Lignans David Barker www.mdpi.com/journal/molecules Edited by Printed Edition of the Special Issue Published in Molecules molecules Lignans Lignans Special Issue Editor David Barker MDPI • Basel • Beijing • Wuhan • Barcelona • Belgrade Special Issue Editor David Barker University of Auckland New Zealand Editorial Office MDPI St. Alban-Anlage 66 4052 Basel, Switzerland This is a reprint of articles from the Special Issue published online in the open access journal Molecules (ISSN 1420-3049) from 2018 to 2019 (available at: https://www.mdpi.com/journal/molecules/ special issues/lignans). For citation purposes, cite each article independently as indicated on the article page online and as indicated below: LastName, A.A.; LastName, B.B.; LastName, C.C. Article Title. Journal Name Year , Article Number , Page Range. ISBN 978-3-03897-908-1 (Pbk) ISBN 978-3-03897-909-8 (PDF) c © 2019 by the authors. 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Contents About the Special Issue Editor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix Preface to ”Lignans” . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xi David Barker Lignans Reprinted from: Molecules 2019 , 24 , 1424, doi:10.3390/molecules24071424 . . . . . . . . . . . . . . 1 Yongbei Liu, Yupei Yang, Shumaila Tasneem, Nusrat Hussain, Muhammad Daniyal, Hanwen Yuan, Qingling Xie, Bin Liu, Jing Sun, Yuqing Jian, Bin Li, Shenghuang Chen and Wei Wang Lignans from Tujia Ethnomedicine Heilaohu: Chemical Characterization and Evaluation of Their Cytotoxicity and Antioxidant Activities Reprinted from: Molecules 2018 , 23 , 2147, doi:10.3390/molecules23092147 . . . . . . . . . . . . . . 5 Jiwon Baek, Tae Kyoung Lee, Jae-Hyoung Song, Eunyong Choi, Hyun-Jeong Ko, Sanghyun Lee, Sang Un Choi, Seong Lee, Sang-Woo Yoo, Seon-Hee Kim and Ki Hyun Kim Lignan Glycosides and Flavonoid Glycosides from the Aerial Portion of Lespedeza cuneata and Their Biological Evaluations Reprinted from: Molecules 2018 , 23 , 1920, doi:10.3390/molecules23081920 . . . . . . . . . . . . . . 16 Ya Li, Shuhan Xie, Jinchuan Ying, Wenjun Wei and Kun Gao Chemical Structures of Lignans and Neolignans Isolated from Lauraceae Reprinted from: Molecules 2018 , 23 , 3164, doi:10.3390/molecules23123164 . . . . . . . . . . . . . . 27 Maria Carla Marcotullio, Massimo Curini and Judith X. Becerra An Ethnopharmacological, Phytochemical and Pharmacological Review on Lignans from Mexican Bursera spp. Reprinted from: Molecules 2018 , 23 , 1976, doi:10.3390/molecules23081976 . . . . . . . . . . . . . . 45 Patrik Eklund and Jan-Erik Raitanen 9-Norlignans: Occurrence, Properties and Their Semisynthetic Preparation from Hydroxymatairesinol Reprinted from: Molecules 2019 , 24 , 220, doi:10.3390/molecules24020220 . . . . . . . . . . . . . . 65 Xianhe Fang and Xiangdong Hu Advances in the Synthesis of Lignan Natural Products Reprinted from: Molecules 2018 , 23 , 3385, doi:10.3390/molecules23123385 . . . . . . . . . . . . . . 77 Samuel J. Davidson, Lisa I. Pilkington, Nina C. Dempsey-Hibbert, Mohamed El-Mohtadi, Shiying Tang, Thomas Wainwright, Kathryn A. Whitehead and David Barker Modular Synthesis and Biological Investigation of 5-Hydroxymethyl Dibenzyl Butyrolactones and Related Lignans Reprinted from: Molecules 2018 , 23 , 3057, doi:10.3390/molecules23123057 . . . . . . . . . . . . . . 99 Jian Xiao, Guangming Nan, Ya-Wen Wang and Yu Peng Concise Synthesis of (+)- β - and γ -Apopicropodophyllins, and Dehydrodesoxypodophyllotoxin Reprinted from: Molecules 2018 , 23 , 3037, doi:10.3390/molecules23113037 . . . . . . . . . . . . . . 125 Patrik A. Runeberg, Yury Brusentsev, Sabine M. K. Rendon and Patrik C. Eklund Oxidative Transformations of Lignans Reprinted from: Molecules 2019 , 24 , 300, doi:10.3390/molecules24020300 . . . . . . . . . . . . . . 132 v Mayra Ant ́ unez-Mojica, Andr ́ es M. Rojas-Sep ́ ulveda, Mario A. Mendieta-Serrano, Leticia Gonzalez-Maya, Silvia Marquina, Enrique Salas-Vidal and Laura Alvarez Lignans from Bursera fagaroides Affect In Vivo Cell Behavior by Disturbing the Tubulin Cytoskeleton in Zebrafish Embryos Reprinted from: Molecules 2019 , 24 , 8, doi:10.3390/molecules24010008 . . . . . . . . . . . . . . . . 170 Marina Pereira Rocha, Priscilla Rodrigues Valadares Campana, Denise de Oliveira Scoaris, Vera Lucia de Almeida, Julio Cesar Dias Lopes, Julian Mark Hugh Shaw and Claudia Gontijo Silva Combined In Vitro Studies and in Silico Target Fishing for the Evaluation of the Biological Activities of Diphylleia cymosa and Podophyllum hexandrum Reprinted from: Molecules 2018 , 23 , 3303, doi:10.3390/molecules23123303 . . . . . . . . . . . . . . 183 Agnieszka Szopa, Michał Dziurka, Angelika Warzecha, Paweł Kubica, Marta Klimek-Szczykutowicz and Halina Ekiert Targeted Lignan Profiling and Anti-Inflammatory Properties of Schisandra rubriflora and Schisandra chinensis Extracts Reprinted from: Molecules 2018 , 23 , 3103, doi:10.3390/molecules23123103 . . . . . . . . . . . . . . 214 Shuyu Chen, Jingjing Shi, Lisi Zou, Xunhong Liu, Renmao Tang, Jimei Ma, Chengcheng Wang, Mengxia Tan and Jiali Chen Quality Evaluation of Wild and Cultivated Schisandrae Chinensis Fructus Based on Simultaneous Determination of Multiple Bioactive Constituents Combined with Multivariate Statistical Analysis Reprinted from: Molecules 2019 , 24 , 1335, doi:10.3390/molecules24071335 . . . . . . . . . . . . . . 230 Lisa I. Pilkington Lignans: A Chemometric Analysis Reprinted from: Molecules 2018 , 23 , 1666, doi:10.3390/molecules23071666 . . . . . . . . . . . . . . 245 Carmen Rodr ́ ıguez-Garc ́ ıa, Cristina S ́ anchez-Quesada, Estefan ́ ıa Toledo, Miguel Delgado-Rodr ́ ıguez and Jos ́ e J. Gaforio Naturally Lignan-Rich Foods: A Dietary Tool for Health Promotion? Reprinted from: Molecules 2019 , 24 , 917, doi:10.3390/molecules24050917 . . . . . . . . . . . . . . 269 Laurine Garros, Samantha Drouet, Cyrielle Corbin, C ́ edric Decourtil, Thibaud Fidel, Julie Lebas de Lacour, Emilie A. Leclerc, Sullivan Renouard, Duangjai Tungmunnithum, Jo ̈ el Doussot, et al. Insight into the Influence of Cultivar Type, Cultivation Year, and Site on the Lignans and Related Phenolic Profiles, and the Health-Promoting Antioxidant Potential of Flax ( Linum usitatissimum L.) Seeds Reprinted from: Molecules 2018 , 23 , 2636, doi:10.3390/molecules23102636 . . . . . . . . . . . . . . 294 Andr ́ e F. Brito and Yu Zang A Review of Lignan Metabolism, Milk Enterolactone Concentration, and Antioxidant Status of Dairy Cows Fed Flaxseed Reprinted from: Molecules 2019 , 24 , 41, doi:10.3390/molecules24010041 . . . . . . . . . . . . . . . 309 Alessandra Durazzo, Massimo Lucarini, Emanuela Camilli, Stefania Marconi, Paolo Gabrielli, Silvia Lisciani, Loretta Gambelli, Altero Aguzzi, Ettore Novellino, Antonello Santini, Aida Turrini and Luisa Marletta Dietary Lignans: Definition, Description and Research Trends in Databases Development Reprinted from: Molecules 2018 , 23 , 3251, doi:10.3390/molecules23123251 . . . . . . . . . . . . . . 330 vi Delphine Winstel and Axel Marchal Lignans in Spirits: Chemical Diversity, Quantification, and Sensory Impact of ( ± )-Lyoniresinol Reprinted from: Molecules 2019 , 24 , 117, doi:10.3390/molecules24010117 . . . . . . . . . . . . . . 344 Thomas Olof Sandberg, Christian Weinberger and Jan-Henrik Sm ̊ att Molecular Dynamics on Wood-Derived Lignans Analyzed by Intermolecular Network Theory Reprinted from: Molecules 2018 , 23 , 1990, doi:10.3390/molecules23081990 . . . . . . . . . . . . . . 359 vii About the Special Issue Editor David Barker , Associate Professor in Organic and Medicinal Chemistry. David Barker was born in Altrincham, UK. After moving to Australia, he graduated from the University of Sydney with a BSc degree (Honours, First Class) and then completed his PhD in 2002 at the same university, under the supervision of Prof. Margaret Brimble and Associate Professor Malcolm McLeod. After post-doctoral research at the School of Medical Sciences at the University of New South Wales working with Prof. Larry Wakelin, in 2004 he joined the University of Auckland as a lecturer. He is currently Associate Professor in Organic and Medicinal Chemistry and he has a diverse range of synthetic interests, including biologically active natural products, especially lignans and molecules of a marine origin. He also works on a range of drug discovery projects, particularly targeting cancer, and on the development of novel polymeric scaffolds. ix Preface to ”Lignans” Lignans are traditionally defined as a class of secondary metabolites that are derived from the dimersation of two or more phenylpropanoid units. Despite their common biosynthetic origins, they boast a vast structural diversity. It is also well-established that this class of compounds exhibits a range of potent biological activities. Owing to these factors, lignans have proven to be a challenging and desirable synthetic target and have instigated the development of a number of different synthetic methods, advancing our collective knowledge towards the synthesis of complex and unique structures. Lignans are also well-known components of a number of widely eaten foods and are frequently studied for their dietary impact. This book is based on the Special Issue of the journal Molecules on ‘Lignans’. This collection of research and review articles describe topics ranging in scope from recent isolation and structural elucidation of novel lignans, total syntheses and strategies towards lignan synthesis, assessment of their biological activities and potential for further therapeutic development. Research showing the impact of lignans in the food and agricultural industries is also presented. David Barker Special Issue Editor xi molecules Editorial Lignans David Barker School of Chemical Sciences, University of Auckland, Private Bag, Auckland 92019, New Zealand; d.barker@auckland.ac.nz Received: 8 April 2019; Accepted: 10 April 2019; Published: 11 April 2019 The 13 research articles/communications, six reviews, and one perspective that comprise this Special Issue on Lignans, highlight the most recent research and investigations into this diverse and important class of bioactive natural products. Lignans are traditionally defined as a class of secondary metabolites that are derived from the oxidative dimerization of two or more phenylpropanoid units. Despite their common biosynthetic origins, they boast a vast structural diversity. It is also well-established that this class of compounds exhibit a range of potent biological activities. Owing to these factors, lignans have proven to be a challenging and desirable synthetic target that has instigated the development of a number of different synthetic methods, advancing our collective knowledge towards the synthesis of complex and unique structures. New lignans are constantly being found and this Special Issue details some of the most recently discovered novel lignans—Liu et al. isolated three new dibenzocyclooctadiene lignans, heilaohulignans A–C from Heilaohu, the roots of Kadsura coccinea, which have a long history of use in Tujia ethnomedicine for the treatment of rheumatoid arthritis and gastroenteric disorders [ 1 ]. Heilaohulignan C, in particular, demonstrated cytotoxic activity in a number of human cancer cell lines. Two new lignan glycosides have also been found in the aerial portion of Lespedeza cuneata (Fabaceae), known as Chinese bushclover, a plant that has been used in traditional medicine for the treatment of diseases including diabetes, hematuria, and insomnia [ 2 ]. These newly-discovered compounds were tested for their biological activities against human breast cancer cell lines, showing some cytotoxic activity. A review detailing over 270 lignans isolated from Lauraceae, a valuable source of lignans and neolignans is also presented, compiled by Li et al. [ 3 ]. Furthermore, Mexican Bursera plants have been used in traditional medicine for treating various pathophysiological disorders and are a rich source of lignans. An Italian research group have summarized the biological activities of lignans isolated from selected Mexican Bursera plants in their review [4]. A subclass of lignans, norlignans lack a carbon present in the parent lignan structure, with 9-norlignans lacking a terminal carbon (C-9). An overview of the occurrence and biological activity of all the 9-norlignans reported to date are given in the article by Eklund and Raitanen, which also reports the semisynthetic preparation of a number of 9-norlignans using the natural lignan hydroxymatairesinol, obtained from spruce knots, as the starting material [5]. As stated above, owing to their potent biological activities, lignans are a popular synthetic target. A summary of the advances in lignan natural product synthesis over the last decade is outlined in the review by Fang and Hu [6]. Davidson et al. have presented their work on their novel, efficient, convergent, and modular synthesis of the well-known dibenzyl butyrolactone lignans through the use of the acyl-Claisen rearrangement to stereoselectively prepare a key intermediate [ 7 ]. Not only were the natural products able to be obtained, but the reported synthetic route also enabled the modification of these lignans to give rise to 5-hydroxymethyl derivatives, which were then shown to have an excellent cytotoxic profile which resulted in programmed cell death of Jurkat T-leukemia cells with less than 2% of the incubated cells entering a necrotic cell death pathway. Molecules 2019 , 24 , 1424; doi:10.3390/molecules24071424 www.mdpi.com/journal/molecules 1 Molecules 2019 , 24 , 1424 Advances in the synthesis of aryldihydronaphthalene and arylnapthalene lignans are also detailed in this Special Issue through the concise synthesis of (+)- β - and γ -apopicropodophyllins and dehydrodesoxypodophyllotoxin [ 8 ]. This was achieved using the key reaction involving regiocontrolled oxidations of stereodivergent aryltetralin lactones, which were easily accessed from a nickel-catalyzed reductive cascade approach. As stated, lignans are formed from the oxidative dimerization of two or more phenyl propanoid units. However, numerous oxidative transformations of lignans themselves have been reported in the literature. Runeberg et al. provide an overview on the current findings in this field, focusing on transformations targeting a specific structure, reaction, or an interconversion of the lignan skeleton [ 9 ]. The extensive analysis of the potent biological activities of lignans remains a popular avenue of investigation. Antunez-Mojica et al. used a zebrafish embryo model to guide the chromatographic fractionation of antimitotic secondary metabolites, ultimately leading to the isolation of several podophyllotoxin-type lignans from the steam bark of Bursera fagaroides [ 10 ]. Subsequent to their isolation, the biological effects on mitosis, cell migration, and microtubule cytoskeleton remodeling of the isolated lignans were then further evaluated in zebrafish embryos through various methods. Ultimately, it was demonstrated that the zebrafish model can be a fast and inexpensive in vivo model to identify antimitotic natural products through bioassay-guided fractionation. Pereira Rocha et al. combined the in silico prediction of biological activities of lignans from Diphylleia cymosa and Podophyllum hexandrum with in vitro bioassays testing the antibacterial, anticholinesterasic, antioxidant, and cytotoxic activities of these lignans [ 11 ]. In this study, the in silico approach was validated and several ethnopharmacological uses and known biological activities of lignans were confirmed, whilst it was shown that others should be investigated for new drugs with potential clinical use. To explore the differences in lignan composition profiles between various parts and genders of Schisandra rubriflora and Schisandra chinesis (wuweizi), Szopa et al. used UHPLC-MS/MS [ 12 ]. Additionally, the anti-inflammatory activity of plant extracts and individual lignans was tested in vitro for the inhibition of 15-lipooxygenase (15-LOX), phospholipases A2 (sPLA2), cyclooxygenase 1 and 2 (COX-1; COX-2) enzyme activities. The results of anti-inflammatory assays revealed higher activity of S. rubriflora extracts, while individual lignans showed significant inhibitory activity against 15-LOX, COX-1 and COX-2 enzymes. Closely related, Chen et al. evaluated the quality and effect of cultivated and wild growing methods on the lignan composition of Schisandra chinesis through the use of UFLC-QTRAP-MS/MS in combination with multivariate statistical analysis, demonstrating that the composition differs between plants grown in these conditions and the quality of cultivated wuweizi was not as good as wild wuweizi [13]. While lignans have been shown to exhibit extensive potent biological activities, other factors need to be considered for them to be potential drugs. The physicochemical properties of various lignans subclasses were analyzed by Dr Lisa Pilkington to assess their Absorption, Distribution, Metabolism, Excretion and Toxicity (ADMET) profiles and establish if these compounds are lead-like/drug-like and thus have potential to be or act as leads in the development of future therapeutics [ 14 ]. Overall, she established that lignans show a particularly high level of drug-likeness, an observation that, coupled with their potent biological activities, demands future pursuit into their potential for use as therapeutics. Traditionally, health benefits attributed to lignans have included a lowered risk of heart disease, menopausal symptoms, osteoporosis, and breast cancer. Rodriguez-Garcia et al. present a review that focuses on the potential health benefits attributable to the consumption of different diets containing naturally lignan-rich foods [ 15 ]. Current evidence endorses lignans as human health-promoting molecules and, therefore, dietary intake of lignan-rich foods could be a useful way to bolster the prevention of chronic illness, such as certain types of cancers and cardiovascular disease. Lignan composition profiles of flaxseed, the richest grain source of lignans, was also studied, assessing the relative impact of genetic and geographic parameters on the phytochemical yield and 2 Molecules 2019 , 24 , 1424 composition [ 16 ]. It was found that cultivar is more influential than geographic parameters on the flaxseed phytochemical accumulation yield and composition. In addition, the corresponding antioxidant activity of these flaxseed extracts was evaluated using both in vitro , and in vivo methods, which confirmed that flaxseed extracts are an effective protector against oxidative stress and that secoisolariciresinol diglucoside, caffeic acid glucoside, and p -coumaric acid glucoside are the main contributors to the antioxidant capacity. A review of the use and effect of flaxseed as a food source for dairy cows has also been presented [ 17 ], covering the gastrointestinal tract metabolism of lignans in humans and animals. The review also provided an in-depth assessment of research towards the impacts of flaxseed products on milk enterolactone concentration and animal health, and the pharmacokinetics of enterolactone consumed through milk, which may have implications to both ruminants and humans’ health. With the rise in exploration of dietary lignans and their various effects, exemplified by the aforementioned studies, the study by Durazzo et al. provides assessment and analysis of the development and management of databases on dietary lignans, which includes a description of the occurrence of lignans in food groups, the initial construction of the first lignan databases, and their inclusion in harmonized databases at national and/or European level [18]. In addition to work into their notable biological activities, there has been a recent increase in investigations exploring lignans in other roles. This includes gaining insight into the effects of barrel-aging on spirits, whereby lignans present in the wooden barrels are released into the aging spirit. To evaluate the impact of lignans in spirits, screening of a number of lignans was set up and served to validate their presence in the spirit and release by oak wood during aging [ 19 ]. The most abundant, and also the bitterest, lignan, ( ± )-lyoniresinol was detected and quantified in a large number of samples to be above the gustatory threshold, suggesting its effect of increased bitterness in spirit taste. Related to this, the molecular dynamics on wood-derived lignans were analyzed by intramolecular network theory by Sandberg et al. [ 20 ]. These wood-derived lignan-based ligands called LIGNOLs were studied, where it was found in the hydration studies that tetramethyl 1,4-diol is the LIGNOL which was most likely to form hydrogen bonds to TIP4P solvent. In summary, it can be seen in this Special Issue that research in natural lignans and lignin-derived compounds continues to be a fruitful area of research. Scientists working across a large number of disciplines continue to be attracted to work on lignans due to their relatively high natural abundance, coupled with their highly potent and diverse range of biological activities. Conflicts of Interest: The author declares no conflict of interest. References 1. Liu, Y.; Yang, Y.; Tasneem, S.; Hussain, N.; Daniyal, M.; Yuan, H.; Xie, Q.; Liu, B.; Sun, J.; Jian, Y.; et al. Lignans from Tujia Ethnomedicine Heilaohu: Chemical Characterization and Evaluation of Their Cytotoxicity and Antioxidant Activities. Molecules 2018 , 23 , 2147. [CrossRef] [PubMed] 2. Baek, J.; Lee, T.K.; Song, J.-H.; Choi, E.; Ko, H.-J.; Lee, S.; Choi, S.U.; Lee, S.; Yoo, S.-W.; Kim, S.-H.; et al. Lignan Glycosides and Flavonoid Glycosides from the Aerial Portion of Lespedeza cuneata and Their Biological Evaluations. Molecules 2018 , 23 , 1920. [CrossRef] [PubMed] 3. Li, Y.; Xie, S.; Ying, J.; Wei, W.; Gao, K. Chemical Structures of Lignans and Neolignans Isolated from Lauraceae. Molecules 2018 , 23 , 3164. [CrossRef] [PubMed] 4. Marcotullio, M.C.; Curini, M.; Becerra, J.X. An Ethnopharmacological, Phytochemical and Pharmacological Review on Lignans from Mexican Bursera spp. Molecules 2018 , 23 , 1976. [CrossRef] [PubMed] 5. Eklund, P.; Raitanen, J.-E. 9-Norlignans: Occurrence, Properties and Their Semisynthetic Preparation from Hydroxymatairesinol. Molecules 2019 , 24 , 220. [CrossRef] [PubMed] 6. Fang, X.; Hu, X. Advances in the Synthesis of Lignan Natural Products. Molecules 2018 , 23 , 3385. [CrossRef] [PubMed] 3 Molecules 2019 , 24 , 1424 7. Davidson, S.J.; Pilkington, L.I.; Dempsey-Hibbert, N.C.; El-Mohtadi, M.; Tang, S.; Wainwright, T.; Whitehead, K.A.; Barker, D. Modular Synthesis and Biological Investigation of 5-Hydroxymethyl Dibenzyl Butyrolactones and Related Lignans. Molecules 2018 , 23 , 3057. [CrossRef] [PubMed] 8. Xiao, J.; Nan, G.; Wang, Y.-W.; Peng, Y. Concise Synthesis of (+)- β - and γ -Apopicropodophyllins, and Dehydrodesoxypodophyllotoxin. Molecules 2018 , 23 , 3037. [CrossRef] [PubMed] 9. Runeberg, P.A.; Brusentsev, Y.; Rendon, S.M.K.; Eklund, P.C. Oxidative Transformations of Lignans. Molecules 2019 , 24 , 300. [CrossRef] [PubMed] 10. Ant ú nez-Mojica, M.; Rojas-Sep ú lveda, A.M.; Mendieta-Serrano, M.A.; Gonzalez-Maya, L.; Marquina, S.; Salas-Vidal, E.; Alvarez, L. Lignans from Bursera fagaroides Affect in Vivo Cell Behavior by Disturbing the Tubulin Cytoskeleton in Zebrafish Embryos. Molecules 2019 , 24 , 8. [CrossRef] [PubMed] 11. Pereira Rocha, M.; Valadares Campana, P.R.; de Oliveira Scoaris, D.; de Almeida, V.L.; Dias Lopes, J.C.; Shaw, J.M.H.; Gontijo Silva, C. Combined in Vitro Studies and in Silico Target Fishing for the Evaluation of the Biological Activities of Diphylleia cymosa and Podophyllum hexandrum. Molecules 2018 , 23 , 3303. [CrossRef] [PubMed] 12. Szopa, A.; Dziurka, M.; Warzecha, A.; Kubica, P.; Klimek-Szczykutowicz, M.; Ekiert, H. Targeted Lignan Profiling and Anti-Inflammatory Properties of Schisandra rubriflora and Schisandra chinensis Extracts. Molecules 2018 , 23 , 3103. [CrossRef] 13. Chen, S.; Shi, J.; Zou, L.; Liu, X.; Tang, R.; Ma, J.; Wang, C.; Tan, M.; Chen, J. Quality Evaluation of Wild and Cultivated Schisandrae Chinensis Fructus Based on Simultaneous Determination of Multiple Bioactive Constituents Combined with Multivariate Statistical Analysis. Molecules 2019 , 24 , 1335. [CrossRef] 14. Pilkington, L.I. Lignans: A Chemometric Analysis. Molecules 2018 , 23 , 1666. [CrossRef] [PubMed] 15. Rodr í guez-Garc í a, C.; S á nchez-Quesada, C.; Toledo, E.; Delgado-Rodr í guez, M.; Gaforio, J.J. Naturally Lignan-Rich Foods: A Dietary Tool for Health Promotion? Molecules 2019 , 24 , 917. [CrossRef] [PubMed] 16. Garros, L.; Drouet, S.; Corbin, C.; Decourtil, C.; Fidel, T.; Lebas de Lacour, J.; Leclerc, E.A.; Renouard, S.; Tungmunnithum, D.; Doussot, J.; et al. Insight into the Influence of Cultivar Type, Cultivation Year, and Site on the Lignans and Related Phenolic Profiles, and the Health-Promoting Antioxidant Potential of Flax (Linum usitatissimum L.) Seeds. Molecules 2018 , 23 , 2636. [CrossRef] [PubMed] 17. Brito, A.F.; Zang, Y. A Review of Lignan Metabolism, Milk Enterolactone Concentration, and Antioxidant Status of Dairy Cows Fed Flaxseed. Molecules 2019 , 24 , 41. [CrossRef] [PubMed] 18. Durazzo, A.; Lucarini, M.; Camilli, E.; Marconi, S.; Gabrielli, P.; Lisciani, S.; Gambelli, L.; Aguzzi, A.; Novellino, E.; Santini, A.; et al. Dietary Lignans: Definition, Description and Research Trends in Databases Development. Molecules 2018 , 23 , 3251. [CrossRef] [PubMed] 19. Winstel, D.; Marchal, A. Lignans in Spirits: Chemical Diversity, Quantification, and Sensory Impact of ( ± )-Lyoniresinol. Molecules 2019 , 24 , 117. [CrossRef] [PubMed] 20. Sandberg, T.O.; Weinberger, C.; Smatt, J.-H. Molecular Dynamics on Wood-Derived Lignans Analyzed by Intermolecular Network Theory. Molecules 2018 , 23 , 1990. [CrossRef] [PubMed] © 2019 by the author. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/). 4 molecules Article Lignans from Tujia Ethnomedicine Heilaohu: Chemical Characterization and Evaluation of Their Cytotoxicity and Antioxidant Activities Yongbei Liu 1,† , Yupei Yang 1,† , Shumaila Tasneem 1 , Nusrat Hussain 1,2 , Muhammad Daniyal 1 ID , Hanwen Yuan 1 , Qingling Xie 1 , Bin Liu 3 , Jing Sun 4 , Yuqing Jian 1 , Bin Li 1 , Shenghuang Chen 1 and Wei Wang 1,2,4, * 1 TCM and Ethnomedicine Innovation & Development International Laboratory, Innovative Drug Research Institute, School of Pharmacy, Hunan University of Chinese Medicine, Changsha, 410208, China; ybliu2018@163.com (Y.L.); yangyupei24@163.com (Y.Y.); tasneemshum@gmail.com (S.T.); nusrat_hussain42@yahoo.com (N.H.) daniyaldani151@yahoo.com (M.D.); hanwyuan@hotmail.com (H.Y.); XieQL1992@163.com (Q.X.); cpujyq2010@163.com (Y.J.); libin_hucm@hotmail.com (B.L.); cshtyh@163.com (S.C.) 2 H.E.J. Research Institute of Chemistry, International Center for Chemical and Biological Sciences, University of Karachi, Karachi-75270, Pakistan 3 Hunan Province Key Laboratory of Plant Functional Genomics and Developmental Regulation, College of Biology, Hunan University, Changsha 410082, China; binliu2001@hotmail.com 4 Shaanxi Key Laboratory of Basic and New herbal Medicament Research, Shaanxi Collaborative Innovation Center of Chinese Medicinal Resource Industrialization, Shaanxi University of Chinese Medicine, Xianyang 712046, China; ph.175@163.com * Correspondence: wangwei402@hotmail.com; Tel.: +86-136-5743-8606 † These authors contributed equally to this work. Received: 30 July 2018; Accepted: 24 August 2018; Published: 27 August 2018 Abstract: Heilaohu, the roots of Kadsura coccinea, has a long history of use in Tujia ethnomedicine for the treatment of rheumatoid arthritis and gastroenteric disorders, and a lot of work has been done in order to know the material basis of its pharmacological activities. The chemical investigation led to the isolation and characterization of three new ( 1 – 3 ) and twenty known ( 4 – 23 ) lignans. Three new heilaohulignans A-C ( 1 – 3 ) and seventeen known ( 4 – 20 ) lignans possessed dibenzocyclooctadiene skeletons. Similarly, one was a diarylbutane ( 21 ) and two were spirobenzofuranoid dibenzocyclooctadiene ( 22 – 23 ) lignans. Among the known compounds, 4 – 5 , 7 , 13 – 15 and 17 – 22 were isolated from this species for the first time. The structures were established, using IR, UV, MS and NMR data. The absolute configurations of the new compounds were determined by circular dichroism (CD) spectra. The isolated lignans were further evaluated for their cytotoxicity and antioxidant activities. Compound 3 demonstrated strong cytotoxic activity with an IC 50 value of 9.92 μ M, compounds 9 and 13 revealed weak cytotoxicity with IC 50 values of 21.72 μ M and 18.72 μ M, respectively in the HepG-2 human liver cancer cell line. Compound 3 also showed weak cytotoxicity against the BGC-823 human gastric cancer cell line and the HCT-116 human colon cancer cell line with IC 50 values of 16.75 μ M and 16.59 μ M, respectively. A chemiluminescence assay for antioxidant status of isolated compounds implied compounds 11 and 20, which showed weak activity with IC 50 values of 25.56 μ M and 21.20 μ M, respectively. Keywords: lignans; heilaohu; tujia ethnomedicine; chemical characterization; cytotoxicity; antioxidant Molecules 2018 , 23 , 2147; doi:10.3390/molecules23092147 www.mdpi.com/journal/molecules 5 Molecules 2018 , 23 , 2147 1. Introduction Kadsura coccinea (Lem.) A. C. Smith belongs to the medicinally important genus Kadsura from the Schisandraceae family. It is an evergreen climbing shrub, which is mainly distributed in south-western provinces of P. R. China [ 1 ]. Its leaves, fruits, stems and roots are used as medicine. The fruits have unique shapes and high nutritional and medicinal values [ 2 ]. The stems and roots are called Heilaohu in Tujia ethnomedicine for looking swarthy while dispelling wind effectively. The isolates of this plant mainly contain lignans, triterpenoids and essential oils. Bioactive lignans and triterpenoids from this plant are of special interest [ 3 ]. The compounds from genus Kadsura have been reported with different bioactivities including anti-tumor [ 4 , 5 ], anti-HIV [ 6 –8 ], anti-inflammatory [ 9 , 10 ], inhibition of nitric oxide (NO) production [11,12] and other pharmacological effects. The lignans from Heilaohu are very important due to their bioactivities and structural diversity. The lignans from this plant can be divided into four different categories on the basis of skeleton types: dibenzocyclooctadienes, spirobenzofuranoid dibenzocyclooctadienes, diarylbutanes, and aryltetralins lignans. Dibenzocyclooctadiene (two benzene rings sharing an eight membered ring neighborhood) is the most common basic skeleton in Heilaohu. Methoxy, hydroxyl and methylenedioxy are the most frequently found substituents at benzene rings, while other important substituents including acetyl-, angeloyl-, tigloyl-, propanoyl-, benzoyl-, cinnamoyl- and butyryl- groups are invariably presented at C-1, C-6 or C-9 [ 13 – 15 ]. Spirobenzofuranoid dibenzocyclooctadienes are rare in other genera and can be considered as the characteristic chemical constituents of genus Kadsura . This category features a furan-ring at C-14, C-15 and C-16 positions and a ketonic group at the C-1 or C-3 position [ 3 ], and the same connections on the eight membered ring located at the C-6 or C-9 position. Diarylbutanes and aryltetralins have previously been reported but are not very common in genus Kadsura , and most of them were found in the DCM (CHCl 3 ) layer and EtOAc layer. This work was conducted to further explore lignans from Heilaohu. The chemical investigation led to the isolation and characterization of three new ( 1 – 3 ) and twenty known ( 4 – 23 ) lignans. The three new Kadsura lignans A–C ( 1 – 3 ) and seventeen known lignans, schizandrin ( 4 ) [ 16 ], binankadsurin A ( 5 ) [ 17 ], acetylbinankadsurin A ( 6 ) [ 18 ], isobutyroylbinankadsurin A ( 7 ) [ 19 ], isovaleroylbinankadsurin A ( 8 ) [ 19 ], kadsuralignan I ( 9 ) [ 20 ], kadsuralignan J ( 10 ) [ 20 ], kadsuralignan L ( 11 ) [ 21 ], kadsulignan N ( 12 ) [ 22 ], longipedunin B ( 13 ) [ 15 ], schisantherin F ( 14 ) [ 23 ], schizanrin D ( 15 ) [ 23 ], acetylgomisin R ( 16 ) [ 24 ], intermedin A ( 17 ) [ 25 ], kadsurarin ( 18 ) [ 14 ], kadsutherin A ( 19 ) [ 25 ] and kadsuphilol A ( 20 ) [ 26 ] possessed dibenzocyclooctadiene skeletons. Similarly, meso-dihydroguaiaretic acid dimethyl ( 21 ) [ 27 ] had a diarylbutane type. Schiarianrin E ( 22 ) [ 28 ] and schiarisanrin A ( 23 ) [ 29 ] contained spirobenzofuranoid dibenzocyclooctadiene lignan skeletons. A literature survey revealed that kadsulignan I ( 9 ) exhibited inhibitory effects on LPS-induced NO production in BV-2 cells with IC 50 value of 21.00 μ M [ 30 ]. Kadsuralignan L ( 11 ) demonstrated moderate NO production inhibitory activity with an IC 50 value of 52.50 μ M [ 21 ]. Heilaohu has been used for the treatment of rheumatoid arthritis in traditional medicine for a long time, and a few of its isolated compounds have been used for their anti-inflammatory and cytotoxic activities [ 3 ]. With the aim of searching for natural compounds which are responsible for folk efficacy and medicinal application as anti-cancer agents and as anti-inflammatory agents, we employed a chemiluminescence assay for anti-oxidant activity to find out the anti-inflammatory properties of a compound. We also used a cytotoxicity assay against cancer cell lines, namely HepG-2 human liver cancer cells, BGC-823 human gastric cancer cells and HCT-116 human colon cancer cells, after the chemical characterization of compounds. 2. Results and Discussion 2.1. Structure Characterization of the Isolated Compounds from Heilaohu Heilaohulignan A ( 1 ) (Figure 1) was obtained as an amorphous powder. Its molecular formula, C 26 H 32 O 8 , was determined by [M + Na] + ion peak at m / z 495.1998 (calcd. 495.1995) in HR-ESI-MS, 6 Molecules 2018 , 23 , 2147 showing 11 degrees of unsaturation. The UV data, with absorption maxima at λ max 242 nm, and its IR spectrum, with absorption bands at 3419 (OH), 1645 (C=C) and 1463 cm − 1 (aromatic moiety), suggested that 1 is a dibenzocyclooctadiene lignan with a hydroxyl substitution. Figure 1. Structures of heilaohulignans A–C ( 1 – 3 ). The 1 H- and 13 C-NMR spectra of 1 (Table 1) indicated the presence of 12 aromatic carbons ( δ C 141.7 (C-1), 138.8 (C-2), 151.5 (C-3), 113.0 (C-4), 134.8 (C-5), 102.4 (C-11), 148.8 (C-12), 135.0 (C-13), 140.3 (C-14), 118.2 (C-15) and 122.9 (C-16)) and two aromatic proton singlets at δ H 6.71 (1H, s) and 6.32 (1H, s), which were assignable to H-4 and H-11, respectively. A butane chain was deduced on the cross-peaks of H-6 ( δ H 2.65, m), H-7 ( δ H 2.01, m), H-8 ( δ H 1.81, m) and H-9 ( δ H 4.73, s) in the 1 H- 1 H COSY spectrum. In addition, in the HMBC spectrum, correlations were found between H-9 and C-10, C-8 and C-15, and between H-6 and C-4, C-7 and C-16. The functional moieties evident from the 1 H- and 13 C-NMR data included one methylenedioxy, three methoxy groups and four methyl groups; the presence of signals at δ H 0.97 (d, J = 7.0 Hz, 3H), 1.09 (d, J = 7.0 Hz, 3H) and 2.61 (m, 1H), and δ C 176.7 (C=O), 18.7 (CH 3 ), 18.7 (CH 3 ), 34.0 (CH) suggested the presence of an isobutyroyl group. Table 1. 1 H- (600 MHz) and 13 C-NMR (150 MHz) data of compounds 1 , 2 , and 3 (CDCl 3 ). Number 1 2 3 δ H (ppm) J (Hz) δ C (ppm) δ H (ppm) J (Hz) δ C (ppm) δ H (ppm) J (Hz) δ C (ppm) 1 − 141.7 − 143.0 − 147.0 2 − 138.8 − 140.2 − 133.6 3 − 151.5 − 152.2 − 150.5 4 6.71 s 113.0 6.58 s 113.5 6.41 s 106.9 5 − 134.8 − 131.3 − 133.5 6 2.65 m 38.9 2.50 m, 3.03 m 34.6 2.66 m 38.6 7 2.01 m 35.1 2.04 m 43.0 2.12 m 34.8 8 1.81 m 43.0 − 80.9 2.10 m 41.7 9 4.73 s 82.8 − 207.3 5.62 s 82.9 10 − 134.8 − 135.4 − 136.0 11 6.32 s 102.4 6.52 s 100.7 6.54 s 103.0 12 − 148.8 − 148.7 − 148.9 13 − 135.0 − 136.9 − 136.1 14 − 140.3 − 141.7 − 141.1 15 − 118.2 − 117.7 − 119.0 16 − 122.9 − 121.6 − 117.1 17 1.01 d (7.3) 15.3 1.33 s 23.3 1.09 d (7.0) 19.8 18 1.17 d (7.3) 20.0 0.89 d (7.1) 14.8 1.61 dd (7.1, 1.1) 14.2 19 5.93 dd (8.9, 1.4) 101.0 5.96 s, 6.02 s 101.6 5.98 s, 5.93 s 101.2 1 ′ − 176.7 − 173.1 − 167.5 2 ′ 2.61 dt (13.9, 6.9) 34.0 2.43 m 41.5 − 127.6 3 ′ 0.97 d (7.0) 18.7 1.40 m, 1.62 m 26.8 6.02 d (1.5) 137.2 4 ′ 1.09 d (7.0) 18.7 0.86 t (7.4) 11.7 1.47 s 11.8 5 ′ − − 1.02 d (7.0) 16.9 0.97 d (7.1) 15.0 2-OCH 3 3.96 s 59.6 3.80 s 60.6 3.84 s 60.7 3-OCH 3 3.78 s 61.1 3.86 s 56.1 3.84 s 59.8 14-OCH 3 3.89 s 56.0 3.88 s 59.8 3.90 s 55.8 7