC, a supercritical fluid is used instead of the liquid mobile phase. This technique is considered a good alternative to HPLC, as the properties of the supercritical fluid used in the mobile phase is between a liquid and a gas. Supercritical CO 2 is commonly employed owing to its low cost, the fact that it is safe to use in the food industry, and also because it has no negative impact on the environment [149]. Supercritical fluid chromatography has been used for the analysis of the hydrolysis products of indole GLS. Buskov et al. [150] used SFC for the determination of ascorbigens in the autolysates of broccoli, white cabbage, red cabbage, cauliflower, Brussels sprouts, and various Portuguese cabbages, while Buskov et al. [151] studied the degradation products of 4-hydroxybenzylglucosinolate. The study of the hydrolysis products of indol-3-ylmethyl-glucosinolates in broccoli heads by SFC has been also reported [152].  [94] and Pilipczuk [153] analyzed ITCs with Ultra-High-Performance Liquid Chromatography-Mass Spectrometry (UHPLC-MS), applying a derivatization method using N-acetyl-L-cysteine (NAC) (Figure 5), achieving very low detection limits (0.9-2.6 µM) [94]. High resolution mass spectrometry has been used for the qualitative and quantitative determination of ITCs and indoles. A mass spectrometry study of SFN and indole-3-carbinol has been performed by Kokotou et al. [42] using Ultraperformance Liquid Chromatography-High Resolution Mass Spectrometry/Mass Spectrometry (UPLC-HRMS/MS) avoiding a derivatization step. The researchers also developed a method for the simultaneous determination of compounds in broccoli extracts.

Other Techniques
Gonda et al., reported the development of a capillary electrophoresis (CE) method for the simultaneous quantification of sinigrin, gluconasturtiin, and allyl-ITC [162]. The ITCs are determined after the myrosinase hydrolysis of GLS and derivatization of ITCs in-vial, utilizing mercaptoacetic acid ( Figure 6). The analysis was performed in extracts of cruciferous vegetables such as Brussels sprouts, radish, horseradish, and watercress. This method has the advantage that it can be used to determine both GLS and ITCs, and it also requires a minimum sample size.

Conclusions
Since ITCs and indoles are products of enzymatic hydrolysis, the sample preparation procedure is considered crucial for their determination. The hydrolysis of glucosinolates by myrosinase should be complete and the conditions must be in favor of producing ITCs and indoles, rather than other compounds with an increased toxicity. Following the sample preparation, the extraction solvents that are commonly used are chlorinated. The toxicity of the extraction solvents poses limitations in the use of extracts in the development of food products enriched with these health-promoting compounds. Considering the solvent toxicity, future research should be focused on the employment of eco-friendly solvents, such as natural deep eutectic solvents, which are still unexplored in this field. These efforts may be enhanced by the use of non-conventional techniques, such as ultrasound and microwaveassisted extraction. Further research on the development of non-conventional techniques is required towards the control and optimization of the processing conditions, as the treatment efficiency may be affected by the nature of the raw materials. The analytical determination of ITCs and indoles is commonly performed by liquid or gas chromatography. Nevertheless, the determination of ITCs presents analytical problems such as the precipitation in the liquid chromatography column and the weak DAD signal, while in gas chromatography certain ITCs can be degraded. These problems are attributed to the high volatility, instability, and the lack of chromophores which can be addressed by the use of derivatization reagents and the employment of hyphenated mass spectrometry methods. Due to the differences in the chemical properties between ITCs and indoles, the analytical methods for the simultaneous determination of these compounds in cruciferous vegetables are scarce, and therefore additional analytical methods must be developed for this purpose.

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