Phenolic Compounds from Sesame Cake and Antioxidant Activity: A New Insight for Agri-Food Residues’ Significance for Sustainable Development

Agri-food residues represent a rich source of nutrients and bioactive secondary metabolites, including phenolic compounds. The effective utilization of these by-products in food supplements and the nutraceuticals industry could provide a way of valorization in the transition to becoming more sustainable. In this context, the present study describes the phenolic profiling of sesame (Sesamum indicum L.) cake using reversed-phase high-performance liquid chromatography coupled to diode array detection and quadrupole-time-of-flight-mass spectrometry. Compounds were characterized based on their retention time, UV spectra, accurate mass spectrometry (MS) and MS/MS data along with comparison with standards, whenever possible, and the relevant literature. The characterized compounds (112 metabolites) belong to several classes, namely, phenolic acids (hydroxybenzoic acids and hydroxycinnamic acids), flavonoids, and lignans. Moreover, organic acids and some nitrogenous compounds were characterized. The total phenol content and the antioxidant activity of the cake extract were determined. This study provides useful information for the valorization of by-products from the sesame oil industry.


Introduction
Pedaliaceae is considered a small family, with 14 genera and 70 species. It is natively distributed in the Old World and commonly known as the sesame family [1]. Sesame (Sesamum indicum L.) is a prominent oil crop that is cultivated all over the world. It is believed that sesame originates from India [2]. Nevertheless, it has been present in the Ancient Egyptian civilization since the third century BC, where the ancient Egyptian used it for soothing asthma [3].
Sesame seeds are considered a rich source of proteins, dietary fibers, carbohydrates, fats, and vitamins [2,4]. Several studies investigated the phytochemical composition of sesame seeds and/or oil and homogenized in the aforementioned magnetic stirrer at room temperature for 60 min. The mixture was finally centrifuged at 7155× g (8000 rpm) for 15 min and 5 • C using Sorvall ST 16 (Thermo Sci., ThermoFisher, Waltham, MA, USA) and the supernatant was collected. The pellet was re-extracted with 25 mL acetone/water (70: 30, v/v). Both supernatants were combined and evaporated under vacuum using a rotary evaporator at 38 • C (Rotavapor R-200, BüchiLabortechnik, AG, Switzerland). Three independent extractions were performed. Finally, the dry cake extracts were dissolved in methanol/water (80:20, v/v), filtered (0.45 µm syringe filter, regenerated cellulose) and stored at −20 • C until analysis.

Analysis by RP-HPLC-DAD-ESI-QTOF-MS and -MS/MS
Analyses were made with an Agilent 1200 series rapid resolution (Santa Clara, CA, USA) equipped with a binary pump, an autosampler, and a diode array detector (DAD). Separation was carried out with a core-shell Halo C18 analytical column (150 mm × 4.6 mm, 2.7 µm particle size). The system was coupled to a 6540 Agilent Ultra-High-Definition (UHD) Accurate-Mass Q-TOF LC/MS equipped with an Agilent Dual Jet Stream electrospray ionization (Dual AJS ESI) interface.
Gradient elution was conducted with two mobile phases consisting of acidified water (0.5% acetic acid, v/v) (phase A) and acetonitrile (phase B) with a constant flow rate of 0.5 mL/min according to [13]. The gradient program was 0 min, 99% A and 1% B; 5.50 min, 93% A and 7% B; 11 min, 86% A and 14% B; 17.5 min, 76% A and 24% B; 22.50 min, 60% A and 40% B; 27.50 min, 0% A and 100% B; 28.5 min 0% A and 100% B; 29.5 min, initial conditions, which were finally maintained for 5.50 min for column equilibration (total run 35 min). The injection volume was 15 µL and three analyses were performed.
The operating conditions briefly were drying nitrogen gas temperature, 325 • C with a flow of 10 L/min; nebulizer pressure, 20 psig; sheath gas temperature, 400 • C with a flow of 12 L/min; capillary voltage, 4000 V; nozzle voltage, 500 V; fragmentor voltage, 130 V; skimmer voltage, 45 V; octapole radiofrequency voltage, 750 V. Data acquisition (2.5 Hz) in profile mode was governed via MassHunter Workstation software (Agilent technologies). The spectra were acquired in negative-ion mode over a mass-to-charge (m/z) range from 70 to 1500. The detection window was set to 100 ppm. Reference mass correction on each sample was performed with a continuous infusion of Agilent TOF biopolymer analysis mixture containing trifluoroacetic acid ammonium salt (m/z 112.9856 corresponding to the trifluoroacetic acid ion) and hexakis (1H, 1H, 3H-tetrafluoropropoxy) phosphazine (m/z 1033.9881 corresponding to the trifluoroacetic acid ammonium salt adduct ion). MS/MS experiments were performed in automatic mode, using the following collision energy values: m/z 100, 40 eV; m/z 500, 45 eV; m/z 1000, 50 eV; and m/z 1500, 55 eV.
MassHunter Qualitative Analysis B.06.00 (Agilent technologies) was used for data analysis following the strategy proposed by [10,12,14]. The characterization of compounds was performed by generation of candidate formula with a mass accuracy limit of 5 ppm. The MS score related to the contribution to mass accuracy, isotope abundance and isotope spacing for the generated molecular formula was set at ≥90. After the generation of the molecular formula, retention time (Rt), UV, and MS/MS spectra were also considered and compared with literature. Consequently, the following chemical structure databases were consulted: ChemSpider (http://www.chemspider.com), SciFinder Scholar (https://scifinder.cas.org), Reaxys (http://www.reaxys.com), PubChem (http://pubchem.ncbi. nlm.nih.gov), KNApSAcK Core System database (http://www.knapsackfamily.com/knapsack_jsp/ top.html), METLIN Metabolite Database (http://metlin.scripps.edu), Phenol-Explorer (www.phenolexplorer.eu), Dictionary of Natural Products (http://dnp.chemnetbase.com), Phytochemical dictionary of natural products database [15] and tracing available literature via Egyptian Knowledge Bank (https://www.ekb.eg/). Confirmation was made through a comparison with standards, whenever these were available in-house. Moreover, the peak area obtained by MS for each compound was determined to estimate the abundance of each characterized phenolic compound.

Total Phenol Content (TPC) Assay
The TPC of the extracts was determined in triplicate colorimetrically by Folin-Ciocalteu reagent [16] and modified according to [17] in 96-well polystyrene microplates (Thermo Fisher) and using a Synergy MxMonochromator-Based Multi-Mode Microplate reader (Bio-Tek Instruments Inc., Winooski, VT, USA). The absorbance of the solution was measured at a wavelength of 760 nm after incubation for 2 h in the dark and compared with a calibration curve of serially diluted gallic acid (GA). The results were expressed as GA equivalents (GAE). Analyses were done in triplicate.

Trolox Equivalent Antioxidant Capacity Assay
TEAC absorbance measurements were performed using the aforementioned microplate reader and following the procedure described by [12]. Briefly, ABTS + was produced by reacting ABTS stock solution with 2.45 mM potassium persulfate (final concentration). The mixture was kept in dark at room temperature for 24 h and the solution diluted with water until reaching an absorbance value of 0.70 (±0.03) at 734 nm. Afterwards, 300 µL of this solution and 30 µL of the cake extract were mixed and measured. Absorbance reading was compared to a standard calibration curve of Trolox and the results expressed as equivalents of Trolox (TE). Caffeic acid was used as a positive control. Analyses were done in triplicate.

Metabolic Profiling of Sesame Cake by RP-HPLC-DAD-QTOF-MS and -MS/MS
The sesame cake was subjected to core-shell RP-HPLC-DAD-ESI-QTOF-MS and -MS/MS analysis in negative ionization mode, providing Rt, experimental m/z, generated molecular formulae, mass error in ppm, mass score, double bond equivalents (DBE), UV maxima, and tandem mass fragments. For clarification, Tables 1 and 2 illustrate the aforementioned parameters for phenolic compounds and non-phenolic compounds, respectively. Moreover, Tables S1 and S2 (supplementary material) also detail metabolites class/subclass, plant species, family, and previously reported literature for phenolic compounds and non-phenolic compounds, respectively. The total number of characterized metabolites was 112, including 92 metabolites that were reported for the first time in sesame, with 20 new proposed structures. Figure 1 shows the base peak chromatogram of the cake extract, showing its complexity.

Total Phenol Content (TPC) Assay
The TPC of the extracts was determined in triplicate colorimetrically by Folin-Ciocalteu reagent [16] and modified according to [17] in 96-well polystyrene microplates (Thermo Fisher) and using a Synergy MxMonochromator-Based Multi-Mode Microplate reader (Bio-Tek Instruments Inc., Winooski, VT, USA). The absorbance of the solution was measured at a wavelength of 760 nm after incubation for 2 h in the dark and compared with a calibration curve of serially diluted gallic acid (GA). The results were expressed as GA equivalents (GAE). Analyses were done in triplicate.

Trolox Equivalent Antioxidant Capacity Assay
TEAC absorbance measurements were performed using the aforementioned microplate reader and following the procedure described by [12]. Briefly, ABTS + was produced by reacting ABTS stock solution with 2.45 mM potassium persulfate (final concentration). The mixture was kept in dark at room temperature for 24 h and the solution diluted with water until reaching an absorbance value of 0.70 (±0.03) at 734 nm. Afterwards, 300 µL of this solution and 30 µL of the cake extract were mixed and measured. Absorbance reading was compared to a standard calibration curve of Trolox and the results expressed as equivalents of Trolox (TE). Caffeic acid was used as a positive control. Analyses were done in triplicate.

Metabolic Profiling of Sesame Cake by RP-HPLC-DAD-QTOF-MS and -MS/MS
The sesame cake was subjected to core-shell RP-HPLC-DAD-ESI-QTOF-MS and -MS/MS analysis in negative ionization mode, providing Rt, experimental m/z, generated molecular formulae, mass error in ppm, mass score, double bond equivalents (DBE), UV maxima, and tandem mass fragments. For clarification, Tables 1 and 2 illustrate the aforementioned parameters for phenolic compounds and non-phenolic compounds, respectively. Moreover, Tables S1 and S2 (supplementary material) also detail metabolites class/subclass, plant species, family, and previously reported literature for phenolic compounds and non-phenolic compounds, respectively. The total number of characterized metabolites was 112, including 92 metabolites that were reported for the first time in sesame, with 20 new proposed structures. Figure 1 shows the base peak chromatogram of the cake extract, showing its complexity.

Hydroxybenzoic Acids
Qualitatively, phenolic acids were the most abundant phenolic compounds by 40 metabolites. They are divided into hydroxybenzoic acids (13) and hydroxycinnamic acids (27).

Hydroxybenzoic Acids
Qualitatively, phenolic acids were the most abundant phenolic compounds by 40 metabolites. They are divided into hydroxybenzoic acids (13) and hydroxycinnamic acids (27).
Concerning hydroxybenzoic acids, they can be classified into non-hydroxylated (benzoic acid), mono-hydroxylated (p-hydroxybenzoic acid), di-hydroxylated (gentisic, protocatechuic, and vanillic acid derivatives), and tri-hydroxylated (gallic acid and syringic acid derivatives). It bears noting that five of them were confirmed with standards. In brief, m/z 121.03 exerted a neutral loss of CO and UV absorbance at λ max 272 nm. It was characterized as benzoic acid and has been previously reported in sesame [4]. The compound with a m/z value of 137.02 was confirmed to be p-hydroxybenzoic acid upon comparison with a standard. Similarly, two compounds showed a molecular formula of C 7 H 6 O 4 and m/z of 153.02. By comparing with standards, they were confirmed as protocatechuic acid (3,4-dihydroxybenzoic acid) and gentisic acid (2,5-dihydroxybenzoic acid).
In regard to O-methylated derivatives of dihydroxybenzoic acids, five derivatives of vanillic acid were characterized. Vanillic acid was observed with m/z 167.04, and was confirmed with standard. It has been reported before in sesame [18]. In addition, three isomers of vanillic acid hexoside (C 14 H 18 O 9 ) were observed at Rt 9.02, 10.28, and 10.66 min. They revealed the neutral loss of the hexose moiety (162 Da) and CO 2 (44 Da), complying with the typical decarboxylation of phenolic acids [10,12,17]. They were reported for the first time in S. indicum, nevertheless, they were reported in the genus Sesamum in accordance with the Dictionary of Natural Products database (Table S1). Vanillic acid pentoside hexoside was characterized with m/z 461.13 and sequential loss of pentose (m/z 329.09), hexose (m/z 167.03, i.e., vanillic acid ion), and methyl (m/z 153.02) moieties. It was detected for the first time in sesame (Table 1 and Table S1). Concerning tri-hydroxylated benzoic acids, both gallic and syringic acids were observed with m/z 169.01 and 197.05, respectively. These assignments were confirmed with standards. It is worth noting that the compound with an m/z value of 329.09 and molecular formula C 14 H 18 O 9 exerted the neutral loss of pentose with an aglycone fragment of (m/z 197.05) followed by fragmentation of syringic acid. It was tentatively identified as syringic acid pentoside and proposed as a new structure. Similarly, compound at m/z 359.10 (C 15 H 20 O 10 ) showed a neutral loss of a hexose moiety with syringic acid fragmentation pattern. In this way, two consecutive losses of CH 3 were observed until fragment 166.10, indicating methoxy substituents. Moreover, the loss of CO 2 from the carboxyl moiety was also observed (m/z 153.06 and 138.03) followed by losses of CH 3 (Figure 2a) [17]. Therefore, it was identified as syringic acid hexoside, which was described for the first time in sesame.

Hydroxycinnamic Acids
Regarding hydroxycinnamic acid, it was found in free form, conjugated with quinic acid or with sugars. The occurrence of p-coumaric acid, m-coumaric acid, chlorogenic acid (caffeoylquinic acid), caffeic acid, ferulic acid, and sinapic acid were unequivocally confirmed with standards enabling characterization validation, which was in agreement with previous studies [19]. It is worth noting that both m-coumaric acid and chlorogenic acid were described for the first time in sesame. Moreover, cinnamic acid (21, m/z 147.05) was observed with the neutral loss of CO 2 from the carboxylate moiety and water (Table 1 and Table S1).
Five isomers of p-coumaric acid hexosides were detected expressing the neutral loss of hexose moieties releasing aglycones of m/z 163.04 followed by decarboxylation (m/z 119.05), except for the hydrated form (M−H+H 2 O) and, hence, dehydration occurred firstly [13]. In the same manner, three isomers of ferulic acid hexoside (C 16 H 20 O 9 , m/z 355.10) and sinapic acid hexoside (C 17 H 22 O 10 , m/z 385.11) were observed ( Table 1 and Table S1). As an example, the fragmentation of the latter compound is shown in Figure 2b. Another compound was described as ferulic acid dihexoside (sibiricose A5). In addition, seven novel hydrocinnamic acids were tentatively identified as ferulic acid pentoside isomers (I-III), sinapic acid deoxyhexoside hexoside isomers (I-III), and diferuloyl hexoside (Table 1 and Table S1). The latter was characterized by the presence of fragment ions at m/z 337.0929 (C 16 (Figure 2c), as for sinapic acid. Remarkably, most of these compounds are described for the first time in sesame (Table S1). Moreover, caffeoyl derivatives were found. Based on molecular formula (C21H28O13) and fragmentation pattern with successive loss of deoxyhexose and hexose, the caffeic acid derivate cistanoside F (C21H28O13) was characterized. Furthermore, three caffeoylphenylethanoid derivatives were observed. Verbascoside and isoverbascoside expressed the neutral loss of caffeoyl moieties and deoxyhexoses through the presence of the ion m/z 461.17 and m/z 315.11, respectively. β-Hydroxyverbascoside was identified with similar fragmentation pattern as of verbascoside with additional loss of hydroxyl group (16 Da) [19]. These caffeoyl derivatives were isolated before from sesame [20,21].

Lignans
Lignans are natural phenolic compounds that possess several biological activities, especially antioxidant and estrogenic activities. A simple lignan is composed of two phenyl propanoid Moreover, caffeoyl derivatives were found. Based on molecular formula (C 21 H 28 O 13 ) and fragmentation pattern with successive loss of deoxyhexose and hexose, the caffeic acid derivate cistanoside F (C 21 H 28 O 13 ) was characterized. Furthermore, three caffeoylphenylethanoid derivatives were observed. Verbascoside and isoverbascoside expressed the neutral loss of caffeoyl moieties and deoxyhexoses through the presence of the ion m/z 461.17 and m/z 315.11, respectively. β-Hydroxyverbascoside was identified with similar fragmentation pattern as of verbascoside with additional loss of hydroxyl group (16 Da) [19]. These caffeoyl derivatives were isolated before from sesame [20,21].

Lignans
Lignans are natural phenolic compounds that possess several biological activities, especially antioxidant and estrogenic activities. A simple lignan is composed of two phenyl propanoid derivatives (C6-C3) linked through a β-β-linkage [22]. In this context, 18 lignan derivatives were identified in cake of sesame, which belonged to the furofuran subclass of lignans. As a matter of fact, the in-depth analysis of tandem MS data made it possible to preliminary predict lignan structures, whose presence in sesame were not discovered and could yet contribute to its bioactivity ( Table 1). For that, studies using tandem MS/MS were consulted for analog structures [22][23][24][25]. All of them were glycosides, and the loss of each sugar was observed until the product ions of the aglycones were released and hence observed [22].
Briefly, three isomers of pinoresinol dihexoside were detected showing subsequent losses of two hexosyl moieties with aglycone fragmentation showing the fragment m/z 151.04 (C 8 H 7 O 3 − ) due to cleavage of the tetrahydrofuran ring followed by methyl loss from the guaiacyl moiety [22,24,25] (Table 1 and Table S1, Figure 3a). Similarly, two isomers, (m/z 767. 24 Figure 3b illustrates the fragmentation pattern of xanthoxylol malonyl trihexoside. Although these glycosides have been reported here for the first time, and aglycone was characterized by Fukuda et al. [25]. In regard to sesaminol derivatives, two isomers of sesaminol trihexoside (I-II) and sesaminol tetrahexoside (I-II) were identified with the observation of m/z 149.05 characterizing furofurano lignans [23], as commented upon before, as well as the counterpart at m/z 219.06 (C 12 H 11 O 4 − ).
Moreover, two isomers of sesaminol dipentoside were identified, also showing a fragment at m/z 135.03; i.e.,methylenedioxyphenyl-CH 2 (C 8 H 7 O 2 − ) [23]. The acetic acid adduct of sesamolinol hexoside (m/z 593.19, C 28 H 34 O 14 ) and three new isomers of sesamolinol dipentoside were observed with the observation of the aglycone at m/z 371.11 (Table 1 and Table S1). In the first case, fragment ions at m/z 138.0323 (C

Flavonoids
A total of 26 flavonoids were characterized in the sesame cake extract, being classified mainly into a flavan-3-ol, flavanones (2), flavones (15), and flavonols (8) ( Table 1). It is worth noting that (−)-epicatechin, naringenin, luteolin 7-O-β-d-glucopyranoside, luteolin, apigenin, quercetin, rutin, quercetin 3-O-β-d-glucopyranoside, quercetin 3-O-β-d-galactopyranoside, quercetin 3-O-rhamnopyranoside, myricetin, kaempferol, and kaempferide were identified through comparison with standards. All of them were described for the first time in the genus Sesamum, except for apigenin and luteolin 7-O-β-d-glucopyranoside, according to the phytochemical dictionary database [15]. m/z 151.00 (C7H3O4) ( 1,3 A − ) by this type of compound [26]. Moreover, the ion fragment with m/z at 103.05 (C8H7 − ) could be derived from band B ( 1,3 B − ). Besides the fragmentation pattern, it showed UV absorbance at λmax 288 nm, suggesting a flavanone nucleus [12]. Therefore, it was tentatively identified as pinocembrin, which was described for the first time in sesame. The occurrence of C-glycosides of flavones was noticed with 12 derivatives of either luteolin or apigenin, which were observed for the first time in sesame. They were characterized by the presence of prominent fragment ions after the characteristic sequential loss of 90 (C3H6O3) and/or 120 Da (C4H8O4), in agreement with previous studies [12,14,17,19,27]. As an example, two isomers of luteolin C-hexoside were identified, exerting characteristic fragments at m/z 357.06 and 327.05, respectively. Similarly, three isomers of luteolin C-deoxyhexoside-C-hexoside were tentatively identified based on comparing their fragmentation pattern and UV absorbance with reported literature [27]. As for apigenin derivative, five isomers of apigenin C-pentoside-C-hexoside were Additionally, the fragmentation pattern of compound at m/z 255.07 (Rt 29.81 min, C 15 H 12 O 4 ) revealed the common fragment ion released after retro Diels-Alder fission and retrocyclization at m/z 151.00 (C 7 H 3 O 4 ) ( 1,3 A − ) by this type of compound [26]. Moreover, the ion fragment with m/z at 103.05 (C 8 H 7 − ) could be derived from band B ( 1,3 B − ). Besides the fragmentation pattern, it showed UV absorbance at λ max 288 nm, suggesting a flavanone nucleus [12]. Therefore, it was tentatively identified as pinocembrin, which was described for the first time in sesame.
The occurrence of C-glycosides of flavones was noticed with 12 derivatives of either luteolin or apigenin, which were observed for the first time in sesame. They were characterized by the presence of prominent fragment ions after the characteristic sequential loss of 90 (C 3 H 6 O 3 ) and/or 120 Da (C 4 H 8 O 4 ), in agreement with previous studies [12,14,17,19,27]. As an example, two isomers of luteolin C-hexoside were identified, exerting characteristic fragments at m/z 357.06 and 327.05, respectively. Similarly, three isomers of luteolin C-deoxyhexoside-C-hexoside were tentatively identified based on comparing their fragmentation pattern and UV absorbance with reported literature [27]. As for apigenin derivative, five isomers of apigenin C-pentoside-C-hexoside were observed, showing fragmentation patterns and UV absorbance of C-flavones as described in reported studies [15,17]. Moreover, a minor fragment ion at m/z 117.03 (C 8 H 5 O) ( 1,3 B − ) was observed, suggesting that the aglycone is apigenin. Similarly, two isomers of apigenin di-C-pentoside were tentatively characterized. As Figure 3c shows, the consequent neutral loss of sugar fragments (30-180 Da) was observed (Table 1 and Table S1, Figure 3c).
Regarding nitrogenous compounds, it is worth mentioning that five amino acids were characterized, viz. asparagine, leucine/isoleucine, tyrosine, and phenylalanine. Their fragmentation patterns were characterized by deamination and/or decarboxylation [13,15,17]. In addition, both tyrosine and phenylalanine were confirmed with standards. Furthermore, a peptide was observed (Rt 6.29 min, m/z 611.1454, C 20 H 32 N 6 O 12 S 2 ) exerting the loss of a glutathione moiety (m/z 306.08) followed by a loss of SH 2 from the cysteinyl group. Finally, the product ion of the glutamyl moiety was observed at m/z 128.04. It was compared with data on the METLIN database to be described as oxidized glutathione (GSSG), indicating the presence of reduced glutathione (GSH) in the cake of sesame, which is easily auto-oxidized to GSSG during sample preparation and/or analysis [31]. In fact, GSH is considered to be a powerful cellular antioxidant that prevents oxidative stress in biological systems and, hence, prevents the onset and progression of many serious diseases such as diabetes mellitus, cancer, and Alzheimer's disease [31]. Furthermore, a derivative of tryptophan (m/z 529.18, C 26 H 30 N 2 O 10 ) was observed as well as succinyladenosine (m/z 382.10, C 14 H 17 N 5 O 8 ), a nucleoside derivative. It bears noting that the characteristic tetrasaccharide sesamose could be the ion with m/z 665.21, presenting subsequent losses of hexosyl moieties in MS/MS (Table 2 and Table S2).

TPC, TEAC, and Phenolic Abundance
The extract of sesame cake showed a total phenol content of 1.9 ± 0.3 mg GAE/g cake extract.
In fact, this value is even beyond results by Mohadaly et al. [18], where total phenol contents were assayed of single different solvents cake extracts of the Egyptian cultivar 'Shandweel-3'. The value of TPC ranged from 0.1 (petroleum ether extract) to 0.8 (methanol extract) mg GAE/g cake extract. This could be attributed to the combined solvent extraction accompanied with ultra-sonication, which enhances the extraction process [12,32]. In regard to the TEAC assay, the extract expressed a value of 2.65 ± 0.08 µmol TE/g of cake extract. In fact, Janu et al. [33] focused on the antioxidant activity of the sesame oil, which was found to be 0.004 µg TE/mL oil (i.e., around 0.02 µmol TE/g oil) indicating the value of the cake as an agri-industrial by-product that needs further attentions for its antioxidant potential as well as other biological activities.
To evaluate the contribution of phenolic compounds, a summary of the characterization results is shown in Figure 4. In the perspective of subclasses, flavonoids were the most abundant, representing 38.3% of the total characterized phenolic metabolites followed by hydroxycinnamic acids and then lignans (Figure 4a). Similarly, flavonoids and hydroxycinnamic acids were also the most representative families in qualitative terms (Figure 4b).
potential as well as other biological activities.
To evaluate the contribution of phenolic compounds, a summary of the characterization results is shown in Figure 4. In the perspective of subclasses, flavonoids were the most abundant, representing 38.3% of the total characterized phenolic metabolites followed by hydroxycinnamic acids and then lignans (Figure 4a). Similarly, flavonoids and hydroxycinnamic acids were also the most representative families in qualitative terms (Figure 4b). The exploration of alternative strategies for the treatment of many chronic diseases such as cancer, diabetes, and heart and liver diseases continues to attract scientists in discovering drugs derived from plant origins [34][35][36]. In fact, there is growing attention in the valorization of agri-food residues to provide new functional ingredients with bioactivities for sustainability of the agri-industry. It bears noting that such by-products represent around 40% of total plant foods [10,37]. For that, the elucidation of the potential bioactive phytochemicals is a requirement. In this regard, the application of UHPLC-QTOF-MS enabled us to characterize 86 phenolic compounds in sesame cake and, hence, as far as we know, this is the first study providing comprehensive phenolic profiling of sesame cake. In addition, the antioxidant activity of the sesame cake extract was determined by the TEAC method. In this regard, furofurano lignans possess anticancer, cardiovasculoprotective, neuroprotective, antioxidant, and anti-inflammatory activities. Moreover, they are metabolized by gut microflora into enterolactone and enterodiol, which are considered phytoestrogens [38]. Thus, these compounds could contribute to the antioxidant activity of the extract.
In this line, a previous study on sesame cake showed that the main contributors to the antioxidant activity were sesamol and water-(sesaminol tri-and di-glucoside) and lipid-soluble lignans (sesamin and sesamolin), but the extraction procedure was based on Soxhlet extraction with methanol [39]. In this sense, our results revealed the presence of sesamol and a wider range of lignan The exploration of alternative strategies for the treatment of many chronic diseases such as cancer, diabetes, and heart and liver diseases continues to attract scientists in discovering drugs derived from plant origins [34][35][36]. In fact, there is growing attention in the valorization of agri-food residues to provide new functional ingredients with bioactivities for sustainability of the agri-industry. It bears noting that such by-products represent around 40% of total plant foods [10,37]. For that, the elucidation of the potential bioactive phytochemicals is a requirement. In this regard, the application of UHPLC-QTOF-MS enabled us to characterize 86 phenolic compounds in sesame cake and, hence, as far as we know, this is the first study providing comprehensive phenolic profiling of sesame cake. In addition, the antioxidant activity of the sesame cake extract was determined by the TEAC method. In this regard, furofurano lignans possess anticancer, cardiovasculoprotective, neuroprotective, antioxidant, and anti-inflammatory activities. Moreover, they are metabolized by gut microflora into enterolactone and enterodiol, which are considered phytoestrogens [38]. Thus, these compounds could contribute to the antioxidant activity of the extract.
In this line, a previous study on sesame cake showed that the main contributors to the antioxidant activity were sesamol and water-(sesaminol tri-and di-glucoside) and lipid-soluble lignans (sesamin and sesamolin), but the extraction procedure was based on Soxhlet extraction with methanol [39]. In this sense, our results revealed the presence of sesamol and a wider range of lignan glycosides, but there were no free lignans, which could be due to the use of more polar extraction conditions and the removal of the fatty phase. Moreover, C-glycosides were the most abundant compounds both as a subclass, accounting for around 37.3% in relative abundance, and individually, i.e., apigenin C-pentoside-C-hexoside I (8.8%) followed by luteolin C-deoxyhexoside-C-hexoside III (7.0%), luteolin C-deoxyhexoside-C-hexoside II (6.7%), and apigenin C-pentoside-C-hexoside IV (5.1%). As a matter of fact, it seems that C-glycosylation enhances antioxidant capacity, where the hydroxyl group and metal chelation sites of flavones are free [14,40]. Thus, these compounds could be the highest contributors to the antioxidant activity of the extract, agreeing with Zhou et al. [41]. These authors highlighted that the antioxidant activity of sesame cake extracts was associated with the total content of flavonoids, but these compounds were not characterized. Furthermore, oxidized glutathione was detected for the first time in the cake of sesame, which could be produced from reduced glutathione during sample preparations. It is considered a strong marker for the antioxidant potential of this agri-industrial byproduct.

Conclusions
In this study, core-shell RP-HPLC-DAD-ESI-QTOF-MS and -MS/MS methods were employed to analyze the cake of the Egyptian cultivar of sesame 'Giza 32'. A total of 112 metabolites were characterized in sesame cake, and among them, 86 were phenolic compounds. The observed lignans were of furofurano type and, among them, 12 lignans are considered to be new proposed structures. Moreover, this is the first report showing the conjugation of malonyl moieties to lignans in Pedaliaceae. With regard to the characterized flavonoids, they were classified into flavones (15), flavonols (8), flavanones (2), and a flavan-3-ol. C-Glycosides of flavones have been reported here for the first time in sesame. This type of flavonoid was the most abundant. Furthermore, the antioxidant activity of the sesame cake extract was determined by TEAC method and, hence, our results suggest that not only sesamol and lignans, but also C-glycosides and other compounds could contribute to this bioactivity. Consequently, further studies are required for the development of food supplements and nutraceuticals from sesame cake to widen its applicability and to move into a more sustainable industry with zero waste.