Bromination of eudesmin isolated from araucaria araucana induces epimerization and give bromine derivatives with loss of anti-Candida activity

Abstract Furofuran lignanes show important biological activities for the treatment of infectious diseases, inflammatory and metabolic pathologies. They have been isolated from leaves and barks of many plants. In Chile the native conifer Araucaria araucana produces eudesmin, matairesinol, secoisolariciresinol and lariciresinol in stemwood, branchwood and knotwood. These compounds were previously isolated by laborious flash chromatography on silica gel. Here we report the easy isolation of eudesmin by soxhlet extraction from milled knots of Araucaria araucana with hexane, followed by cryo-crystallization at −20 °C. Upon bromination of the isolated eudesmin epimerization at one benzylic position occurs, giving epieudesmin and the corresponding mono and di-brominated derivatives. The structures were determined by 1D, 2D NMR and X-ray diffraction. The analysis of products against Candida yeast showed that eudesmin has a moderate activity against different strains of Candida from 62.5 to 500 µg/mL. This activity decreases for epieudesmin, while bromine derivatives are not active. Graphical Abstract


Introduction
Lignans are a large family of natural compounds found in many vascular plants. They are formed by two phenylpropane units (C6C3), linked by the coupling b-b 0 of the propyl side chains. Lignans are classified in eight groups as dibenzylbutane, aryltetralin, arylnaphthalene, dibenzocyclooctene, furan, furofuran, dibenzylbutyrolactol and dibenzylbutyrolactone (Teponno et al. 2016). Furofuran lignans are one of the major groups due to the variety of substituents at aryl groups and the configurations at the furofuran ring. Xu et al. reviewed in 2019 the structure and activity of 137 furofurans isolated from 53 plants in the last 20 years (Xu et al. 2019), evidencing the wide range of biological activities of these compounds as antivirals, anticancer or anti-inflammatory agents (Cui et al. 2020;Xu et al., 2019). Eudesmin is a furofuran isolated from leafs of Metrodorea flavida (Baetas et al. 1996); Zanthoxylum armatum (Bhatt et al. 2017), Raulinoa echinata (Biavatti et al. 2001), from root and stem of Zanthoxylum armatum (Guo et al. 2012), from barks of Magnolia kobus (Seo et al. 2008), from leafs and barks of Zanthoxylum armatum (Kumar et al. 2014), from flower buds of Magnolia fargesii (Kim et al. 2009). Eudesmin blocks adipogenesis in mesenchymal stem cells, suggesting its use as a natural therapy for treatment of obesity and metabolic diseases (Nam et al. 2018). It has also been shown to control the growing of Helicobacter pylori in vitro and in vivo (Yang et al. 2018). On the other hand, epieudesmin is a diasteromer of eudesmin with opposite configuration at one benzylic position. Epieudesmin is also a natural compound isolated from many plants (reviewed by Xu et al. 2019), for example, from Mitrephora teysmannii (Rayanil et al. 2016) with antitrypanosomal activity (Sartorelli et al. 2010).
Araucaria araucana is a native conifer of the centre south of Chile that grows up over 2000 m. The phytochemistry and ethnopharmacology of the Araucariaceae family was reviwed by Frezza et al. 2020. Knots of A. araucana are commonly known as 'picoyo', which can survive to fungal attack for many years in the forest, due to a high concentration of lignans such as secoisolariciresinol, lariciresinol, matairesinol and eudesmin (Bravo-Arrepol et al. 2020). We describe herein the isolation of eudesmin from knots of Araucaria araucana and its bromination reaction, in search for new bromine lignanes with antifungal activity. The epimerization of furane ring is characterised, together with the inhibitory assay against C. albicans, C. tropicalis, C. glabrata, C. krusei, C. parapsilosis, Rhodotorula rubra, Cryptococcus neoformans and Geotrichum candidum of all products.

Isolation and bromination of eudesmin
Eudesmin was isolated by continuous extraction of knots of A. araucana with boiling hexane and further cryocristallization at À20 C, producing crystals of eudesmin (1.4% yield). Eudesmin was identified by NMR and GC-MS (NMR in Suppl. Figs. S12 to S14).

Anti-fungal activity assay
All compounds were analysed against clinical strains of C. albicans, C. tropicalis, C. glabrata, C. krusei, C. parapsilosis, Rhodotorula rubra, Cryptococcus neoformans and Geotrichum candidum. The results show that eudesmin has moderate activity against all fungal strains, the minimum inhibitory concentration values expressed in mg/mL are summarised in the Figure 1. Although epieudesmin is structurally closely related to eudesmin, this compound loses the antifungal activity, and moreover, all bromo derivatives are not active below 1000 mg/mL.

Discussion
We show that eudesmin can be extracted in boiling hexane and crystallised at À20 C. This method allows us to obtain easily 1.8 g of pure eudesmin from 80 g of knots of A. araucana, avoiding laborious, time-consuming and expensive chromatographic methods.
Eudesmin is a furofuran lignan with various biological activities, including anti-fungal activity. We have shown that eudesmin can control Candida yeast with moderate activity, but that the furan ring is susceptible to epimerization in acidic media, producing the corresponding epimer. Similar results were obtained for sesamin, a furofuran lignan isolated from sesame seeds, with anti-cancer properties (Majdalawieh et al. 2017) which maintains an equilibrium with its epimer asarinin (Li et al. 2005). The bromination of eudesmin produces mono and di bromo derivatives in ortho-position of the phenyl ring. These products were crystallised to give suitable crystals for X-ray diffraction (XRD) Suppl. Fig. S2. to S11, allowing the determination of the absolute configuration of the brominated derivatives.
Eudesmin has potent activities against H. pylori; for example its MBC against antibiotic resistant strains of H. pylori is between 2.5 and 10 mM, while for other bacteria such as Streptococcus aureus, Pseudomonas aeruginosa, Salmonella enterica serovar Typhimurium and Escherichia coli the activity of eudesmin decreases (MBC > 320 mM) (Yang et al. 2018). The activity of eudesmin against H. pylori is remarkable, considering that this is a natural product usually found in seeds or fruits, but the activity of this product can be affected due to the epimerization equilibrium induced by the stomach environment where H. pylori grows. Here we have seen that eudesmin has a moderate activity against parasitic fungus strains, but its epimer loses this activity, suggesting that biological studies of furofuran lignan should be assessed considering the epimer and the pH of the environment.

Conclusions
Overall, this study contributes to understanding the stability of eudesmin in a bromination reaction and the epimerization induced in acidic media. Also, the activity against different parasitic fungus show that eudesmin is more active than epieudesmin, while the bromine derivatives were found to be inactive.

Disclosure Statement
The authors declare no conflict of interest.

Funding
This research was funded by ANID of Chile government with the grant number Fondecyt 11181076 to CP.