Content of Two Major Steroidal Glycoalkaloids in Tomato (Solanum lycopersicum cv. Micro-Tom) Mutant Lines at Different Ripening Stages

Esculeoside A and tomatine are two major steroidal alkaloids in tomato fruit (Solanum lycopersicum) that exhibit anti-inflammatory, anticancer, and anti-hyperlipidemia activities. Tomatine contained in immature tomato fruit is converted to esculeoside A as the fruit matures. To develop new tomato varieties based on the content analysis of functional secondary metabolites, 184 mutant lines were generated from the original cultivar (S. lycopersicum cv. Micro-Tom) by radiation breeding. Ultra-performance liquid chromatography coupled with evaporative light scattering detector was used to identify the mutant lines with good traits by analyzing tomatine and esculeoside A content. Compared with the original cultivar, candidates for highly functional cultivars with high esculeoside A content were identified in the mature fruit of the mutant lines. The mutant lines with low and high tomatine content at an immature stage were selected as edible cultivars due to toxicity reduction and as a source of tomatine with various pharmacological activities, respectively. During the process of ripening from green to red tomatoes, the rate of conversion of tomatine to esculeoside A was high in the green tomatoes with a low tomatine content, whereas green tomatoes with a high tomatine content exhibited a low conversion rate. Using methanol extracts prepared from unripe and ripe fruits of the original cultivar and its mutant lines and two major compounds, we examined their cytotoxicity against FaDu human hypopharynx squamous carcinoma cells. Only tomatine exhibited cytotoxicity with an IC50 value of 5.589 μM, whereas the other samples did not exhibit cytotoxicity. Therefore, radiation breeding represents a useful tool for developing new cultivars with high quality, and metabolite analysis is applicable for the rapid and objective selection of potential mutant lines.


Introduction
Tomato (Solanum lycopersicum L.; formerly Lycopersicon esculentum Mill.; Solanaceae) is the second most important vegetable crop after potato (Solanum tuberosum L.), with around 182.3 million tons of tomato fruit produced annually on 4.85 million hectares [1]. Tomato is a fruit containing a lot of health-promoting compounds and is widely consumed fresh and used in cooking. Therefore, several techniques are being used to breed excellent tomato varieties, focusing on improving tomato fruit quality as important as improving yield and stress tolerance [2].
Tomatine content decreases during the ripening process, and it is completely converted to esculeosides and lycoperosides once the tomato fruit turns red [5,8]. The degradation of tomatine involves several steps, including the hydroxylation of α-tomatine to hydroxytomatine, the acetylation of hydroxytomatine to acetoxy-hydroxytomatine, and the glycosylation of acetoxy-hydroxytomatine to esculeoside A, mediated by several enzymes related in the metabolic conversion of α-tomatine to esculeoside A [9][10][11][12]. Other studies have demonstrated that biosynthesis of esculeoside A from α-tomatine is associated with the ripening hormone, ethylene [13][14][15]. Tomatine is a solanum alkaloid similar to solanine, which is a well-known toxic glycoalkaloid in potatoes [16]; however, in vivo studies have demonstrated that oral tomatine administration is less toxic or non-toxic compared with other glycoalkaloids [17]. Tomatine has also been reported to exhibit several biological activities including anticancer [18,19], immunostimulatory [20], anti-inflammatory [21], and antibiotic [22] effects. Esculeoside A was isolated and identified as the major constituent in ripe red tomatoes by Fujiwara et al. [6,7]. Esculeoside A also has various pharmacological effects including anti-atherosclerosis [23], anti-hyaluronidase [24,25], anti-arthritis [26], and hypoglycemic [27] activity.
As part of our efforts to develop new varieties of S. lycopersicum cv. Micro-Tom with high functionality and improved quality, we generated mutant lines by radiation breeding with improved yields of phytochemicals. A comparative study of tomatine and esculeoside A content at two ripening stages of the original cultivar and the mutant lines was measured using ultra-performance liquid chromatography coupled with evaporative light scattering detector (UPLC-ELSD). In addition, the conversion rate of tomatine to esculeoside A was also determined at two ripening stages per line based on their concentrations in each sample.

Quantitative Analysis of Two Steroidal Glycoalkaloids in Micro-Tom Mutant Lines at Two Different Ripening Stages
To establish steroidal glycoalkaloid profiles based on the fruit maturation period between the original cultivar (S. lycopersicum cv. Micro-Tom) and its mutant lines, UPLC-ELSD analysis was conducted with two standards, tomatine and esculeoside A, along with methanol extracts prepared from the tomatoes. Esculeoside A was isolated from methanol extracts of commercially available red micro-tomatoes and tomatine was obtained from the Sigma Chemical Co. (St. Louis, MO, USA). The structures were identified by analysis of the 1 H and 13 C NMR spectra and by comparing the data with published values [6]. Chromatographic conditions were optimized with regard to the separation of the peaks for the two compounds by UPLC-ELSD, which may be applied as a quick, universal, and convenient analytical method for the evaluation of these compounds, which are UV transparent. A major peak for tomatine and esculeoside A were observed in the chromatograms of the unripe green tomato samples and the ripe red tomato samples, respectively. For quantitation, calibration curves of tomatine and esculeoside A were prepared using five concentrations (tomatine: 31.25, 62.50, 125.00, 250.00, and 500.00 µg/mL; esculeoside A: 15.63, 31.25, 62.50, 125.00, and 250.00 µg/mL). As shown Table 1, the polynomial-order 2 equations for tomatine and esculeoside A were y = 7.4421x 2 + 883.2x − 27,793 and y = 25.777x 2 + 1179.1x − 11,719, respectively. The calibration curves were curvilinear with high correlation coefficients (R 2 = 0.999 − 1.000) (Figure 1). This analytical method was used to measure tomatine and esculeoside A levels in the unripe green and ripe red fruits of the original cultivar and its mutant lines. As shown Table 1, the polynomial-order 2 equations for tomatine and esculeoside A were y = 7.4421x 2 + 883.2x − 27793 and y = 25.777x 2 + 1179.1x − 11719, respectively. The calibration curves were curvilinear with high correlation coefficients (R 2 = 0.999 − 1.000) ( Figure 1). This analytical method was used to measure tomatine and esculeoside A levels in the unripe green and ripe red fruits of the original cultivar and its mutant lines. To select target-specific mutant lines, we screened the mutants for high or low relative tomatine content in the immature fruit and high esculeoside A content in the mature fruit. The peak area values were measured by ELSD for the original cultivar and 184 mutant lines at two different ripening stages ( Figure S1). Tomatine has a dual aspect with anti-nutritive properties due to toxicity, along with potential for health promotion [5]. Therefore, selection of a mutant line with reduced toxicity due to a low level of tomatin accumulation and a mutant line with a high level of tomatine accumulation as a source of tomatine showing various pharmacological activities are proposed. To develop tomato cultivars with less toxicity and less bitterness targeting green immature fruits, the top 10 mutant lines with low tomatine content among the immature fruits were selected and designated 14-3, 14-5, 15-4, 18-7, 19-2, 20-5, 22-6, 26-2, 41-1, and 44-5. There have been reports of the consumption of an edible tomato cultivar (cherry tomato, L. esculentum var. cerasiforme) with a very high tomatine content (0.5-5 mg/g of dry weight) and in recipes, such as pickled or fried green tomatoes [28]. Tomatine has also been reported to exhibit various pharmacological activities. For example, tomatine inhibits the growth of human ovarian (Skov3) and breast (MCF-7) cancer cells [18,19]. It also shows immunostimulating effects as a vaccine adjuvant against infectious diseases and cancer immunotherapy [20]. Tomatine exhibits anti-inflammatory effects by suppressing NF-κB and JNK signaling in lipopolysaccharide-stimulated mouse macrophages [21] and it exerts antibiotic activities against microorganisms [22]. Thus, to develop tomato cultivars having value as an important source for extracting tomatin with various To select target-specific mutant lines, we screened the mutants for high or low relative tomatine content in the immature fruit and high esculeoside A content in the mature fruit. The peak area values were measured by ELSD for the original cultivar and 184 mutant lines at two different ripening stages ( Figure S1). Tomatine has a dual aspect with anti-nutritive properties due to toxicity, along with potential for health promotion [5]. Therefore, selection of a mutant line with reduced toxicity due to a low level of tomatin accumulation and a mutant line with a high level of tomatine accumulation as a source of tomatine showing various pharmacological activities are proposed. To develop tomato cultivars with less toxicity and less bitterness targeting green immature fruits, the top 10 mutant lines with low tomatine content among the immature fruits were selected and designated 14-3, 14-5, 15-4, 18-7, 19-2, 20-5, 22-6, 26-2, 41-1, and 44-5. There have been reports of the consumption of an edible tomato cultivar (cherry tomato, L. esculentum var. cerasiforme) with a very high tomatine content (0.5-5 mg/g of dry weight) and in recipes, such as pickled or fried green tomatoes [28]. Tomatine has also been reported to exhibit various pharmacological activities. For example, tomatine inhibits the growth of human ovarian (Skov3) and breast (MCF-7) cancer cells [18,19]. It also shows immunostimulating effects as a vaccine adjuvant against infectious diseases and cancer immunotherapy [20]. Tomatine exhibits anti-inflammatory effects by suppressing NF-κB and JNK signaling in lipopolysaccharide-stimulated mouse macrophages [21] and it exerts antibiotic activities against microorganisms [22]. Thus, to develop tomato cultivars having value as an important source for extracting tomatin with various pharmacological activities, we selected the top 10 mutant lines with a higher content of tomatine compared with the original cultivar, which were designated 13-8, 15-8, 20-7, 26-8, 27-1, 27-7, 37-4, 42-3, 42-5, and 43-2. Esculeoside A is abundant in mature tomatoes and exhibits various biological activities. For example, it was shown to inhibit atherogenesis in apoE-depicient mice [23]. Esculeoside A also inhibited hyaluronidase activity and ameliorated dermatitis in 2,4-dinitro-chlorobenzen-treated mice [24]. Tomato saponin, including esculeoside A ameliorated collagen-induced arthritis in vivo through Th1 inhibition and Th2 activation [24], and it exhibited a hypoglycemic effect in db/db mice through the AMP-activated protein kinase/insulin receptor substrate-1 pathway [27]. Thus, the top 10 mutant lines with the highest esculeoside A content among the mature fruits were selected and designated 13-6, 13-8, 16-7, 17-3, 17-8, 18-2, 22-1, 22-6, 23-1, and 25-2.
To quantify tomatine and esculeoside A in the target-specific mutant lines, an established extraction and analytical method was used. The content of these two compounds in the mutant lines (µg/g of fresh weight) are presented in Tables 1-3. Table 1 shows the content of the two triterpenoid glycosides in unripe green fruits for the selected mutant lines with low tomatine content. The lowest accumulation of tomatine was observed in the mutant line 44-5 with a concentration of 299.1 ± 3.7 µg/g, which was approximately 83.22% lower compared with the wild type (1782.9 ± 7.2 µg/mL). When the same mutant line matured into red fruit, the mutant line 44-5 exhibited an esculeoside A quantity of 444.0 ± 3.2 µg/g, indicating that the conversion rate from tomatine to esculeoside A for the mutant line 44-5 was the highest at 122.16%. Therefore, this mutant line was considered a robust source of tomatine to esculeoside A. Table 2 shows the content of the two compounds in unripe green fruits for the selected mutant lines a high tomatine content. The mutant line 42-3 exhibited the highest accumulation of tomatine with a concentration of 1894.8 ± 7.9 µg/g, which represented 6.29% increase compared with the original cultivar. In the mature red fruit of the mutant line 42-3, the esculeoside A content was 281.7 ± 2.5 µg/g and its conversion rate from tomatine to esculeoside A was very low at 22.62%. This indicates an opposite tendency with respect to the conversion rate of the mutant lines with low tomatine content. Among these mutant lines, the ripe red fruit of the mutant line 37-4 contained tomatine and esculeoside A levels of 510.2 ± 5.5 µg/g and 428.6 ± 2.3 µg/g, respectively. For this mutant line, it was assumed that the metabolic conversion from tomatine to esculeoside A was not an efficient process. Table 3 lists the content of the two compounds in ripe red fruits for the selected mutant lines with high esculeoside A content. The highest value for esculeoside A was observed in line 13-6 at 786.0 ± 4.2 µg/g of fresh weight, which was 196% higher compared with that of the original cultivar (399.8 µg/g of fresh weight). This indicates a positive impact of the irradiation on improving the production of esculeoside A. This was more evident when comparing the quantitation results for esculeoside A content in the fresh ripe tomatoes in the market, which ranged from 92.8 to 456 µg/g of fresh weight [29]. Therefore, this mutant line is a promising candidate as a highly functional cultivar.
We found that the high content of tomatine in immature fruits almost disappeared when the fruits ripened. Instead, the esculeoside A content increased in mature fruits, which was consistent with other studies [6]. According to a recent study on the metabolic pathway of steroid glycosides, glycosylation of tomatidine to tomatin requires four glycosyltransferases: GAME1, GAME17, GAME18 and GAME2 [8]. Additionally, the catalytic action of GAME31 (also known as Sl23DOX) leads to the hydroxylation of tomatine, and GAME5 is involved in the production of esculoside A [9][10][11][12]. The metabolic pathways for steroidal glycosides based on ripening characteristics have been also suggested [13][14][15]. For example, the high level accumulation of tomatine and the absence of esculeoside A were confirmed in tomato fruit tissues of non-ripening and ripening-inhibitor mutants [13].
It has been demonstrated that there is a glycosylation step in the biosynthesis pathway from tomatine to esculeoside A, which is dependent upon ethylene treatment during fruit ripening [14]. The hydroxylation at C-27 of the terpenoid backbone of tomatine result in C22βN→C22αN isomerization, which leads to the biosynthesis of esculeoside A, was proposed by quantum chemical calculations [15]. In addition, the content of various triterpenoid glycosides other than tomatine and esculeoside A in HPLC-ELSD-MS profiles of extracts from several tomato varieties has also been reported, and the total amount in tomato fruit remains somewhat constant during ripening [15]. Based on these studies, we compared the conversion rate of tomatine to esculeoside A in each micro-tomato mutant line (Figure 2). For the mutant lines with a low tomatine content, the conversion rate of tomatine to esculeoside A tended to be high as the fruit maturated, whereas the mutant lines with high tomatine content exhibited a low conversion rate from tomatine to esculeoside A. Mutant lines containing tomatine at the ripening stage were also found: 13-2, 13-5,  Table 4.
which was consistent with other studies [6]. According to a recent study on the metabolic pathway of steroid glycosides, glycosylation of tomatidine to tomatin requires four glycosyltransferases: GAME1, GAME17, GAME18 and GAME2 [8]. Additionally, the catalytic action of GAME31 (also known as Sl23DOX) leads to the hydroxylation of tomatine, and GAME5 is involved in the production of esculoside A [9][10][11][12]. The metabolic pathways for steroidal glycosides based on ripening characteristics have been also suggested [13][14][15]. For example, the high level accumulation of tomatine and the absence of esculeoside A were confirmed in tomato fruit tissues of non-ripening and ripening-inhibitor mutants [13]. It has been demonstrated that there is a glycosylation step in the biosynthesis pathway from tomatine to esculeoside A, which is dependent upon ethylene treatment during fruit ripening [14]. The hydroxylation at C-27 of the terpenoid backbone of tomatine result in C22βN→C22αN isomerization, which leads to the biosynthesis of esculeoside A, was proposed by quantum chemical calculations [15]. In addition, the content of various triterpenoid glycosides other than tomatine and esculeoside A in HPLC-ELSD-MS profiles of extracts from several tomato varieties has also been reported, and the total amount in tomato fruit remains somewhat constant during ripening [15]. Based on these studies, we compared the conversion rate of tomatine to esculeoside A in each micro-tomato mutant line (Figure 2). For the mutant lines with a low tomatine content, the conversion rate of tomatine to esculeoside A tended to be high as the fruit maturated, whereas the mutant lines with high tomatine content exhibited a low conversion rate from tomatine to esculeoside A. Mutant lines containing tomatine at the ripening stage were also found: 13-2, 13-5, 17-7, 22-4, 23-3, 24-5, 24-7, 26-1, 26-5, 27-4, 37-4, 39-5, 40-8, 41-2 and 45-2 (15 out of 184). The quantitative results for these mutant lines are shown in Table 4.

Cytotoxic Activities
Tomato steroidal glycosides have exhibited anti-proliferative activities against various cancer cell lines, including breast MCF-7, melanoma B16F2F, colon CT-26, liver HepG2, prostatic PC3, and lung A549 cancer cell lines [30,31]. However, the anticancer activities of micro-tomato, tomatine, and esculeoside A have not been examined in head and neck squamous cell carcinoma (HNSCC) [32]. Therefore, we measured the cytotoxic activity of methanol extracts from unripe and ripe fruits of the original micro-tomato cultivar and its mutant lines, tomatine, and esculeoside A against FaDu cells. Treatment with the methanol extract of the unripe green fruit and ripe red fruit of the original micro-tomato cultivar resulted in 85.89% ± 5.19% and 85.96% ± 4.97% cell viability at 50 µg/mL, respectively, indicating that their cytotoxic effect was weak (Tables S1 and S2). There were micro-tomato mutant lines that showed lower cell viability compared with that of the original cultivar. Of these, a value of 70.94% ± 2.56% was obtained for the unripe green fruit (14-4) and 70.18% ± 1.77% for the ripe red fruit (18-1) (Tables S1 and S2). Thus, the cytotoxicity of micro-tomato extracts against the FaDu cell line was considered insignificant. However, tomatine exhibited an IC 50 value of 5.589 µM, whereas esculeoside A did not show any activity (Figure 3). Although this finding did not indicate a correlation between the content of tomatine in the extracts of micro-tomato samples and the cytotoxicities of the extracts of the micro-tomato samples, a potential anti-tumor effect of tomatine in FaDu cells may be expected and further studies regarding its mechanism of action and activity in vivo are needed.

General Procedures
A [ 60 Co]-irradiator (150 TBq capacity; AECL, Ottawa, ON, Canada) at the Advanced Radiation Technology Institute (Jeongeup-si, Korea) was used to generate gamma radiation. A proton linear accelerator (TR103) at the Korea Multi-purpose Accelerator Complex (Gyeongju, Korea) was used for proton beam-irradiation. The nuclear magnetic resonance (NMR) experiments were carried out on a 600 MHz Bruker AVANCE NEO NMR spectrometer (Oxford magnet, Bruker Switzerland AG, Fällanden, Switzerland) operated with Bruker TopSpin software (Billerica, MA, USA) at the Core Research Support Center for Natural Products and Medical Materials (CRCNM). An ultra-performance liquid chromatography-evaporative light scattering detector (UPLC-ELSD) was used along with a Waters Acquity UPLC system (Waters, Milford, MA, USA) and an Alltech Model 3300 ELSD detector (BÜCHI, Flawil, Switzerland) equipped with a Kinetex C18 column (2.1 mm × 10 mm, 2.6 mm; Phenomenex, Torrance, CA, USA). A

General Procedures
A [ 60 Co]-irradiator (150 TBq capacity; AECL, Ottawa, ON, Canada) at the Advanced Radiation Technology Institute (Jeongeup-si, Korea) was used to generate gamma radiation. A proton linear accelerator (TR103) at the Korea Multi-purpose Accelerator Complex (Gyeongju, Korea) was used for proton beam-irradiation. The nuclear magnetic resonance (NMR) experiments were carried out on a 600 MHz Bruker AVANCE NEO NMR spectrometer (Oxford magnet, Bruker Switzerland AG, Fällanden, Switzerland) operated with Bruker TopSpin software (Billerica, MA, USA) at the Core Research Support Center for Natural Products and Medical Materials (CRCNM). An ultra-performance liquid chromatographyevaporative light scattering detector (UPLC-ELSD) was used along with a Waters Acquity UPLC system (Waters, Milford, MA, USA) and an Alltech Model 3300 ELSD detector (BÜCHI, Flawil, Switzerland) equipped with a Kinetex C18 column (2.1 mm × 10 mm, 2.6 mm; Phenomenex, Torrance, CA, USA). A highly porous polystyrene gel (Diaion HP-20; Sigma Chemical Co., St Louis, MO, USA), reverse phase C18 (YMC gel ODS-A, 12 nm, S-75 µm, YMC Co., Kyoto, Japan) and Sephadex LH-20 (Cytiva, MA, USA) resin was used for column chromatography (CC). Thin-layer chromatography was performed on a Kieselgel 60 RP-18-F254S device (Merck, Darmstadt, Germany) with visualization under UV light (254 and 365 nm) and by heating at 180 • C for 2 min after spraying with 10% (v/v) sulfuric acid. The standard compound, tomatine, was obtained from Sigma-Aldrich (Sigma Chemical Co., St Louis, MO, USA). All of the other chemicals and solvents used in this study were of analytical grade.

Plant Materials
The mutant tomato fruit lines were generated by irradiating tomato seeds (S.

Isolation and Identification of Esculeoside A from the Ripe Red Micro-Tomato
Fresh fruits (510 g) from commercially available red micro-tomato (S. lycopersicum) were homogenized in water using a mixer and the homogenate was centrifuged and filtered through Whatman No. 2 filter paper. The filtrate was passed through a Diaion HP-20 sequentially by eluting with water and methanol. The methanol eluate was then subjected to reverse phase C18 CC and eluted with a gradient solvent system (40% to 60% methanol in water) to yield 12 fractions (F01-F12). F09 was subjected to an open column packed with sephadex LH-20 using methanol as an eluent. This resulted in the isolation of esculeoside A (2, 15.4 mg).

Preparation of Standard Solutions and Sample Preparation
Fresh unripe or ripe tomato fruits (the mutant lines and its original cultivar) were individually weighed and ground using a Geno/grinder (SPEX, New York, NJ, USA). The ground sample was mixed with methanol at a ratio of 10 mL methanol per 10 g and then extracted by sonication for 1 h. The extracted solutions were centrifuged (12,000× g for 10 min at 20 • C) and the supernatants were filtered through a polyvinylidene fluoride (PVDF) syringe filter (0.45 µm). The filtrates were then freeze-dried. Each of the methanol extracts was accurately weighed and dissolved in methanol at 6 mg/mL and then filtered through a PVDF syringe filter (0.45 µm) for HPLC analysis. Each of the standards, tomatine and esculeoside A, were accurately weighed and dissolved in methanol and 50% methanol in water, respectively, at 1.0 mg/mL. The stock solutions were diluted to prepare a series of standard solutions at five concentrations (tomatine: 31.25, 62.50, 125.00, 250.00, and 500.00 µg/mL; esculeoside A: 15.63, 31.25, 62.50, 125.00, and 250.00 µg/mL) for quantitative analysis.

Quantitative Analysis
Quantitative analyses were performed using a Waters Acquity UPLC system with a Kinetex C18 column. An Alltech Model 3300 ELSD detector was used for analyte detection. The temperature of the drift tube and the flow rate of the carrier gas (nitrogen) were set at 1.5 L/min and 40 • C, respectively. All of the data were processed using Empower 2 software (Waters, Milford, MA, USA). The mobile phase consisted of water containing 0.05% formic acid and 5% acetonitrile (v/v; solvent A) and 0.1% formic acid in acetonitrile (v/v; solvent B), and was run as follows: 0-10 min, 5-52.5% B; 10-11 min, 52.5-100% B; 11-13 min, 100% B; 13-13.01 min, 100-5%; and 13.01-15 min, 5% B. The flow rate was maintained at 0.3 mL/min and the injection volume was 2 µL. Each experiment was conducted in triplicate and all of the data are presented as the mean ± standard deviation (SD).

Cytotoxicity Assay
FaDu human pharynx squamous carcinoma cells were purchased from the Korean Cell Line Bank (Seoul, Korea) and cultured in a Dulbecco's Modified Eagle's Medium (Hyclone, Logan, UT, USA) supplemented with 10% fetal bovine serum (Gibco, Grand Island, NY, USA) in a humidified incubator. The viability of the cells was determined using a crystal violet assay [33]. Briefly, FaDu cells were seeded into 96-well plates at a density of 5 × 10 3 cells/well and incubated at 37 • C for 24 h. Cultured FaDu cells were treated with the indicated concentration of extract (100 µg/mL), tomatine (1.5-100 µg/mL), and esculeoside (1.5-100 µg/mL). After a 48 h incubation, the cells were washed twice with PBS and stained with 0.5% crystal violet (in 20% methanol) for 20 min. The FaDu cells were washed with water 4 times and dried overnight. Thereafter, 200 µL of methanol was added and incubated for 20 min at room temperature. The optical density (OD) was measured at 584 nm using an ELISA reader (Softmax Pro., Molecular Devices, San Jose, CA, USA). The percentage of cell viability was calculated using the following formula: Cell viability (%) = Test OD/Control OD × 100 The 50% inhibitory concentration (IC 50 ) values were calculated from a dose-response analysis performed using GraphPad Prism software (GraphPad Software, La Jolla, CA, USA).

Conclusions
The content of two major steroidal alkaloids in the unripe green and ripe red fruits of S. lycopersicum cv. Micro-Tom, and its mutant lines were analyzed by UPLC-ELSD. Selection of mutant lines with a low content of tomatine may induce the development of green tomatoes, which may increase consumption due to reduced toxicity and less bitterness. Esculeoside A abundant in red tomatoes, contains 4 to 5 times more than lycopene, a powerful antioxidant, and has medicinal effects in preventing arteriosclerosis and regulating blood pressure. Thus, selection of mutant lines with a high content of esculeoside A may lead the development of new red tomato cultivars with high functionality and improved quality. Furthermore, it can be used as industrial materials for the development of cardiovascular-related health functional foods or drugs. In addition, the extracts of the tomato samples and the two compounds were evaluated for cytotoxicity in FaDu cells for the first time in this study. Tomatine exhibited potent cytotoxicity, thus it may represent a lead compound with anticancer effects in HNSCC cancer cells and further studies on its mechanism of action are warranted.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/ 10.3390/plants11212895/s1, Figure S1: The screen for relative content of (a) tomatine ( ) in unripe fruits and (b) esculeoside A ( ) and tomatine ( ) in ripe fruits of the original cultivar of micro-tomato and its mutant lines, Figure S2: 1 H NMR spectrum of esculeoside A (600 MHz, pyridine-d 5 ), Figure  S3: 13 C NMR spectrum of esculeoside A (150 MHz, pyridine-d 5 ), Table S1: Cell viabilities (%) of the methanol extract of unripe green fruit of the micro-tomato original cultivar and mutant lines (n = 4), Table S2: Cell viabilities (%) of the methanol extract of ripe red fruit of the micro-tomato original cultivar and mutant lines (n = 4).