molecules Review Lignans of Sesame (Sesamum indicum L.): A Comprehensive Review Mebeaselassie Andargie 1,*, Maria Vinas 2 , Anna Rathgeb 1, Evelyn Möller 1 and Petr Karlovsky 1,* ���������� ������� Citation: Andargie, M.; Vinas, M.; Rathgeb, A.; Möller, E.; Karlovsky, P. Lignans of Sesame (Sesamum indicum L.): A Comprehensive Review. Molecules 2021, 26, 883. https:// doi.org/10.3390/molecules26040883 Academic Editor: David Barker Received: 13 January 2021 Accepted: 2 February 2021 Published: 7 February 2021 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- iations. Copyright: © 2021 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/). 1 Molecular Phytopathology and Mycotoxin Research, University of Goettingen, Grisebachstrasse 6, 37073 Goettingen, Germany; annarathgeb@gmail.com (A.R.); evelyn.moeller@uni-goettingen.de (E.M.) 2 Centro para Investigaciones en Granos y Semillas (CIGRAS), University of Costa Rica, 2060 San Jose, Costa Rica; maria.vinasmeneses@ucr.ac.cr * Correspondence: mebeaselassie.andargie@agr.uni-goettingen.de (M.A.); pkarlov@gwdg.de (P.K.) Abstract: Major lignans of sesame sesamin and sesamolin are benzodioxol–substituted furofurans. Sesamol, sesaminol, its epimers, and episesamin are transformation products found in processed products. Synthetic routes to all lignans are known but only sesamol is synthesized industrially. Biosynthesis of furofuran lignans begins with the dimerization of coniferyl alcohol, followed by the formation of dioxoles, oxidation, and glycosylation. Most genes of the lignan pathway in sesame have been identified but the inheritance of lignan content is poorly understood. Health-promoting properties make lignans attractive components of functional food. Lignans enhance the efficiency of insecticides and possess antifeedant activity, but their biological function in plants remains hypothetical. In this work, extensive literature including historical texts is reviewed, controversial issues are critically examined, and errors perpetuated in literature are corrected. The following aspects are covered: chemical properties and transformations of lignans; analysis, purification, and total synthesis; occurrence in Seseamum indicum and related plants; biosynthesis and genetics; biological activities; health-promoting properties; and biological functions. Finally, the improvement of lignan content in sesame seeds by breeding and biotechnology and the potential of hairy roots for manufacturing lignans in vitro are outlined. Keywords: sesame lignans; sesamin; sesamolin; sesamol; lignan biosynthesis; lignan glycosides; health-promoting properties; biological function; plant biotechnology 1. Introduction Sesame (Sesamum indicum L.) is an ancient oilseed crop [1] cultivated in subtropical and tropical regions of Africa, Asia, and South America as a source of edible seeds and high-quality oil. The origin of cultivated sesame has not been conclusively identified [2]. Although Africa hosts most wild relatives of cultivated sesame, genetic arguments support the Indian origin of Sesamum indicum [3]. The Indian species S. malabaricum (syn. S. mu- layanum) is the most likely progenitor ([3] and the references therein). To unite the crop and its progenitor under a common species name, Bedigian suggested new combinations S. indicum subsp. malabaricum for S. malabaricum and S. indicum subsp. indicum for the cultivated sesame [4]. In addition to Sesamum indicum, other species of Sesamum and a close relative Ceratotheca sesamoides are grown in Africa for seeds, and their leaves are locally used as vegetables [3]. Only Sesamum indicum (syn. S. indicum subsp. indicum), however, is regarded as a domesticated crop, and only seeds and oil of this species are traded internationally. Major producers of sesame are Tanzania, India, Myanmar, China, Sudan, Ethiopia and Nigeria, in this order [5,6]. Landraces and locally grown varieties of sesame show conspicu- ous diversity, supposedly resulting from selection of variants by farmers and possibly also from repeated domestications [3]. In spite of its age and economical importance for local Molecules 2021, 26, 883. https://doi.org/10.3390/molecules26040883 https://www.mdpi.com/journal/molecules https://www.mdpi.com/journal/molecules https://www.mdpi.com https://orcid.org/0000-0003-0644-0579 https://orcid.org/0000-0002-6532-5856 https://doi.org/10.3390/molecules26040883 https://doi.org/10.3390/molecules26040883 https://creativecommons.org/ https://creativecommons.org/licenses/by/4.0/ https://creativecommons.org/licenses/by/4.0/ https://doi.org/10.3390/molecules26040883 https://www.mdpi.com/journal/molecules https://www.mdpi.com/1420-3049/26/4/883?type=check_update&version=2 Molecules 2021, 26, 883 2 of 52 economies, sesame is regarded as an orphan crop and research devoted to sesame has been scarce; for instance, sesame is not mandated by any international crop research center [6]. Oil of S. indicum is valued for its sensory characteristics and resistance to rancid- ity [2,7,8]. Sesame oil also exerts antioxidative activity and possesses health-promoting properties, which are attributed to tocopherols, tocotrienols, and lignans [9–11]. Major lignans of sesame are sesamin and sesamolin (Figure 1). The total content of these two lignans in sesame seeds may exceed 1.4% (Table 1). Numerous minor lignans present in seeds in low concentrations and/or generated by chemical transformations during seed and oil processing have been described. Among them, sesamol, episesamin and samin (Figure 2) were studied extensively. Sesamol is a degradation product that is present in traces in unroasted seeds but occurs at high concentrations in roasted seeds and processed sesame oil (see Section 2.2). Molecules 2021, 26, x FOR PEER REVIEW 2 of 55 to sesame has been scarce; for instance, sesame is not mandated by any international crop research center [6]. Oil of S. indicum is valued for its sensory characteristics and resistance to rancidity [2,7,8]. Sesame oil also exerts antioxidative activity and possesses health-promoting prop- erties, which are attributed to tocopherols, tocotrienols, and lignans [9–11]. Major lignans of sesame are sesamin and sesamolin (Figure 1). The total content of these two lignans in sesame seeds may exceed 1.4% (Table 1). Numerous minor lignans present in seeds in low concentrations and/or generated by chemical transformations during seed and oil pro- cessing have been described. Among them, sesamol, episesamin and samin (Figure 2) were studied extensively. Sesamol is a degradation product that is present in traces in unroasted seeds but occurs at high concentrations in roasted seeds and processed sesame oil (see Section 2.2). The economic value of lignans is reflected by patents covering purification, chemical transformations, and the use of lignans in health-promoting food additives and skin care components [12–20]. Figure 1. Structures of major lignans of sesame. The concentration of lignans in seeds varies with the variety of sesame. High lignan content is a quality trait and an important target for sesame breeding. Inheritance of lignan content has only recently been systematically investigated (Section 4.3). Molecular markers for high lignan content are not available yet, though markers for the yield and many other agronomic traits have been established [21–25]. Progress in the genomics of sesame [26,27] and high-resolution genetic mapping [26,28–33] raise hopes that marker-assisted selection for lignan content as well as for a desirable ratio of individ- ual lignans and their glycosides will be possible soon. Figure 1. Structures of major lignans of sesame. The economic value of lignans is reflected by patents covering purification, chemical transformations, and the use of lignans in health-promoting food additives and skin care components [12–20]. The concentration of lignans in seeds varies with the variety of sesame. High lignan content is a quality trait and an important target for sesame breeding. Inheritance of lignan content has only recently been systematically investigated (Section 4.3). Molecular markers for high lignan content are not available yet, though markers for the yield and many other agronomic traits have been established [21–25]. Progress in the genomics of sesame [26,27] and high-resolution genetic mapping [26,28–33] raise hopes that marker-assisted selection for lignan content as well as for a desirable ratio of individual lignans and their glycosides will be possible soon. Lignans are metabolites formed from two molecules of phenylpropanoids. In sesame, the synthesis of lignans involves the fusion of oxopropane side chains of cinnamyl alcohol into a furofuran core (3,7-dioxabicyclo[3.3.0.]octane) (Figure 3). These metabolites are desig- nated furofuran lignans. Seven enzymes involved in the biosynthesis of lignans in sesame have been characterized (Section 3.1). Some of these enzymes catalyze multiple steps of the pathway. The genes encoding further enzymes, such as the dirigent protein involved in the initial step of the pathway and enzymes related to the formation of lignans that do not belong to the furofuran group (e.g., lariciresinol, secoisolariciresinol and matairesinol), were putatively identified in the genome of sesame based on sequence similarity. Molecules 2021, 26, 883 3 of 52Molecules 2021, 26, x FOR PEER REVIEW 3 of 55 Figure 2. Minor lignans of sesame (first two rows) and transformation products (bottom row). Figure 2. Minor lignans of sesame (first two rows) and transformation products (bottom row). Molecules 2021, 26, x FOR PEER REVIEW 4 of 55 Lignans are metabolites formed from two molecules of phenylpropanoids. In sesame, the synthesis of lignans involves the fusion of oxopropane side chains of cinnamyl alcohol into a furofuran core (3,7-dioxabicyclo[3.3.0.]octane) (Figure 3). These metabolites are des- ignated furofuran lignans. Seven enzymes involved in the biosynthesis of lignans in ses- ame have been characterized (Section 3.1). Some of these enzymes catalyze multiple steps of the pathway. The genes encoding further enzymes, such as the dirigent protein in- volved in the initial step of the pathway and enzymes related to the formation of lignans that do not belong to the furofuran group (e.g., lariciresinol, secoisolariciresinol and ma- tairesinol), were putatively identified in the genome of sesame based on sequence simi- larity. Figure 3. Dimerization of cinnamyl alcohol forms pinoresinol. Numbering according to IUPAC recommendation [34]. Lignans of sesame attracted interest of nutritional scientists and health professionals because of their health-promoting activities (see Sections 5.1 and 5.2) such as lowering blood glucose and cholesterol levels, prevention against cardiovascular diseases and can- cer, and alleviation of postmenopausal syndrome [35]. The ability of sesamin to suppress tumor growth [36,37] suggests that sesamin might even be developed into a therapeutic agent. Sesame oil and lignans are components of creams and body oils [38,39]. Apart from the nutritional, cosmetic, and health-promoting use, sesame lignans and especially syn- thetically available sesamol served as potentiators of insecticides (see Section 5.4). Several reviews of plant lignans are available [17,40–42]. Because of their nutritional importance, information about food lignans has been collected in several databases [43]. As these databases and reviews covered lignans from many plant species, space devoted to lignans of sesame was limited. In 2013, Dar and Arumugam [44] published a review entitled “Lignans of sesame: purification methods, biological activities and biosynthesis— a review” but they, too, covered lignans from many plants while the coverage of sesame was limited. A comprehensive review focusing on the lignans of sesame has been missing. This work attempts to review chemical, biological, and applied aspects of sesame lignans comprehensively. The literature was surveyed from the first reports on sesame lignans in the 1890th, including scarcely accessible historical texts. Representative examples were selected for finding documented in many reports. Controversial issues were critically ex- amined, and several errors perpetuated in literature were corrected. The Section 2 on lignan chemistry provides an overview of the chemical properties of lignans; their transformation and degradation during processing; the variation of lignan content in S. indicum and related species; and the purification, analysis, and total synthesis of lignans. In the Sections 3 and 4 on biosynthesis and genetics, enzymes and genes of the lignan pathway are described, and published information on the inheritance of lignan content in sesame seeds is reviewed. In the Section 5, the biological activities and health-promoting properties of lignans are reviewed and their therapeutic potential is as- sessed. In the Section 6, biological functions of lignans in sesame plants are discussed. Finally, the potential of plant breeding and biotechnology for the improvement of lignan Figure 3. Dimerization of cinnamyl alcohol forms pinoresinol. Numbering according to IUPAC recommendation [34]. Lignans of sesame attracted interest of nutritional scientists and health professionals because of their health-promoting activities (see Sections 5.1 and 5.2) such as lowering blood glucose and cholesterol levels, prevention against cardiovascular diseases and cancer, and alleviation of postmenopausal syndrome [35]. The ability of sesamin to suppress tumor growth [36,37] suggests that sesamin might even be developed into a therapeutic agent. Sesame oil and lignans are components of creams and body oils [38,39]. Apart from the nutritional, cosmetic, and health-promoting use, sesame lignans and especially synthetically available sesamol served as potentiators of insecticides (see Section 5.4). Several reviews of plant lignans are available [17,40–42]. Because of their nutritional importance, information about food lignans has been collected in several databases [43]. As these databases and reviews covered lignans from many plant species, space devoted to lignans of sesame was limited. In 2013, Dar and Arumugam [44] published a review entitled “Lignans of sesame: purification methods, biological activities and biosynthesis—a review” but they, too, covered lignans from many plants while the coverage of sesame was limited. A comprehensive review focusing on the lignans of sesame has been missing. Molecules 2021, 26, 883 4 of 52 This work attempts to review chemical, biological, and applied aspects of sesame lignans comprehensively. The literature was surveyed from the first reports on sesame lignans in the 1890th, including scarcely accessible historical texts. Representative examples were selected for finding documented in many reports. Controversial issues were critically examined, and several errors perpetuated in literature were corrected. The Section 2 on lignan chemistry provides an overview of the chemical properties of lignans; their transformation and degradation during processing; the variation of lignan content in S. indicum and related species; and the purification, analysis, and total synthesis of lignans. In the Sections 3 and 4 on biosynthesis and genetics, enzymes and genes of the lignan pathway are described, and published information on the inheritance of lignan content in sesame seeds is reviewed. In the Section 5, the biological activities and health- promoting properties of lignans are reviewed and their therapeutic potential is assessed. In the Section 6, biological functions of lignans in sesame plants are discussed. Finally, the potential of plant breeding and biotechnology for the improvement of lignan content in sesame seeds and for the production of lignans in vitro is assessed in the Section 7. 2. Chemistry of Sesame Lignans 2.1. Structures and Chemical Properties of Lignans of Sesamum indicum 2.1.1. Aglycons Haworth introduced the term lignan in 1936 for phenolic metabolites of plants that consist of two n-propylbenzene moieties [45]. Phenylpropanoid monomers are connected via β-atoms of the propane chains [34]. According to the cyclization pattern and the pres- ence and location of oxygens, lignans are divided into eight classes [34]. Major lignans of sesame (Figure 1) belong to the furofuran family. In furofuran lignans, the oxopropane side chains of phenylpropanoid building units are fused into 3,7-dioxabicyclo[3.3.0]octane (Figure 3). Minor lignans of Sesamum indicum (Figure 2) belong to the furofuran, tetrahy- drofuran, and butyrolactone classes; further lignans are produced by the degradation and transformation of furofuran lignans. Chemical properties of lignans of S. indicum and their content in seeds of sesame are shown in Table 1. Table 1. Aglycones of lignans found in Sesamum indicum and related species. Lignan MW Monoisotopic Mass UV Maxima Extinction Coefficient [g−1 L] Content in Seeds [mg/100 g] Reference Sesamin 354.35 354.1103 287/236 23.03/26.01 4 77–930 [46–67] Sesamolin 370.35 370.1053 288/235 21.79/24.85 4 61–530 [53–72] Sesaminol 370.40 370.1053 238/295 3.99/3.85 5 0–1.4 [73,74] Sesamolinol 372.40 372.1209 231/287 3.95/3.80 5 6–29 [53,75] Pinoresinol 358.38 358.1416 232/280 - 29–38 [52,76–78] Sesamol 1 138.12 138.0316 296/233 29.74/21.18 4 0–30 1 [62,63,79–81] Lariciresinol 360.40 360.1572 230/280 - 0.8 3 [77,78,82] Matairesinol 358.40 358.1416 230/282 - 0.4 3 [77] Piperitol 356.40 356.1259 - - 1.3 3 [78] Episesamin 2 354.40 354.1103 - - 9–270 [54,73] Samin 250.25 250.0841 287/238 - 50 2 [83,84] 1 Only found in roasted seeds or oil. 2 Only found in bleached oil. 3 Only a single value reported. 4 In isooctane, 5 in chloroform. All Sesamum species studied so far produce lignans. Sesamin and sesamolin were detected in most species and we assume that all Sesamum species produce these two lignans, though most species accumulate them in lower levels than S. indicum. In addition to sesamin and sesamolin, some sesame species produce unique lignans not occurring in S. indicum (Table 2). These species are of commercial interest as a source of enzymes and genes for the engineering of lignan biosynthesis (K. Dossa, personal communication). The major lignans of sesame—sesamin and sesamolin—are also the oldest lignans described. Sesamin was isolated by James Fowler Tocher in 1890th in Aberdeen, Scotland, Molecules 2021, 26, 883 5 of 52 from acetic acid extract of sesame oil [46–48]. The structure of sesamin was elucidated in 1939 at the University of Würzburg, Germany [49] and its absolute configuration was determined in 1960 in Heidelberg, Germany [51]. The second major lignan of sesame is sesamolin [50,72]. The name sesamolin was coined in 1928 by W. Adriani [69] for a crystalline compound with a melting point of 94 ◦C, which was isolated from sesame oil for the first time in 1903 in Italy [68]. The structure of sesamolin was determined in 1955 at the University of Sheffield [70]. Further lignans often reported from sesame, that are are present in smaller amounts, are pinoresinol [76], sesaminol [73,85], sesamolinol [75], episesaminone [86], matairesinol [77], and episesamin [73]. Table 2. Lignans detected in Sesamum species other than S. indicum. The first report of each lignan in each species is cited. Source (+)-Sesamin (+)-Sesamolin (+)-Sesan-Golin (+)-ala-Tumin (+)-2-epi-Sesalatin (+)-7′-epi-Sesantalin S. alatum [87] 1 [87] 2 [88] [89] [90] S. angolense [66] [66] [91] S. angustifolium [66] [66] [87] S. orientalevar. malabaricum [66] 3 [66] 3 S. calycinum [66] [66] S. latifolium [66] [87] 4 S. radiatum [66] [72] 4,5 [92] [92] S. schinzianum [72] 6 [72] 6 S. mulayanum [93] 7 [93] 7 S. laciniatum [93] 8 [93] 8 S. capense [66] 9 S. pedalioides [66] 9 1 [87] reported 0.01% in oil; [94] reported 0.14 mg g−1 seed; acc. to [93], the ratio of sesamin to sesamolin was 1:3. 2 [87] reported 0.01% in oil; [94] reported 0.38 mg g−1 seed. 3 Acc. to [93], the ratio of sesamin to sesamolin was 4:1. 4 In her publication from 2010 [95], Kamal-Eldin noted that S. latifolium was mistakenly named as S. radiatum in her previous publications, especially in [87]. For further details, see Section 2.8. 5 Acc. to [72], the ratio of sesamin to sesamolin was 7:1. 6 The ratio of sesamin to sesamolin was 5:1. 7 The ratio of sesamin to sesamolin was 5:1; authors indicated that S. mulayanum was a synonym for S. malabaricum. 8 The ratio of sesamin to sesamolin was 1:3. 9 Only trace amounts were detected in a single herbarium sample by TLC. Mixtures of lignans containing sesamolactol were extracted from several plants other than sesame since the 1980th but the structure of (-)-sesamolactol and its presence in the perisperm of sesame seeds were only established in 2006 [96]. Lariciresinol was originally purified from resin of spruce [97], but in 2005 it was also found in sesame seeds [77]. Sesamol, sesaminol, sesaminol epimers, and episesamin are degradation products, which are found only in traces in unroasted seeds and oil (see Section 2.2). Sesaminol was also reported in unroasted fully matured and germinating seeds [85]. 2.1.2. Lignan Glucosides Sesaminol and pinoresinol occur in sesame seeds mainly in glycosylated forms (Table 3 and Figure 4). Sesame oil contains aglycons and some monoglycosylated lignans, while most of di- and tri-glycosylated lignans remain in oil-free meal after oil extraction. Major glycosylated lignans in sesame seeds are di- and triglucosides of pinoresinol [76,98], mono-, di- and triglucosides of sesaminol [99], and sesamolinol diglucoside [100]. The structures of glucosylated lignans of sesame are shown in Figure 4. Enzymatic deglucosylation of lignan glucosides is reviewed in Section 2.2.2. Molecules 2021, 26, 883 6 of 52 Table 3. Major glycosylated lignans found in Sesamum indicum and related species. Lignan MW Monoisotopic Mass Content in Seeds [mg/100 g] Reference Sesaminol monoglucoside 532.5 532.1581 5–20 [53,98,99] Sesaminol diglucoside 694.6 694.2109 8–18 [53,98–100] Sesaminol triglucoside 830.7 830.2481 14–91 [53,99,100] Sesamolinol diglucoside 696.6 696.2265 5–232 [100] 1 Pinoresinol diglucoside 2 682.7 682.2473 1.4–2.1 [76,98] Pinoresinol triglucoside 844.8 844.3001 5.22 [98] 1 First description and structure elucidation. 2 A mixture of three isomers: pinoresinol 4′-O-β-D-glucopyranosyl (l→6)-β-D-glucopyranoside, 4′-O-β-D-glucopyranosyl(l→2)-β-D-glucopyranoside, and di-O-β-D-glucoside. Molecules 2021, 26, x FOR PEER REVIEW 7 of 55 Figure 4. Structures of major lignan glycosides of sesame. Figure 4. Structures of major lignan glycosides of sesame. 2.1.3. Does Sesame Contain Pinoresinol Monoglucoside? The presence of pinoresinol monoglucoside in sesame is the first out of two contro- versial questions regarding pinoresinol glucosides in sesame. Some authors claimed that sesame seeds contained pinoresinol monoglucoside, but we were unable to find published data substantiating their claim. Moazzami et al. [100] listed pinoresinol mono-, di- and triglucosides as components of sesame seeds in the introduction section of their paper, citing Katsuzaki et al. [76], but the cited paper does not support the presence of pinoresinol monoglucoside in sesame. The authors repeated the claim in another paper published in the same year [101], citing the same work by Katsuzaki et al. [76] and two papers of their own [100,102]. None of the cited papers supported the claim. Pathak et al. [103] listed pinoresinol monoglucoside among the component of sesame seeds in their review of bioactive compounds in sesame, citing the seminal work by Katsuzaki et al. [52] and the publication by Moazzami et al. [102] mentioned above. None of these publications dealt with pinoresinol monoglucoside. Dar and Arumugan [44] listed pinoresinol monoglucoside among the lignans of sesame, citing several publications, yet Molecules 2021, 26, 883 7 of 52 none of the cited papers supported the claim. Gerstenmeyer and coworkers [104] reported that they presumably detected pinoresinol monoglucoside in sesame seed by HPLC-MS based on the m/z value of its molecular ion, yet they could not verify the identity of the analyte due to the lack of a standard. In summary, the critical review of literature revealed that the presence of pinoresinol monoglucoside in sesame is not supported by data. We hypothesize that a sensitive analytical method will identify traces of pinoresinol monoglucoside in sesame seeds as intermediate of the synthesis of di- and triglucosides or as a product of partial hydrolysis. Pinoresinol monoglucoside is produced in plants other than sesame such as Forsythia [105], and prune [106]. 2.1.4. Bias of Biomedical Research on Pinoresinol Diglucoside In their seminal work, Katsuzaki and coworkers [52] characterized three digluco- sides of pinoresinol: two diglucosides with both glucose molecules attached to the same methoxyphenol moiety and another diglucoside with glucose molecules attached to differ- ent methoxyphenols. These three diglucosides were present in sesame seeds in comparable concentrations. However, only the latter compound, with glucose molecules attached to different methoxyphenol moieties of pinoresinol, was used in all biomedical studies on pinoresinol diglucoside that we are aware of (e.g., [107–109]). Pinoresinol di-O-β-D-glucoside was used as the only standard in many analytical methods for the quantification of pinoresinol diglucosides (e.g., [110]). Furthermore, protocols for the quantification of total pinoresinol relying on enzymatic deglucosylation that were validated only with pinoresinol di-O-β-D-glucoside (e.g., [111]) might fail with other pinoresinol diglucoside isomers. The use of a single isomer as a standard for the quantification of pinoresinol digluco- sides likely lead to an underestimation of pinoresinol diglucoside content in sesame [112,113] and other species [114]. Pinoresinol diglucosides other than di-O-β-D-glucoside were rarely analyzed [102]. What is the reason for this bias? To our knowledge, only pinoresinol di-O-β-D- glucoside is available commercially, e.g., from Sigma-Aldrich, Selleckchem, Cayman Chem- icals, ChemFaces, Adooq Bioscience, Apexbio, and other suppliers. The companies that specified the source always named Eucommia ulmoides. We speculate that all commercial pinoresinol diglucoside originates from the bark of E. ulmoides rather than sesame [115]. E. ulmoides is a tree cultivated in China for the production of gutta-percha [116]. Extracts of the bark are used in Chinese traditional medicine (杜仲 [dù zhòng], in English known as tu-chung) [117]. While all isomers of sesaminol diglucosides are available from Japanese companies Nakalai Tesque and Nagara Science, we are not aware of a commercial source of pinoresinol 4′-O-β-D-glucopyranosyl-β-D-glucopyranosides. We assume that the avail- ability of pinoresinol di-O-β-D-glucoside from E. ulmoides on the market accounts for the bias of research on biomedical effects of pinoresinol diglucoside. Comparative studies with all three diglucosides are highly desirable. 2.2. Transformation and Degradation of Sesame Lignans 2.2.1. Transformation and Degradation of Lignans during Seed and Oil Processing Seeds of sesame are often roasted to improve their sensory properties and sesame oil is subjected to industrial raffination, which includes alkaline saponification and bleaching with acidic clay. Several chemical transformations of lignans occur during these processes (Figure 5). Molecules 2021, 26, 883 8 of 52Molecules 2021, 26, x FOR PEER REVIEW 10 of 55 Figure 5. Degradation and transformation products of sesame lignans during industrial pro- cessing. (A) Degradation products of sesamolin. (B) Degradation products of sesamin. Yoshida and Tagaki [126] investigated the effect of temperature on the conversion of sesamolin to sesamol in sesame seeds. Essentially all sesamolin was converted to sesamol after 25 min at 250 °C (Figure 6). At high temperatures, sesamol dimerizes into sesamol dimer (Figure 5), which possesses antioxidative activity [127], but the concentration of sesamol dimer in refined oil is very low [123]. In the presence of FeCl3, oxidation of sesa- mol by oxygen lead to a mixture of complex conjugated dimers [128], which exerted cyto- toxic activities [128,129]. However, the reaction conditions used (10 days at 40 °C in the presence of 10 mol % FeCl3) do not occur during processing and storage of sesame oil. Realistic conditions for industrial-scale epimerization of sesamin, an apparatus, and a pro- cedure for preparative enrichment of episesamin were protected by a patent [130]. Figure 5. Degradation and transformation products of sesame lignans during industrial processing. (A) Degradation products of sesamolin. (B) Degradation products of sesamin. The most important transformation is the production of sesamol from sesamolin. The term sesamol was coined by Hans Kreis in 1903 for yet unidentified phenolic product of sesame responsible for color tests used to identify sesame oil [118] (see also Section 2.5.2). Kreis was apparently unaware of Villavecchia and Fabris work from 1893 [119]. The structure of sesamol was established four years later [79,80]. In unroasted seeds and raw oil, sesamol is present in traces or undetectable [73,120,121]. Conversion of sesamolin to sesamol is catalyzed by acidic clays used for decoloration (bleaching) of sesame oil and by heating [87,122–125]. Yoshida and Tagaki [126] investigated the effect of temperature on the conversion of sesamolin to sesamol in sesame seeds. Essentially all sesamolin was converted to sesamol after 25 min at 250 ◦C (Figure 6). At high temperatures, sesamol dimerizes into sesamol dimer (Figure 5), which possesses antioxidative activity [127], but the concentration of sesamol dimer in refined oil is very low [123]. In the presence of FeCl3, oxidation of sesamol by oxygen lead to a mixture of complex conjugated dimers [128], which exerted cytotoxic activities [128,129]. However, the reaction conditions used (10 days at 40 ◦C in the presence of 10 mol % FeCl3) do not occur during processing and storage of sesame oil. Realistic conditions for industrial-scale epimerization of sesamin, an apparatus, and a procedure for preparative enrichment of episesamin were protected by a patent [130]. Molecules 2021, 26, 883 9 of 52Molecules 2021, 26, x FOR PEER REVIEW 11 of 55 Figure 6. Conversion of sesamolin into sesamol by roasting. Seeds were roasted for 25 min in an electric oven at a designated temperature. The graph was constructed using data published by Yoshida and Tagaki [126]. Sesamolin is transformed into sesaminol and its epimers during bleaching of sesame oil [73,99] (Figure 5A). Bleaching also leads to the epimerization of sesamin (Figure 5B) [73,123]. The mechanism of these transformations has not been conclusively established. Based on their study on the conversion of sesamolin to sesaminol under anhydrous con- ditions catalyzed by sulfonic acid and on Fukuda’s original work [131], Huang and coworkers [132] suggested a two-step mechanism. According to their hypothesis, proto- nated sesamolin brakes down into sesamol and oxonium ion, both of which subsequently recombine into the products. Depending on the orientation of molecules engaged in the reaction, sesaminol, its epimers, or sesamolin epimer is produced [132]. We suggest that in sesame oil highly reactive oxonium ions would react with major components of the matrix, which are present in large excess, before re-joining with sesamol. We assume that an intramolecular rearrangement of protonated sesamolin might account for the transfor- mation; in any case, the mechanism has to be elucidated experimentally. Sesamolin by itself has no antioxidative activity yet its transformation products ses- aminol and sesamol are strong antioxidants, believed to be responsible for a large part of antioxidative effects of sesame oil. Heating as part of industrial processing was therefore investigated with the aim of increasing the content of antioxidative lignans [126]. 2.2.2. Enzymatic and Alkaline Hydrolysis of Lignan Glucosides Deglucosylation of lignan glucosides by intestinal bacteria [133,134] is the first step of lignan metabolism in the digestion track of mammals (Section 2.2.3). In the laboratory, deglucosylation is used to determine total aglycon concentration (Section 2.5.2) and to maximize the yield of aglycons in preparative purification (Section 2.5.3). Deyama [115] reported successful hydrolysis of pinoresinol diglucoside to aglycon by a commercial β-glucosidase. Mono- and diglucoside of sesamolinol cannot be hydro- lyzed by commercial β-glucosidases, probably because steric hindrance prevented enzy- matic catalysis [98]. Glucosides of lignans other than sesamolinol were successfully hy- drolyzed by glycosidases [52,76]. Deglycosylation of lignan glucosides was also achieved with a mixture of glycosidases and cellulases [54,135]. Park and coworkers reported that the main hydrolysis product of sesaminol triglucoside in their hands was monoglucoside rather than aglycone even after prolonged treatment with a mixture of β-glucosidase and cellulase [136]. Peng and coworkers, in spite of thorough optimization of the procedure with the same combination of enzymes, achieved only a 50% yield of pure aglycone [137]. Different properties of the enzymes used in different labs may explain the contradiction; unfortunately, the sources of the enzyme have often not been reported. Gerstenmeyer and Figure 6. Conversion of sesamolin into sesamol by roasting. Seeds were roasted for 25 min in an electric oven at a designated temperature. The graph was constructed using data published by Yoshida and Tagaki [126]. Sesamolin is transformed into sesaminol and its epimers during bleaching of sesame oil [73,99] (Figure 5A). Bleaching also leads to the epimerization of sesamin (Figure 5B) [73,123]. The mechanism of these transformations has not been conclusively established. Based on their study on the conversion of sesamolin to sesaminol under anhydrous conditions cat- alyzed by sulfonic acid and on Fukuda’s original work [131], Huang and coworkers [132] suggested a two-step mechanism. According to their hypothesis, protonated sesamolin brakes down into sesamol and oxonium ion, both of which subsequently recombine into the products. Depending on the orientation of molecules engaged in the reaction, sesaminol, its epimers, or sesamolin epimer is produced [132]. We suggest that in sesame oil highly reactive oxonium ions would react with major components of the matrix, which are present in large excess, before re-joining with sesamol. We assume that an intramolecular rear- rangement of protonated sesamolin might account for the transformation; in any case, the mechanism has to be elucidated experimentally. Sesamolin by itself has no antioxidative activity yet its transformation products sesaminol and sesamol are strong antioxidants, believed to be responsible for a large part of antioxidative effects of sesame oil. Heating as part of industrial processing was therefore investigated with the aim of increasing the content of antioxidative lignans [126]. 2.2.2. Enzymatic and Alkaline Hydrolysis of Lignan Glucosides Deglucosylation of lignan glucosides by intestinal bacteria [133,134] is the first step of lignan metabolism in the digestion track of mammals (Section 2.2.3). In the laboratory, deglucosylation is used to determine total aglycon concentration (Section 2.5.2) and to maximize the yield of aglycons in preparative purification (Section 2.5.3). Deyama [115] reported successful hydrolysis of pinoresinol diglucoside to aglycon by a commercial β-glucosidase. Mono- and diglucoside of sesamolinol cannot be hydrolyzed by commercial β-glucosidases, probably because steric hindrance prevented enzymatic catalysis [98]. Glucosides of lignans other than sesamolinol were successfully hydrolyzed by glycosidases [52,76]. Deglycosylation of lignan glucosides was also achieved with a mixture of glycosidases and cellulases [54,135]. Park and coworkers reported that the main hydrolysis product of sesaminol triglucoside in their hands was monoglucoside rather than aglycone even after prolonged treatment with a mixture of β-glucosidase and cellulase [136]. Peng and coworkers, in spite of thorough optimization of the procedure with the same combination of enzymes, achieved only a 50% yield of pure aglycone [137]. Different properties of the enzymes used in different labs may explain the contradiction; unfortunately, the sources of the enzyme have often not been reported. Gerstenmeyer and coworkers [104] reported that prolonged incubation with β-glucuronidase/arylsulphatase from Helix pomatia (Roche, Mannheim, Germany) and cellulase Onozuka R-10 (Merck, Molecules 2021, 26, 883 10 of 52 Darmstadt, Germany) completely hydrolysed all lignan glucosides, while treatment with cellulase alone was not sufficient. To overcome the limitation of commercially available glycosylases, researchers from Kiyomoto Co. in collaboration with two universities in Japan designed a smart screening strategy to identify microbial sources of a glucosidases suitable for the hydrolysis of lignan glycosides, especially sesaminol triglucoside [138]. They collected samples of decaying sesame oil cake, extracted them with chloroform, and analyzed the extract for traces of sesaminol. One of the positive samples yielded a strain of Paenibacillus sp. that hydrolyzed sesaminol triglucoside. The glucosidase responsible for the reaction was purified, partial amino acid sequence of the protein was determined, and the gene was cloned and expressed in E. coli. The recombinant enzyme hydrolyzed sesaminol triglucoside to pure aglycone. The authors applied for patent protection [139]. Later on, they identified a second glucosylase produced by the same bacterium, which was specific for the β-1,2-glucosidic bond of sesaminol triglucoside, producing sesaminol diglucoside [140]. Gaya and coworkers [141] investigated β-glucosidase from Lactobacillus mucosae, which deglucosylated secoisolariciresinol diglucoside into secoisolariciresinol, and successfully expressed the gene in food-grade lactic bacteria for the deglycosylation of lignans in food. Instead of treatment with glycosidases, alkaline hydrolysis can be used to convert lignan glucosides to aglycons. Various conditions for alkaline hydrolysis have been re- ported in literature, such as refluxing with 1 M potassium hydroxide in ethanol [142], treatment with 9 M sodium hydroxide in water at room temperature overnight [143], and treatment with sodium methoxide in pure methanol (3 h at 40 ◦C with sonication) [104]. A combination of alkaline hydrolysis (0.3 M NaOH in 70% methanol, incubated for 1 h at 60 ◦C) with a subsequent enzymatic hydrolysis using β-glucuronidase/sulfatase from Helix pomatia has also been used [144]. Secoisolariciresinol diglucoside was hydrolyzed successfully by incubation with 1 M sodium or potassium hydroxide for 4 to 24 h at room temperature [145]. 2.2.3. Transformation of Ligans in the Body of Mammals In the digestive track of animals, intestinal bacteria remove glucose from lignan glucosides [133,134] and transform lignan aglycons into metabolites designated enterolig- nans [40,146]. Major enterolignans produced by microorganisms in the human digestive track are enterodiol and enterolactone [147–151]. The conversion of lignans to enterolignans was also demonstrated in vitro in cultures inoculated with human fecal inoculum [152] and in axenic cultures of bacteria isolated from human intestine (e.g., [151,153]). The conversion involves four steps: deglycosylation, demethylation, dehydrogenation, and dehydroxylation. In addition, one or two reduction steps are involved, depending on the type of lignan [108,151]. Because of the potential of phytoestrogens to ameliorate menopausal syndrome (Section 5.2.2), the conversion of food lignans into enteroligans by female intestinal microflora attracted research interests. Corona and coworkers [144] compared the conversion of secoisolariciresinol, lariciresinol, pinoresinol, and matairesinol from an oilseed mixture by fecal microflora of young and premenopausal women. They found that the fecal microflora of young women generated mostly enterolactone while the microflora of premenopausal women generated mostly enterodiol. The results were reported with a precision of up to six significant figures, yet the relative standard deviations of most values exceeded 100%, suggesting that the results should not be overrated. Different steps of lignan transformation in the human gut are catalyzed by different bacterial species including Clostridium spp., Bacterioides spp., Eubacterium spp. [150]. The final dehydrogenation of enterodiol into enterolactone was catalyzed by a new strictly anaerobic bacterium [148], which was later characterized and named Lactonifactor longovi- formis [154]. Ruminococcus sp. isolated from human intestine also transformed enterodiol into enterolactone [155]. Molecules 2021, 26, 883 11 of 52 Cytochrome P450 oxidases in the liver of mammals transform lignans by opening and demethylating methylenedioxy-moieties, converting them to vicinal dihydroxyphenol (catechol) derivatives. These reactions were studied in liver homogenates of rats [156], in human liver microsomes, and with human liver enzymes expressed in yeast [157]. Sesamin monocatechol is eventually glucuronated and methylated [158], and the metabolites are excreted in the bile and urine [159]. 2.3. Total Synthesis and Industrial Production Total synthesis of lignans will be briefly outlined in this section. Industrial synthesis of sesamol, introduced in the 1950th [12], provides sesamol at a much lower price than the extraction from sesame oil. Several synthetic strategies were exploited, continuously improving the industrial production [13,160]. A number of synthetic routes for native furofuran lignans have been reported since the 1990th, but none proved suitable for industrial use. The main challenge for the chemical synthesis of lignans was the control of stereochemistry of the furofuran core. The synthesis of tetrahydrofuran lignans is less complex; lariciresinol [82] was synthesized in 1994. Lignans of both tetrahydrofuran and furofuran families including pinoresinol, piperitol, and sesamin were obtained by radical cyclization of epoxides [78]. In the last decade, furofuran lignans have been the target of new synthetic efforts. Electrochemical asymmetric oxidative dimerization of cinnamic acid was used in a biomimetic approach to synthesize sesamin [161]. An elegant bioinspired yet not biomimetic approach to furofuran lignans was the exocyclization of biaryl cyclobutane, which afforded pinoresinol [162]. Sesamin and sesaminol were also synthesized by crossed aldol reaction with a quinomethide intermediate [163]. Asymmetric synthesis of furofuran skeleton [164], the total synthesis of tetrahydrofuran lignans [165], and general synthetic approaches to furofuran lignans were recently reviewed [166]. Patent protection for the total synthesis of (+)-sesamin and other furofuran lignans based on the alkylation of chiral epoxides have recently be sought [167], indicating that the chemical synthesis of lignans matures towards industrial use. 2.4. Stabilization of Fats by Sesame Oil and Unexpected Discovery of Sesame Lignans in Diverse Oils Antioxidative activity of lignans and tocopherol protects fats from spoilage by rancidi- fication. Therefore, small amounts of sesame oil used to be added to animal fats, vegetative oils, and shortenings to stabilize them [168–170]. The method was protected by numerous patents [171–174]. A recent revelation of the presence of sesame lignans in diverse vegetable oils puts a new spin on the topic [175]. Caraway, rapeseed, hemp, peanut, sunflower, pumpkin, poppy, and other edible oils were found to contain sesamin and sesamolin at the same ratio that is known from sesame. The oils were not declared to contain any sesame oil. The authors of the study suggested that sesame lignans in these oils resulted from unintended contamination; pointed out the risk for consumers allergic to sesame; and recommended cleaning the processing equipment thoroughly [175]. We would not rule out that certain companies may add undeclared sesame oil to their products intentionally to extend their shelf life, which would violate food law and possibly also infringe patent rights. Therefore, including sesame lignans in food safety monitoring appears advisable even for oils and fats that are not declared to contain sesame oil. Large number of suspicious products can be pre-tested using a simple colorimetric assay (e.g., [176], see also Section 2.5.2). This may be particularly useful in environments where adulteration of fats is a common practice [177]. 2.5. Extraction, Analysis, and Purification 2.5.1. Extraction of Lignans from Seeds and Oil of Sesame The polarity of lignan molecules is low to medium, but extraction protocols have to take into account that most lignans are glycosylated and the solubility of di- and tri- glycosides in organic solvents is limited. The extraction of sesame seeds with 80% ethanol, Molecules 2021, 26, 883 12 of 52 which is suitable for aglycons as well as glycosylated lignans, was developed in 1998 [53]. In this work, Ryu et al., however, defatted crashed seeds with n-hexane before extraction [53]. Defatting seeds before extraction has sporadically been used until recently (e.g., [178]). The levels on unglycosylated lignans reported in these studies were likely underestimated because lignans dissolve in n-hexane to a certain extent. Many groups actually extracted lignans from sesame into n-hexane [54,87,179,180], or used lignan solutions in n-hexane for crystallization [181]. The solubility of lignans in pure n-hexane is limited, as shown by [142], who reported precipitation of sesamin and episesalatin from n-hexane. Therefore, refluxing in Soxhlet apparatus [55,142,180,182] or repeated extractions with n-hexane [56,183] were typically used. n-hexane is certainly not an ideal solvent for lignans. Defatting seeds with n-hexane (or cyclohexane, which has similar properties and is preferable because of lower toxicity) can be safely used before extraction of glycosylated lignans [52,57,100,102]. Several solvents have been used for the extraction of lignans for analytical purposes. Pure methanol [88], mixtures of methanol and chloroform [121,126] (which do not ex- tract glycosides), a mixture of ethanol with water [58], and a mixture of ethanol with acetate buffer [104] have been used. Other extraction solvents used in the past included heptane or hexane with isopropanol in a 1:3 ratio [54,101], and acetone/water [147,184]. Extraction with 80% ethanol without previous defatting [58] is the standard protocol today (e.g., [59–61]). 80% methanol is also occasionally used [94,185]. Apart from agly- cons and monoglucosides, 80% ethanol (and likely 80% methanol) extracts di-glucosides. Tri-glucosides were successfully extracted into 80% ethanol by some authors [52,76,100] while other found tri-glucosides in the insoluble residues after extracting seeds with 80% ethanol [53]. 70% acetone is an interesting alternative to 80% ethanol because it is suitable for the extraction of all lignans [186] as well as their conjugates [147]. 2.5.2. Color Tests and Chromatographic Methods for Lignans Analysis Before chromatography became established in the analysis of sesame oil, color reac- tions and photometry in UV light were used to detect certain lignans and semi-quantitatively estimate their concentration. Villavecchia’s colorimetric test [119] was established to dis- tinguish sesame oil from other oils and to differentiate butter from margarine (Figure 7), which was labeled with 5–10% sesame oil in the first half of the 19th century [122,187]. Villavecchia test became the official method of the American Oil Chemists’ Society for the detection of sesame oil in vegetable and animal oils and fats [188]. Because Villavecchia test responds to sesamol, it is suitable for the quantification of this lignan [189,190]. When Budowski and coworkers [81] recognized that sesamolin produced the same color reaction because it was converted into sesamol under acidic conditions of the test, they developed a photometric test based on light absorption of the product of furfural-sulfuric acid reaction with sesamol at 518 nm. They also established an analytical method for sesamin based on UV absorption of oil after removal of sesamol by treatment with alkali [50]. Suarez et al. [62] combined their method with the Villavecchia reaction for the determination of the content of sesamol, sesamolin and sesamin in oil. A new method for rapid estimation of total lignans in sesame oil relying merely on the light absorption at 288 nm was developed as recently as in 2015 [180]. After chromatography became widely available as an analytical method, colorimetric methods became obsolete [191]. In spite of that, the Villavecchia test was used until recently for the estimation of the content of sesame oil in pharmaceuticals because it allowed for a high throughput. Even the Budowski/Suarez test was occasionally used for the determination of sesamol in oil until recently [183]. A new colorimetric test was developed in 2005 for the detection of adulteration of edible fats with sesame oil [176]. The test relies on the reaction of thiophene carboxaldehyde with sesamol under acidic contions, and it can detect 0.1% sesame oil in other oils or fats. Molecules 2021, 26, 883 13 of 52 Molecules 2021, 26, x FOR PEER REVIEW 15 of 55 for a high throughput. Even the Budowski/Suarez test was occasionally used for the de- termination of sesamol in oil until recently [183]. A new colorimetric test was developed in 2005 for the detection of adulteration of edible fats with sesame oil [176]. The test relies on the reaction of thiophene carboxaldehyde with sesamol under acidic contions, and it can detect 0.1% sesame oil in other oils or fats. Figure 7. Colorimeter for Villavecchia test used in Aarhus Oliefabrik (Aarhus, Denmark). Normal-phase chromatography on analytical columns for the analysis of sesamin, sesamol and sesamolin was available since the 1950’s [63]. Thin-layer chromatography (TLC) and especially two-dimensional TLC [192] and HPTLC [193] were used in parallel with normal-phase liquid chromatography [123] untill recently. Gas chromatography cou- pled with mass spectrometric detection (GC-MS) was introduced into lignan analysis in the 1990s [76,142] and is still used [194]. A thorough comparison of TLC, GC-MS, and HPLC-UV was carried out by Kamal-Eldin and coworkers [142]. Normal-phase HPLC is still used occasionally, as recently shown for sesamin, sesamol and sesamolin [195], but reverse-phase columns eluted with water-methanol or water-acetonitrile gradients have superseded normal-phase chromatography in the meantime in most methods for lignan analysis [101,142,194,196]. Fluorescence and UV light absorption as detection signals [53] are being gradually replaced by tandem mass spectrometry (e.g., [104,196,197]). A com- prehensive HPLC-MS/MS method covering all major lignans of sesame has not been de- veloped yet. Electrochemical methods for sesame lignans have recently been established. For instance, voltametric methods for the determination of sesamol directly in sesame oil [198] and in acidic solutions [199] were described. Whether these methods can compete with HPLC-MS/MS in routine analysis remains to be seen. 2.5.3. Purification of Sesame Lignans All solvents used for the extraction of lignan for analytical purposes are suitable for preparative purification, but other solvents have also been used, especially in industrial production. 80% ethanol was used for aglycons as well as glycosides of lignans [52,76]. Acetone [71,200], hot pure methanol [57,201–203], and 80% methanol [57] were also used. Standard methods of natural product chemistry used for the purification of lignans in- cluded differential crystallization [64,200,204], column chromatography [52,53,182,205], counter-current chromatography [83,178,196], and preparative TLC [64,206]. In spite of the limited suitability of n-hexane for the extraction of lignans (see Section 2.5.1), hexane was occasionally used for preparative purification of lignans (e.g., [207]). The limited sol- ubility of the target compounds in hexane can be compensated by a large solvent-to-sam- ple ratio and protracted extraction in Soxhlet apparatus, which continuously exposes the sample to pure solvent generated from condensing vapors. Figure 7. Colorimeter for Villavecchia test used in Aarhus Oliefabrik (Aarhus, Denmark). Normal-phase chromatography on analytical columns for the analysis of sesamin, sesamol and sesamolin was available since the 1950’s [63]. Thin-layer chromatography (TLC) and especially two-dimensional TLC [192] and HPTLC [193] were used in paral- lel with normal-phase liquid chromatography [123] untill recently. Gas chromatography coupled with mass spectrometric detection (GC-MS) was introduced into lignan analysis in the 1990s [76,142] and is still used [194]. A thorough comparison of TLC, GC-MS, and HPLC-UV was carried out by Kamal-Eldin and coworkers [142]. Normal-phase HPLC is still used occasionally, as recently shown for sesamin, sesamol and sesamolin [195], but reverse-phase columns eluted with water-methanol or water-acetonitrile gradients have superseded normal-phase chromatography in the meantime in most methods for lignan analysis [101,142,194,196]. Fluorescence and UV light absorption as detection sig- nals [53] are being gradually replaced by tandem mass spectrometry (e.g., [104,196,197]). A comprehensive HPLC-MS/MS method covering all major lignans of sesame has not been developed yet. Electrochemical methods for sesame lignans have recently been established. For instance, voltametric methods for the determination of sesamol directly in sesame oil [198] and in acidic solutions [199] were described. Whether these methods can compete with HPLC-MS/MS in routine analysis remains to be seen. 2.5.3. Purification of Sesame Lignans All solvents used for the extraction of lignan for analytical purposes are suitable for preparative purification, but other solvents have also been used, especially in industrial production. 80% ethanol was used for aglycons as well as glycosides of lignans [52,76]. Acetone [71,200], hot pure methanol [57,201–203], and 80% methanol [57] were also used. Standard methods of natural product chemistry used for the purification of lignans in- cluded differential crystallization [64,200,204], column chromatography [52,53,182,205], counter-current chromatography [83,178,196], and preparative TLC [64,206]. In spite of the limited suitability of n-hexane for the extraction of lignans (see Section 2.5.1), hexane was occasionally used for preparative purification of lignans (e.g., [207]). The limited solubility of the target compounds in hexane can be compensated by a large solvent-to-sample ratio and protracted extraction in Soxhlet apparatus, which continuously exposes the sample to pure solvent generated from condensing vapors. Liquid-liquid extraction can be used to enrich particular lignans in extracts before purification. For instance, partition between n-hexane and water enriched piperitol and pinoresinol in the water phase, and subsequent partition between water and ethyl acetate enriched both compounds in the ethyl acetate phase [202]. Liquid-liquid extraction into ethyl acetate was used for the purification of aglycons and mono- and diglucosides but triglucosides cannot be extracted efficiently into ethyl acetate (see Section 2.5.1). Molecules 2021, 26, 883 14 of 52 Some researchers added butylated hydroxytoluene to extraction solvents to pre- vent oxidation [121,126,179] but most labs did not regard this as necessary. For instance, Williamson and coworkers [208] have not added butylated hydroxytoluene to extracts of sesame seeds for the purification of lignans, though they added butylated hydroxytoluene to the same extract for the purification of other oxidation-sensitive metabolites. An interesting liquid-liquid extraction of lignans from sesame oil into unconventional solvent γ-butyrolactone was developed for the production of pyrethrin synergists by the Norda Essential Oil and Chemical Company in New York [209]. According to this method, a mixture of sesame oil and γ-butyrolactone is heated to 130 ◦C until a homogeneous solution is obtained. After cooling to 60 ◦C, solvent and oil separate. Lignans are obtained from the solvent layer after removal of γ-butyrolactone by distillation. Supercritical extraction of sesame lignans with carbon dioxide liquefied by a high pressure (150 to 350 bar) [210] was developed for industrial production of lignans. The ex- traction is carried out at ambient temperature under nearly anaerobic conditions. Extraction of lignans with supercritical butane is protected by a Chinese patent [211]. Seed or oil extracts for the purification of lignans can be treated with glucosidases or exposed to alkaline conditions to hydrolyze lignan glucans, increasing hereby the yield of aglycons (see Section 2.2.2). 2.6. Variation in Lignan Content among Accessions of Sesame The content of lignans in sesame seeds varies by an order of magnitude. Differences in lignan content among varieties and accessions have been documented in dozens of studies (e.g., [54,65,73,101,152]), but a comprehensive overview of these results is missing. All furofuran lignans in sesame originate from the same pathway, therefore their concentrations are supposed to correlate. Within a set of 65 varieties developed by the sesame breeding company Sesaco Corporation, which included varieties with white, yellow, brown and black seeds, strong positive correlations were established between the content of sesamin and sesamolin (R2 = 0.69), as well as sesaminol and sesamolinol (R2 = 0.53) [101]. Interestingly, correlations between sesamin and sesaminol, sesamin and sesamolinol, sesamolin and sesaminol, and sesamolin and sesamolinol were negative (R2 = 0.37, 0.36, 0.35, and 0.46, respectively) [101]. Our results obtained with a set of 25 sesame varieties from different parts of the world have not confirmed negative correla- tions of sesamin with sesaminol nor sesamolin with sesaminol (unpublished data). The comparison of sesamin and sesamolin content in 21 hybrids of Thailand varieties of sesame revealed a strong positive correlation [185]. Similar results were reported from Japan [212], China [60], and India [61,94,213]. Tashiro and coworkers [214] reported very tight positive correlations between sesamin and sesamolin content in 42 varieties from Asia and Latin America, covering a wide range of agronomic characteristics. The content of sesamin was significantly higher in white and brown seeds than in black seeds; no consistent trend was found for sesamolin. The correlation between the content of sesamin and sesamolin in black and brown seeds was tight (r = 0.778 and 0.903, respectively); weaker correlation between sesamin and sesamolin was found in white seeds (r = 0.386). Comparison of lignan content in 43 varieties of sesame from all climatic zones in India showed that the content of sesamin and sesamolin was higher in black seeds than in white and brown ones [61]. Another study from India found no relationship between seed color and lignan content [94]. In Chinese varieties of sesame, lignan content was higher in white seeds than in black ones [60]. Thus, it appears that seed color and lignan content in sesame are unrelated; the associations reported in some studies may be due to limited genetic diversity in the germplasm collections used. Figure 8 shows the content of sesamin and sesamolin as reported for varieties and accessions from three continents. The source data and references for this overview are provided in Supplementary Information. The overview confirms a positive correlation between the content of sesamin and sesamolin. No relationship between the geographical Molecules 2021, 26, 883 15 of 52 origin and lignan content is apparent, which was expected because sesame is an old crop with a long history of seeds trade [1,215]. Molecules 2021, 26, x FOR PEER REVIEW 17 of 55 sesame are unrelated; the associations reported in some studies may be due to limited genetic diversity in the germplasm collections used. Figure 8 shows the content of sesamin and sesamolin as reported for varieties and accessions from three continents. The source data and references for this overview are provided in Supplementary Information. The overview confirms a positive correlation between the content of sesamin and sesamolin. No relationship between the geographical origin and lignan content is apparent, which was expected because sesame is an old crop with a long history of seeds trade [1,215]. The relationship between the genetic relatedness of sesame accessions and their lignan production has not been investigated. It is only known that patterns of metabolic diversity in general are incongruent with the genetic relatedness in sesame [216]. This sit- uation is common in plants [217–220] and we assume that it holds for lignans of sesame, too. Secondary metabolite production is subjected to a strong selection pressure while surveys of genetic diversity rely on neutral markers such as amplified fragment length polymorphism AFLP [221] and microsatellites [222]. Patterns of genetic diversity obtained with these methods cannot be used as a predictor of lignan production. Molecular markers linked to the loci affecting lignan synthesis are needed; the development of such markers is described in Section 4.3. Comparison of varieties regarding their lignan content in tissues other than seeds has only been reported in a single study. According to Kareem and coworkers [223], the dif- ferences in sesamin content of roots and hairy root among accessions and varieties of Sesamum indicum were larger than the differences among the sesamin content in seeds (Table 4). Lignans other than sesamin were not detected in roots or hairy roots [223]. Figure 8. Variation of lignan content in sesame seeds. Lignan content is given in mg/g. Asterisk indicates that sesamolin content was not determined. 2.7. Lignans in Other Tissues and Organs of Sesamum indicum Virtually all research on the lignans of sesame was carried out on seeds and oil ob- tained from seeds. The synthesis of lignans is, however, not limited to seeds (Table 4). Callus cultures from Sesamum indicum accumulated sesamin [224–226] and sesamolin [225,226]. According to Ogasawara and coworkers [226], sesamin and sesamolin were re- ported from callus cultures for the first time in 1987 by the Biotechnology Research Labor- atory of Kobe Steel Ltd., Tsukuba, Japan [227–229] (in Japanese). Sesamol, sesaminol and sesamolinol were not detectable in callus [225,226]. The lack of sesamol in callus cultures Figure 8. Variation of lignan content in sesame seeds. Lignan content is given in mg/g. Asterisk indicates that sesamolin content was not determined. The relationship between the genetic relatedness of sesame accessions and their lignan production has not been investigated. It is only known that patterns of metabolic diversity in general are incongruent with the genetic relatedness in sesame [216]. This situation is common in plants [217–220] and we assume that it holds for lignans of sesame, too. Secondary metabolite production is subjected to a strong selection pressure while surveys of genetic diversity rely on neutral markers such as amplified fragment length polymorphism AFLP [221] and microsatellites [222]. Patterns of genetic diversity obtained with these methods cannot be used as a predictor of lignan production. Molecular markers linked to the loci affecting lignan synthesis are needed; the development of such markers is described in Section 4.3. Comparison of varieties regarding their lignan content in tissues other than seeds has only been reported in a single study. According to Kareem and coworkers [223], the differences in sesamin content of roots and hairy root among accessions and varieties of Sesamum indicum were larger than the differences among the sesamin content in seeds (Table 4). Lignans other than sesamin were not detected in roots or hairy roots [223]. 2.7. Lignans in Other Tissues and Organs of Sesamum indicum Virtually all research on the lignans of sesame was carried out on seeds and oil ob- tained from seeds. The synthesis of lignans is, however, not limited to seeds (Table 4). Callus cultures from Sesamum indicum accumulated sesamin [224–226] and sesamolin [225,226]. According to Ogasawara and coworkers [226], sesamin and sesamolin were reported from callus cultures for the first time in 1987 by the Biotechnology Research Laboratory of Kobe Steel Ltd., Tsukuba, Japan [227–229] (in Japanese). Sesamol, sesaminol and sesamoli- nol were not detectable in callus [225,226]. The lack of sesamol in callus cultures was confirmed in an independent study [230]. Hairy root cultures produced sesamin but no other lignans [231]. Interestingly, the content of sesamin in hairy roots was higher than in roots [231]. Molecules 2021, 26, 883 16 of 52 Table 4. Lignans of Sesamum indicum found in tissues different from seeds. Tissue Lignans (µg/g) Reference Sesamin Sesamolin Callus 63–460 270–950 [224–226] Leaves 2.6 - [232] Leaves, stem, roots, and flower detected detected [232] * Roots 0–220 not detected [231] Hairy roots 0–75 not detected [231] * The presence of the metabolites was claimed but no analytical data are provided. Sesamin was also reported from sesame leaves [232] and roots [231]. Sesamin concen- trations in leaves were ca. 5000-times lower than the concentrations in seeds of the same varieties [232]. In the roots of most of 25 sesame varieties studied, the concentration of sesamin has not exceeded 10 mg/kg, which is 70 to 900-times less than the sesamin content in seeds (cf. Table 1 and Figure 8). Kareem [231] did not find lignans other than sesamin in the roots. Fuji and coworkers [233], studying non-lignan metabolites of sesame, reported that they found sesamin and sesamolin in leaves, stem, root, and flower of sesame. The analytical method used was HPLC with both UV absorption and ESI-MS detection. The authors claimed that they validated their results using HPLC-ESI-MS/MS. The publication, however, does not show any analytical data on lignans [233]. We have not found sesamin or sesamolin in any part of sesame plants except for capsules with seeds, leaves, and roots (unpublished data). As shown in Table 4, lignans do not accumulate in organs and tissues other than seeds to appreciable levels. The repertoire of lignans in callus tissue is limited to sesamin and sesamolin, while sesamin also accumulated in the roots and hairy roots of sesame [231]. Sesamin content in the leaves and young and old callus amounted to less than 5% of the levels in seeds; similarly, sesamolin content in young and old callus amounted to less than 5% of the levels reported in seeds (Table 4) [225,226,232]. Lignans other than sesamin and sesamolin were not reported from organs other than seeds nor from tissue cultures of sesame. 2.8. Lignans in Wild Relatives of Sesame Several lignans known from S. indicum and new lignans not known from cultivated sesame have been found in wild relatives of sesame (Table 2). In 1951, Pearman and coworkers [234] as cited by [66] reported sesamin from Sesamum angolense. Bedigian and coworkers [66] screened several wild species of sesame for the presence of sesamin and sesamolin. They confirmed the presence of sesamin and sesamolin in S. angolense and found these two lignans also in the seeds of S. angustifolium, S. calycinum, and in S. orientale var. malabaricum. Only sesamin was found in S. latifolium and S. radiatum. In addition, trace amounts of sesamin and sesamolin were also observed in S. petaloides and S. capense, respectively. Bedigian and coworkers [66] also reported the presence of lignans in the seeds of related genera: sesamin was found in Ceratotheca sesamoides, and both sesamin and sesamolin were found in Sesamothamnus busseanus. In addition, the authors found trace amounts of sesamolin in Ceratotheca sesamoides and trace amounts of sesamin in Ceratotheca triloba, Holubia saccata, Pedalium murex, and Pretrea zanguebaricum. Lignans from wild relatives of sesame that are not present in S. indicum are (+)- sesangolin, (+)-alatumin, (+)-2-episesalatin, and (+)-7′-episesantalin (Table 2). Furofuran lignan (+)-sesangolin was purified from S. angolense in a search for new synergists because seed oil of S. angolense possessed unusually high synergistic activity with pyrethrum [91]. Sesangolin was later reported from S. angustifolium [87], S. alatum [88], and S. radiaum [92]. Both alatumin [89] and episesalatin [90] were found in S. alatum. The most recent contri- bution to the diversity of sesame lignans is (+)-7′-episesantalin, which was purified from S. radiatum [92]. Molecules 2021, 26, 883 17 of 52 Apart from a limited specificity of TLC used for lignan analysis in old studies, some studies of lignans in wild relatives of cultivated sesame were plagued by erroneous tax- onomic assignments. S. latifolium was mistaken for S. radiatum in two studies [87,179] published in 1994, as the author noted in her subsequent publication from 2010 [95] (see footnote 4 in Table 2). Thus, papers published between 1994 and 2010 might have spread the wrong data; an example is an industrial compendium on oils [235]. Recently, a Suntory Foundation for Life Sciences and their collaborators in Japan [92] re-investigated the con- tent of lignans in seeds of S. radiatum, confirmed sesamin and sesamolin, and discovered the sesangolin isomer (+)-7′-episesantalin. Likewise, they re-discovered the presence of (+)-sesangolin, apparently not aware of the publication by Su Rho Ruy and coworkers from 1993 in Korean language, in which sesangolin was reported from S. radiatum for the first time [88]. Some controversies about taxonomic assignments in these studies remain unresolved. For instance, in 2010 Kamal-Eldin [95] reported the content of sesamin and sesamolin in S. latifolium as published in 1988 in a Japanese journal [72], but in the cited publication S. radiatum rather than S. latifolium was named. Kamal-Eldin [95] did not explain why she assumed that S. radiatum in this publication [72] was actually S. latifolium. Future studies relying on HPLC coupled with tandem mass spectrometric or high- resolution mass-spectrometric detection will likely render old results based on TLC obsolete. Apart from the accessibility of more powerful analytical techniques, the lesson learned from the past is that chemists analyzing wild relatives of sesame should always seek assistance of plant taxonomists specialized in Pedaliaceae to verify the taxonomic assignment of their samples. Species-specific DNA barcodes, which allow reliable taxonomic assignments to non-specialists, are not available in Sesamum spp. yet. Because the content of lignans varies greatly among accessions of the same species (e.g., [94], see also Section 2.6), reports of phytochemical analysis should include information about the origin of the samples. Many past studies provided limited or no information about their samples, impeding comparison among studies. Discoveries of new lignans in wild relatives of cultivated sesame will motivate research about the genes and enzymes involved in their biosynthesis. 3. Biosynthesis of Lignans 3.1. Biosynthesis of Furofuran Lignans in Sesame The biosynthesis of lignans in plants has been reviewed [236,237], but no current review focusing on sesame is available. Lignans of sesame and other plant species originate from the oxidative dimerization of two molecules of coniferyl alcohol, which is the central metabolite of the phenylpropanoids pathway [238]. The condensation of coniferyl alcohol yields pinoresinol through a laccase enzyme while the stereoselectivity of the reaction is controlled by a dirigent protein [85,239] (Figure 3). Dirigent proteins are widespread in land plants. They are involved in the dimerization of molecules through radical-radical coupling [240,241]. In the case of pinoresinol formation, the first step consists of the formation of radicals of substrate coniferyl alcohol, which is presumably catalyzed by a laccase enzyme, producing free monolignol radicals (CA·). Two CA·molecules bind to a dirigent protein, which enables stereospecific dimerization to either (+) or (-)-pinoresinol, depending on the plant species [239]. Dirigent proteins form pockets with conserved amino acid residues that control the orientation of the substrate [239]. In the case of S. indicum, there is a putative dirigent protein, XP_011080883 [27], in which most of the differing amino acids match those of the (+) enantiomer, in line with the predicted (+)-pinoresinol in this plant species. Until 2006, it was believed that in S. indicum, (+)-pinoresinol was converted to (+)- piperitol and then to (+)-sesamin by two consecutively acting P450 enzymes tentatively designated piperitol synthase and sesamin synthase [242]. However, in 2006 Ono and colleagues showed that in S. indicum, the same cytochrome P450 enzyme (CYP81Q1) cat- alyzes the formation of both, (+)-piperitol and (+)-sesamin, through dual methylenedioxy Molecules 2021, 26, 883 18 of 52 bridge formation (Figure 9, [85]). Therefore, the enzyme was named piperitol/sesamin synthase (Figure 9, Table 5). In 2019, the structure–function relationship of this enzyme from S. indicum was confirmed in a heterologous system, and it was demonstrated that the CYP reductase1 gene product (CPR1) is needed for pinoresinol to sesamin conversion to facilitate electron transfer from NADPH to the CYP81Q1 enzyme [243]. Molecules 2021, 26, x FOR PEER REVIEW 21 of 55 Figure 9. Lignan biosynthetic pathway in Sesamum indicum. 1 = laccase, 2 = dirigent protein, 3 = piperitol/sesamin synthase (CYP81Q1), 4 = sesamolin/sesaminol synthase (CYP92B14), 5 = UGT71A9, 6 = UGT94AG1, 7 = UGT94D1/UGT94AA2. Identified and postulated conversions are represented by solid and broken lines, respectively. The sequential conversion from pinoresinol to secoisolariciresinol and to matairesinol is proposed in sesame based on Forsythia intermedia biosynthetic pathway [244,245]. The convertion of sesamin to 7′-episesantalin has been recently proposed to occur in S. radiatum [92]. Figure 9. Lignan biosynthetic pathway in Sesamum indicum. 1 = laccase, 2 = dirigent protein, 3 = piperitol/sesamin synthase (CYP81Q1), 4 = sesamolin/sesaminol synthase (CYP92B14), 5 = UGT71A9, 6 = UGT94AG1, 7 = UGT94D1/UGT94AA2. Identified and postulated conversions are represented by solid and broken lines, respectively. The sequential conversion from pinoresinol to secoisolariciresinol and to matairesinol is proposed in sesame based on Forsythia intermedia biosynthetic pathway [244,245]. The convertion of sesamin to 7′-episesantalin has been recently proposed to occur in S. radiatum [92]. Table 5. Proteins involved in the biosynthesis of sesame lignans. Protein Name/Accession Type of Enzyme Function in the Pathway EC Number XP_011080883 * Dirigent protein Impart stereoselectivity on the phenoxy radical-coupling reaction - CYP81Q1 Cytochrome P450 Piperitol/sesamin synthase. Formation of dual methylenedioxy bridge 1.14.19.74 CYP92B14 Cytochrome P450 Sesamolin/sesaminol synthase.Converts sesamin to sesamolin or sesaminol 1.14.19.- UGT71A9 Glycosyltransferase Catalyzes the glucosylation at the 2-hydroxyl group of sesaminol 2.4.1.- UGT94D1/UGT94AA2 Glycosyltransferase Catalyzes the β1→6 glucosylation toward the sugar moiety of sesaminol 2-O-glucoside 2.4.1.- UGT94AG1 Glycosyltransferase Catalyzes the β1→2-O-glucosylation of sesaminol mono glucoside and sesaminol 2-O-β-D-glucosyl-(β1→6)-O-β-D-glucoside 2.4.1.- XP_011092597 (SinPLR2) * Bifunctional pinoresinol-lariciresinol reductase Conversion of pinoresinol into lariciresinol and lariciresinol into secoisolariciresinol 1.23.1.1 XP_011094269 * Secoisolariciresinol dehydrogenase-like Conversion of secoisolariciresinol into matairesinol 1.1.1.331 * Predicted based on sequence similarity. Molecules 2021, 26, 883 19 of 52 Ono and coworkers [85] found homologs of CYP81Q1 protein in S. radiatum (CYP81Q2) and S. alatum (CYP81Q3). Recently, it was demonstrated that, unlike CYP81Q1 and CYP81Q2, the CYP81Q3 protein from the wild Sesamum species S. alatum, produces only a single methylenedioxy bridge and has diastereomeric selectivity, i.e., it can catalyze the formation of (+)-pluviatilol from (+)-epipinoresinol but it accepts neither (+) nor (-)- pinoresinol as a substrate [89]. Due to the absence of CYP81Q1, S. alatum cannot synthesize molecules with dual methylenedioxy bridges such as (+)-sesamin and (+)-sesamolin. In- stead, this species accumulates furofuran lignans with single methylenedioxy bridges, e.g., (+)-2-episesalatin and (+)-fargesin (Figure 10). The asymmetric configuration of (+)- epipinoresinol is probably the reason for the inability of the CYP81Q3 enzyme to form the second methylenedioxy bridge, and for the hydroxylation or O-methylation of the second aromatic ring of (+)-pluviatilol to form (+)-2-episesalatin [89]. Murata and coworkers [246] showed that CYP92B14, a P450 monooxygenase, is responsible for the oxygenation of (+)-sesamin to form (+)-sesamolin or (+)-sesaminol via oxidative rearrangement of an α-oxy-substituted aryl group or direct hydroxylation, respectively (Figure 9). They also demonstrated the functional coordination between CYP92B14 and CYP81Q1 (both P450 enzymes), in which the activity of the former enzyme is enhanced by CYP81Q1. Sesamin has been suggested to be a precursor of the keto-lignan (+)-episesaminone, another furofuran lignan [86] (Figure 9). Episesaminone was isolated for the first time from unroasted and unbleached sesame seeds as well as from freshly harvested seeds of S. indicum [86]. In 2006, a diglucoside of episesaminone, episesaminone-9-O-β-D- sophoroside, was found in the perisperm of S. indicum seeds [96]. In S. indicum, the biosynthesis of furofuran lignans continues from (+)-sesaminol through a series of glucosylation steps (Figure 9). The final product is sesaminol trigluco- side (STG), which is a water-soluble lignan that accumulates in high amounts in sesame seeds [102]. The three glucosylation steps of (+)-sesaminol are catalyzed by uridine diphos- phate (UDP)-dependent glycosyltransferases (UGTs) (Table 5). The first glucose is always connected to the lignan aglycon via a β-glycosidic bond while the second and third glucose molecules are connected via 1,4- or 1,6-β-glycosidic bonds. The first two glucosylation steps of (+)-sesaminol were previously identified in S. indicum [247]. Only recently, the last missing step was discovered by Ono and cowork- ers [248]. The first step is the 2-O-glycosylation of (+)-sesaminol to produce sesaminol monoglucoside (SMG) by the enzyme UGT71A9. Homologs of UGT71A9 were found in S. radiatum (UGT71A10) and S. alatum (UGT71A8) [247], evidencing the conservation of these steps in Sesamum spp. The glucosylation of SMG leads to diglucoside SDG(β1→6) [(+)-sesaminol 2-O-β-D-glucosyl-(1→6)-O-β-D-glucoside] or SDG(β1→2) through consec- utive glucosylation of the 6′ or 2′ -hydroxyl group of the sugar moiety by the enzyme UGT94D1 or UGT94AG1, respectively (Figure 9) [248]. Until recently, it was not known which glucosylation step of SMG occurs first, β1→6 or β1→2 glucosylation. Ono and coworkers [248] showed that β1→2 followed by β1→6 glucosylation is the major pathway. With this discovery, the missing last step for STG biosynthesis was established. The same authors found an enzyme similar to UGT94D1, called UGT94AA2, which interacts with UGT71A9, UGT94AG1, and, interestingly, also with CYP81Q1 (the piperitol/sesamin syn- thase) producing, what the authors called, an STG metabolon. In this proposed metabolon for STG biosynthesis, CYP81Q1 acts as a membrane bound protein, which increases the efficiency of sequential glucosylation steps by recruiting three UGT enzymes (UGT71A9, UGT94AA2, and UGT94AG1). Molecules 2021, 26, 883 20 of 52 Molecules 2021, 26, x FOR PEER REVIEW 23 of 55 Figure 10. Biosynthetic pathway of selected lignans in different plant species. 1 = laccase, 2 = dirigent protein, 3 = CYP81Q3 in S. alatum [89], 4 = PLR1 in L. perenne [243], 5 = SDH in F. intermedia [244], 6 = CYP719A23 in P. hexandrum [249], 7-12 = OMT3, CYP71Cu1, OMT1, 2-ODD, CYP71BE54 and CYP82D61 in P. hexandrum, 13-14 = DOP6H, βP6OMT in L. flavum. Identified and postulated conversions are represented by solid and broken lines, respectively. Two arrows indicate several consecutive steps. Figure 10. Biosynthetic pathway of selected lignans in different plant species. 1 = laccase, 2 = dirigent protein, 3 = CYP81Q3 in S. alatum [89], 4 = PLR1 in L. perenne [243], 5 = SDH in F. intermedia [244], 6 = CYP719A23 in P. hexandrum [249], 7–12 = OMT3, CYP71Cu1, OMT1, 2-ODD, CYP71BE54 and CYP82D61 in P. hexandrum, 13–14 = DOP6H, βP6OMT in L. flavum. Identified and postulated conversions are represented by solid and broken lines, respectively. Two arrows indicate several consecutive steps. 3.2. Biosynthesis of Other Lignans in Sesame Pinoresinol is the precursor of the main furofuran lignans in sesame, i.e., the hy- drophobic lignans (+)-sesamin and (+)-sesamolin and the water-soluble STG. However, pinoresinol is also the precursor of dibenzylbutyrolactone class of lignans, which in most plants are produced via reductive cleavage of furofuran rings by pinoresinol–lariciresinol reductases (PLR). These enzymes convert pinoresinol to lariciresinol and then to secoiso- lariciresinol [245] (Figure 9, Table 5). In sesame, SinPLR1 (XP_011092596) and SinPLR2 (XP_011092597) (Table 5) are putative PLRs; the latter one is similar to a PLR in Forsythia intermedia (FiPLR1), which reduces preferably (+)-pinoresinol to (+)-lariciresinol, while Sin- PLR1 is similar to MgPLR1 in Mimulus guttatus [245]. The oxidation of secoisolariciresinol to matairesinol by secoisolariciresinol dehydrogenase (SDH) occurs in some plants such as F. intermedia. In this species, the protein SDH_Fi321 converts (−)-secoisolariciresinol to (−)- matairesinol [244] (Figure 10). We have found a gene encoding similar protein in sesame genome (Table 5, XP_011094269, E-value: 4 × 10−149, percentage identity: 73.8%), which is a candidate for the homolog of SDH_Fi321. The activity remains to be demonstrated experimentally. Interestingly, the gene was upregulated in mature seeds as compared to young seeds (Andargie, Vinas and Karlovsky, unpublished data). 3.3. Biosynthesis of Lignans in Other Plant Species Lignans are not ubiquitous in the plant kingdom, yet they occur in phylogenetically distant vascular plants [250]. The knowledge of lignan pathways in different plants helps understanding the evolution of lignan biosynthesis, and the genes of lignan biosynthesis from other plants facilitate search for yet unknown genes of the pathway in sesame. Plants in which lignans were characterized include species from the orders Asterales (Arctium lappa) [251], Ericales (Lyonia ovalifolia) [252], Lamiales (Sesamum spp., Forsythia spp.) [252,253], Malpighiales (Linum spp.) [254], and Malvales (Wikstroemia sikokiana) [255]. Molecules 2021, 26, 883 21 of 52 Biosynthetic studies have been made mainly in lignan-rich plant species of the genera Sesamum, Forsythia and Linum. In the genus Sesamum, lignan biosynthesis was studied mainly in S. indicum; other lignans have been characterized in S. alatum (e.g., 2-episesalatin and fargesin) [89] (Figure 10). Among the eight classes of lignans [250], the most studied are furofuran, dibenzylbu- tyrolactone, and arylteralin lignans. All of them are produced from pinoresinol through the same initial step, i.e., oxidative dimerization of coniferyl alcohol (Figures 3, 9 and 10). Furofuran lignans are produced by Sesamum spp. (e.g., sesamin, sesamolin, sesaminol and phylligenin) and Forsythia (e.g., epipinoresinol and phylligenin) [256–259]. Dibenzylbuty- rolactone lignans are produced also by Forsythia spp. (e.g., arctigenin) [256], Linum spp. (e.g., yatein) [259] and possibly also by Sesamum spp. [245]. Podophyllum spp. and Linum spp. produce aryltetralin lignans (e.g., podophyllotoxin) [260–262]. Phylligenin is produced from pinoresinol via epipinoresinol in F. intermedia and S. alatum [89,256], whereas 2-episesalatin is produced in S. alatum via pluviatilol through consecutive steps of hydroxylation and O-methylation [89] (Figure 10). Arctigenin is produced from the O-methylation of matairesinol in F. intermedia [256,263]. Podophyl- lotoxin, an aryltetralin lignan with anticancer properties, is produced from matairesinol via pluviatolide in Linum and Podophyllum species [264] (Figure 10). The cytochrome P450 enzyme responsible for matairesinol to pluviatolide via methylenedioxy bridge for- mation were identified in P. hexandrum (CYP719A23) and P. peltatum (CYP719A24) by Marques and collaborators [249]. The next steps from pluviatolide were published for P. hexandrum in 2015 by Lau and Sattely [265]. The enzyme O-methyltransferase (OMT3) catalyzes the methylation of pluviatolide to form 5′-desmethoxy-yatein, which is then hydroxylated by CYP71CU1 to 5′-desmethyl-yatein. The methylation of this intermediate by enzyme OMT1 produces yatein (a native substrate for ring closure) (Figure 10). The biosynthesis of deoxypodophyllotoxin is catalyzed by the enzyme deoxypodophyllotoxin synthase (2-ODD) from yatein forming the core of the aryltetralin scaffold by oxidative ring closure [265]. The next steps to form podophyllotoxin in P. hexandrum have not been identified yet. However, Lau and Sattely [265] demonstrated that two different P450 enzymes (CYP71BE54 and CYP82D61) produce 4-desmethyl-deoxypodophyllotoxin and 4-desmethyl-epipodophyllotoxin (an etoposide lignan) from deoxypodophyllotoxin, re- spectively (Figure 10). In L. flavum, the biosynthesis of 6-methoxypodophyllotoxin (a cytotoxic lignan) occurs through deoxypodophyllotoxin via three steps, including a 6- hydroxylase (DOP6H) enzyme, which is a cytochrome P450-dependent monooxygenase, followed by O-methylation by β-peltatin 6-O-methyltransferase (βP6OMT) enzyme and 7-hydroxylation with a yet unknown enzyme [262,266]. On the other hand, although the enzyme has not been characterized, hydroxylation of carbon 7 of deoxypodophyllotoxin is the putative step that produces podophyllotoxin in various Linum species [261] (Figure 10). Lignans normally accumulate as glucosides. In Forsythia and Linum, the enzyme UGT71A18 is responsible for the glucosylation of furofuran lignans [(+)-pinoresinol, (+)- epipinoresinol and (+)-phylligenin] [267], while UGT74S1 is responsible for the gluco- sylation of the dibenzylbutyrolactone lignan (+)-seicolariciresinol [268]. Enzymes re- sponsible for the glucosylsation of matairesinol and arctigenin are still unknown. In 2016, three Forsythia UGTs were suggested for matairesinol glucosylation (CL10456contig1, CL14684contig1 and CL15275contig1) based on molecular analyses of virtual primer-based sequences assembly (VP-seq) [264]. 4. Genetics of Lignan Synthesis 4.1. Genes Involved in Lignan Synthesis in Sesame The genome of sesame (2n = 26) is composed of 16 linkage groups (LGs). The first two genomes of sesame were sequenced in 2013 [21] and 2014 [27]. Since then, many genomes of S. indicum have been sequenced. In large-scale effort to associate agronomic traits with genetic variations, genomes of 705 sesame accessions were re-sequenced [27]. The assembly Molecules 2021, 26, 883 22 of 52 obtained in 2014 is still used as a reference genome, which is 357 Mb large and contains 27,148 predicted protein-coding genes [27]. Although most enzymes involved in the biosynthesis of furofuran lignans in sesame are known, most of the genes remain uncharacterized. The genes were putatively identi- fied by computational analysis using gene prediction methods (Table 6). These 9 genes are located on 8 LGs. Although secondary metabolite pathways in plants are often scat- tered [269,270] while only few pathways are encoded by gene clusters [271], the scattering of the lignan pathway in S. indicum is remarkable. Table 6. Genes involved in the synthesis of lignans in sesame. NCBI Accession Number or Gene ID Gene Name GC Content in Exons (%) Nc Value * Number of Introns Linkage Group ** LOC105164033 —- 52.0 56.06 0 LG6 AB194714 CYP81Q1 51.2 57.39 1 LG15 LC199944 CYP92B14 43.1 52.34 1 LG2 AB194716 CYP81Q3 51.3 57.02 0 (S. alatum) AB293960 UGT71A9 47.6 59.01 0 LG16 AB333799 UGT94D1 49.8 53.13 0 LG4 LC484014 UGT94AA2 53.6 53.44 0 LG7 LC484013 UGT94AG1 40.6 50.50 0 LG10 LOC105172736 SinPLR2 45.2 57.93 3 LG10 LOC105174016 —- 46.6 56.82 1 LG11 * Nc (effective number of codons) is a measure of the deviation of codon usage from the equal use of all codons [272]. ** The linkage group No. refers to S. indicum. The GC content of the exons of these genes varies from 40.6% to 53.6%. After removal of two genes with an unusually low GC content (Acc. Nos. LC199944 and LC484013), the range narrowed to 45.2–53.6%, which is similar to the average GC content of all protein- coding regions in the genome of sesame [26,27]. The low GC content of LC199944 and LC484013 is reflected by the low effective number of codons (Nc), which is a measure of codon usage bias [272]. Why is the GC content of these two genes so low? Inspection of putative translation products reveals a high content of amino acids with AT-rich codons. For instance, the protein product of LC484013 has the highest relative content of Phe and Ile, and together with the LC199944 the highest content of Asn and Tyr, among all proteins encoded by the genes listed in Table 6. Therefore, protein composition, resulting from a selection pressure on enzyme function, likely accounted for the unusually low GC content of the two genes. 4.2. Expression of Genes of the Lignan Biosynthetic Pathway in Sesame Most studies of the expression of lignans pathway in sesame used seeds of different maturity. For example, the expression of the genes encoding putative dirigent protein (NCBI geneID = LOC105164033) and for piperitol/sesamin synthase (CYP81Q1) was higher in early-mid than in late developmental stages of the seeds (10-20 days post-anthesis) [27]. The expression profile of the CYP92B14 gene coincided with that of CYP81Q1, suggesting a catalytic cooperation between both enzymes, as demonstrated by Murata et al. [246]. These authors also expressed CYP81Q1, CYP92B14, and CPR1 simultaneously in a yeast heterologous system. Using (+)-sesamin as a substrate, high amounts of (+)-sesaminol and (+)-sesamolin were produced in the heterologous system expressing only CYP92B14 and CPR1. CYP81Q1 or CYP92B14 alone did not show any catalytic activity. The gene CPR1, needed for the enzymatic activity of CYP81Q1, is expressed throughout the entire seed development [246]. Wei and coworkers [26] showed that genes related to oil biosynthesis in sesame seeds were strongly associated with sesamin and sesamolin content. For example, a single nu- cleotide polymorphism within the gene SiNST1 (SIN_1005755, NCBI geneID = LOC105173057) defined two alleles, “A” and “C”. The “A” allele was associated with a low content of oil and protein and also with a low content of sesamin and sesamolin, but with a high content of lignin and an accumulation of woody tissue in seeds. The “C” allele was associated with Molecules 2021, 26, 883 23 of 52 a higher content of oil, protein, sesamin and sesamolin, but with a low content of lignin. The gene was strongly expressed in young seeds [26]. In our work, the gene was expressed in mature seeds to a higher level than in young seeds (Andargie, Vinas and Karlovsky, unpublished data). Recently, it was shown that sesamin binds steroleosin B, a membrane protein found in oil bodies of sesame seedlings [273]. The gene encoding steroleosin B was expressed throughout the entire development of seeds. Arabidopsis thaliana accumulating sesamin and steroleosin B due to the expression of sesame synthase and steroleosin B genes from sesame displayed severe growth defects, indicating that the complex of steroldeosin B and sesamin might interact with signal transduction pathways controlling plant development (see Section 6). Our own gene expression experiments with young and mature seeds of sesame corroborated that the genes involved in lignan biosynthesis, i.e., CYP81Q1 catalyzing the synthesis of (+)-sesamin, and CYP92B14 catalyzing the synthesis of (+)-sesaminol and (+)- sesamolin, were expressed to higher levels in young seeds than in mature seeds. In contrast, the gene SiNST1, which apparently affects partition of phenylpropanoids into lignans and monolignols (see above), was stronger expressed in mature seeds. Similarly, we found that the gene putatively involved in the conversion of secoisolariciresinol into matairesinol (NCBI geneID of LOC105174016) was expressed in mature seeds to a higher level than in young seeds (Andargie, Vinas and Karlovsky, unpublished data). The activation of lignan synthesis at later phases of seed development is also supported by the report that lignans were absent from young seeds of sesame [85]. In line with the focus of lignan research in sesame on the seeds, expression of the lignan pathway in sesame was studied nearly exclusively in seeds. In 2006, Ono and coworkers [85] in their characterization of piperitol/sesamin synthase reported that the gene (designated CYP81Q1, see Table 5 and Figure 9) was expressed in maturing seeds and in leaves but not in leaf petioles or stems. They have not found sesamin or any other lignan in the leaves. The homologue of CYP81Q1 in Sesamum radiatum, designated CYP81Q2, was expressed only in seeds [85]. The expression of CYP81Q1 in the leaves of S. indicum was confirmed by Hata and coworkers [232], who also detected sesamin in the leaves for the first time. Comparison of two varieties of sesame revealed large differences in the expression of CYP81Q1 and the content of sesamin in the leaves. Expression of CYP81Q1 at a very low level was also detected in the stems of young plants, though no sesamin was detected in these samples [232]. Continuous light for two weeks dramatically increased the expression of CYP81Q1 in the leaves of sesame, resulting in increased accumulation of lignans in leaves [274]. This finding could be exploited for the commercial production of sesamin from the waste left after threshing, especially if lignan content in seeds can be increased genetically (see Section 7.1). 4.3. Molecular Markers and Heritability of Lignan Synthesis Large differences in lignan content among accessions and varieties (see Section 2.6) indicate the existence of genetic polymorphism, which can be exploited for breeding. For the improvement of sesame as functional food, it is important to understand the heritability of the content of major lignans in sesame seeds. Data on the inheritance of lignan content are limited. Secondary metabolite patterns in sesame are incongruent with the genetic related- ness [216], similarly to other plants [218,219]. Molecular markers are needed for the prediction of secondary metabolite production. A gene associated with sesamin and sesamolin accumulation was identified in a massive GWAS (genome-wide association study) based on full genomes of 705 sesame varieties, which lead to the identification of genes controlling oil yield [29]. One of these genes, designated SiNST1, also controlled lignan synthesis. Seeds of varieties carrying an SiNST1 allele associated with reduced content of oil, sesamin, and sesamolin, also contained more lignin. This observation was Molecules 2021, 26, 883 24 of 52 consistent with the function of a homologous gene NST1 in Arabidopsis thaliana, which controls the synthesis of secondary cell wall [275]. In a study from the National Institute of Crop Science in Tsukuba, Japan [212], F2 pop- ulations derived from accessions with high and low lignan content showed a continuous distribution of sesamin and sesamolin levels, indicating that lignan content was controlled polygenically. The heritability of lignan content was high, hence selection of lines with a high or low lignan content was possible. In a separate study on F5 and F6 recombinant inbred lines (RILs) from a cross between an accession with high lignan content and a sesamolin deficient accession, Yamamoto [276] showed that the content of sesamin and the sum of sesamin, sesamolin and sesaminol triglucoside were inherited as polygenic traits. In contrast, the content of sesamolin and sesaminol triglucoside were controlled by a single gene and several genes, respectively. A dense set of molecular markers was developed for F6 RILs originating from a cross between parents with contrasting metabolic profiles [31]. This material may facilitate further analysis of the loci identified by Yamamoto [276]. The first sesame variety bred for high lignan content was reported in 2017 from Japan [277]. The inheritance and general combining ability (GCA) of sesamin and sesamolin content were investigated at Kalasin University, Thailand [185]. Not surprisingly, the inheritance of sesamin and sesamolin content exhibited high combining ability, with both additive and dominant effects controlling the lignan content. Positive correlation between the content of different furofuran lignans (see Section 2.6) indicates that the rise of the content of any lignan will likely lead to an increase of the content of the other lignans. Breeding efforts to enhance lignan content in sesame were recently reported from India [213]. The authors confirmed a high general combining ability of sesamin and sesamolin content. Unfortunately, high-content parents were only crossed with a low-content tester but not with each other; therefore, none of the progeny reached the lignan levels of the best parents. With systematic efforts targeting lignans just starting, breeding for lignan content in sesame is in its infancy. 5. Biological Activities of Lignans 5.1. Lignans as Health Promoting Agents: From Folk Medicine to Food Additives It has been recognized for a long time that consumption of sesame benefits health [10,11,42,278]. As early as in 1940’s, injection or consumption of sesame oil was reported to prolong the life span, increase the number of pregnancies, and improve the ability to rear progeny in rats [279,280]. Later studies attributed some of these effects to lignans. Since then, plethora of reports supported the assessment of lignan-rich sesame products as functional food that helps preventing diseases an