Lignans and coumarins from the stem bark of Alyxia fascicularis (Wall. ex G. Don) Benth. ex Hook. f.)

Abstract In the first phytochemical investigation of specialized metabolites from the stem bark of Alyxia fascicularis, which is used in different traditional medicines, including those of Myanmar and China, five lignans (1-5) and three coumarins (6-8) were isolated by semipreparative HPLC separations and identified mainly by 1D and 2D NMR spectral analysis. The radical scavenging activity of isolated compounds was tested using the DPPH method. Noteworthy, most lignans exhibited antiradical effects comparable to vitamin C and gallic acid. Instead, compounds 1-8 showed no cytotoxic effect on Hela cell line. A possible biosynthetic pathway to enantiomeric 3 and 4 is suggested. Graphical Abstract


Introduction
Alyxia Banks ex R.Br. is an Australasian genus of flowering plants belonging to the dogbane family, Apocynaceae (Angiosperms).306 scientific plant names of species rank for the genus Alyxia are included in The International Plant Names Index and World Checklist of Selected Plant Families 2022 (Published on the Internet at http:// www.ipni.organd http://apps.kew.org/wcsp/ last accession date: July 15, 2022).Of these 106 are accepted species names.Instead, in the database of 'The World Flora Online (WFO)' (http://www.worldfloraonline.org/search?query=Alyxia/lastaccession date: July 15, 2022) 249 taxa are listed, 116 of which have an accepted name and 130 are synonyms.The native range of these shrubby, climbing, or scrambling plants includes China, the Himalayas, Assam, Thailand, Myanmar, Malaysia, Australia, New Caledonia, and the Pacific Islands (https://wikimili.com/en/Alyxia; last accession date: July 15, 2022).Alyxia fascicularis (Wall.ex G. Don) Benth.ex Hook.f.) is a sprawling shrub or woody vine.The leaves are opposite and arranged in whorls of three, with a petiole that is 3-5 mm long; the leaf blade is 6-10 Â 1.7-3 cm long and glabrous, with bases that are broadly cuneate and apexes that are caudate-acuminate.The acumen is ca.1.5 cm long, and the veins are numerous and parallel.The cymes are short pedunculate, fascicled, glabrous or puberulent; sepals are ovate.The corolla tube measures ca. 3 mm; lobes are tiny and rounded.The fruit is stipitate and moniliform, with cylindric-ellipsoid articles that are about ca. 2 1 cm long.The flowering time is from September to November (cited and adapted from www.eFloras.org.Last accession date: July 15, 2022).The plant is commonly known with the name of Tawnoeyee by the people living in Kachin State of Myanmar, where it is used as a traditional remedy to treat several diseases.For instance, people who have suffered a stroke, consume the plant with alcohol to sustain a good state of health.The plant can also be used for treating arthrogryposis, sore throat, septicemia, and backaches.According to a search on SciFinderV R and PubMed V R databases (last accession date: August 04, 2020) no phytochemical investigation has yet been carried out on specialized metabolites from A. fascicularis.On the other hand, the phytochemistry of only a few Alyxia species has been investigated in detail so far.Triterpenoids and b-sitosterol-D-glucoside were isolated from A. levinei Merr.(Yuan et al. 1991).Chemical components of A. reinwardtii BL include coumarins, pinoresinol-like lignans, flavonoids, phenolic acids, and iridoids (Steffan et al. 2005;Rao et al. 2012;Rattanapan et al. 2012), including the new trimeric iridoid diglucoside pulosarioside (Kitagawa et al. 1988).Long-chain alkanes, anthraquinones, coumarins, common sterols and triterpenoids, and pinoresinol-like lignans were isolated from the stems of A. sinensis (Wang, Hou et al. 2002;Wang, Yan et al. 2002).Analysis of the roots of A. schlechteri afforded three new lignan esters, alyterinates A-C, together with (þ)-pinoresinol-like lignans, coumarins, and two benzaldehyde derivatives (Sriphana et al. 2013).A new benzyl coumarin derivative, named alyterinin, and a new germacrane sesquiterpene, named alyterinone, were isolated from the stems of A. schlechteri, in addition to lignans, a sesquilignan, coumarins, aromatic aldehydes and alcohol, and a d-styrylvalerolactone, named goniothalamin (Sribuhom et al. 2015).Coumarin and a new catechin, named mailein was isolated from A. olivaeformis Gaud (Dority 1965).In this paper the content of the EtOAc partition derived from the methanol extract of A. fascicularis stem bark has been examined.The structures of isolated compounds 1-8 were established by analysis of EI-MS, FAB-MS, IR, 1D ( 1 H and 13 C) and 2D NMR spectral data and comparison with literature data.Moreover, the radical scavenging activity of the isolated compounds was determined by a standard DPPH test.The cytotoxic effects of compounds 1-8 towards Hela cells were assayed by a standard MTT test.
On the other hand, compounds 1-8 showed no significant antiproliferative activity against Hela cancer cell line (IC 50 > 50 mM).We have no conclusive explanation for the negative cytotoxic effect.On the other hand, it is well known that the antiproliferative activity of a compound may depend on several factors, including the enantiomeric form and the type of assay and tested cell line.In this context it is interesting to note that (-)-4 (Kim, Moon et al. 2011), (±)-syringaresinol and (±)-medioresinol showed marginal cytotoxicity/inactivity also against other human cancer cell lines (Chin et al. 2006).Instead, the antiproliferative/cytotoxic effects of pinoresinol strongly depend on the cell line used in the assay and on the enantiomeric form (Chin et al. 2006).In addition to the high radical scavenging properties of compounds 1-3 and 5, it is reported in the literature that furfuran-type lignans 1-5 and coumarins 6-8 display a wide range of biological activities (Sok et al. 2009;Venugopala et al. 2013).For example, the phytoestrogen (þ)-pinoresinol (3) inhibited the enzyme a-glucosidase in vitro and is therefore considered a putative hypoglycemic agent (Wikul et al. 2012).Moreover, pinoresinol showed potent anti-inflammatory properties through the NF-KB signaling pathway, and it exhibited considerable binding affinity with three key SARS CoV's proteins (Allam et al. 2021).Lignans 2 and 3 exhibited strong antifungal activity against Pythium insidiosum, which causes pythiosis in animals and humans (Sriphana et al. 2013).Interestingly, (-)-pinoresinol-4-O-b-D-glucopyranoside (4) was inactive against the bacteria Escherichia coli, Staphylococcus aureus, Micrococcus luteus, and Bacillus cereus (Dong et al. 2006), while the enantiomer (þ)-4 exhibited remarkable anti-inflammatory and antiviral effects against influenza A (H1N1) virus infection (Li et al. 2019).8-Hydroxypinoresinol (5) showed protective effects against peroxynitrite (ONOO -)induced cellular damage (Piao et al. 2009), high antioxidant property, and vasorelaxant activity in rat aorta rings (Piccinelli et al. 2004).Among coumarins, in addition to other biological properties, isofraxidin (8) alleviated myocardial infarction through NLRP3 inflammasome inhibition (Chen et al. 2020) and possessed potent choleretic effects in rats (Borris et al. 1980).
It is interesting to note that lignans 1-3 and 5 belong to a series which is enantiomeric to (-)-pinoresinol-4-O-b-D-glucopyranoside (4), indicating that lignan (-)-4 cannot derive from the direct O-glucosidation of aglycone (þ)-3.To our knowledge, this finding is unprecedented since so far only homochiral 3 and 4 have been isolated from the same plant (for example, Waffo-T eguo et al. 2004;Guo et al. 2007;Ouyang et al. 2007;Liang et al. 2008;Kim, Ha et al. 2011;Kim, Moon et al. 2011).Lignans are believed to be formed by C-C radical coupling of two phenylpropane units of type 9 at the central carbon of the alkyl sidechain (Figure 2), and this coupling is generally considered to be stereochemically controlled by enzymes (Dewick 2009).The presence of lignan enantiomeric series in A. fascicularis may thus be explained by the existence in the plant cells of two biosynthetic pathways controlled by enzymes with opposite enantioselectivities.However, we suggest an alternative explanation for the formation of (þ)-3 and (-)-4, which is based on the initial formation of a racemic mixture, i.e.

Conclusions
Five lignans (1-5) and three coumarins (6-8) were isolated for the first time from A. fascicularis, used as a medicinal plant in Myanmar.Compounds 4 and 8 are new to the genus, as well as is unprecedented the isolation of heterochiral (þ)-3 and (-)-4 from the same plant.The high antiradical activity determined for lignans 1-3 and 5, together with the biological properties reported in literature for the isolated specialized metabolites, indicate that A. fascicularis is a rich source of bioactive compounds and that the use of the plant as an herbal remedy is sustained by scientific evidence.

Disclosure statement
No potential conflicts of interest were reported by the authors.