Unique bibenzyl cannabinoids in the liverwort Radula marginata: parallels with Cannabis chemistry

Abstract

Introduction

Demand for cannabinoid-based products is booming due to the global interest in their potent pharmacological properties (Torkamaneh & Jones, 2022). Plant cannabinoids (phytocannabinoids) are terpenophenolic compounds exerting diverse biological effects in humans via the modulation of the endocannabinoid system (Ligresti et al., 2016). Originally thought to be exclusive to Cannabis sativa L. (Cannabaceae), they have now been discovered in other flowering plants, liverworts, and fungi, where their biosynthesis is thought to have arisen independently on multiple occasions (Gulck & Moeller, 2020). One example of this parallel evolution is in South African Helichrysum umbraculigerum (Asteraceae), which has yielded both C₅ alkyl (e.g. cannabigerol (CBG 12, Fig. 1)) and phenylethyl/ß-aralkyl type (i.e. bibenzyl) cannabinoids (e.g. BB4G 10, Fig. 1) (Bohlmann & Hoffmann, 1979; Hanuš et al., 2016; Pollastro et al., 2017; Berman et al., 2023) from the polyketide pathway.

Fig. 1

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Structures of cannabinoids and other bibenzyls identified in Radula marginata, with main Cannabis sativa cannabinoids for comparison.

Bibenzyl cannabinoids have also been isolated from bryophytes, the plant lineage comprising hornworts, mosses, and liverworts, but only from a few liverwort species in the Radulaceae family (Asakawa et al., 2020).

The first cannabinoid-like compound reported was the bibenzyl-monoterpene hybrid perrottetinene (cis-PET 1, Fig. 1), from Japanese Radula perrottetii (Radulaceae, Marchantiophyta) (Toyota et al., 1994). These authors highlighted the structural similarity of perrottetinene (PET) to tetrahydrocannabinol (trans-THC 3, Fig. 1), the main psychoactive component in many varieties of Cannabis (Andre et al., 2016). Over two decades later, chemically synthesized cis-PET was proven to be psychoactive in mice via interaction with cannabinoid receptor type 1 (CB1) with potentially fewer side effects than THC (Chicca et al., 2018).

This report provoked much interest in Radula species as novel sources of medicinal compounds (Kumar et al., 2019; Gulck & Moeller, 2020; Arif et al., 2021). A review of Radula natural products world-wide (Asakawa et al., 2020) reported PET in Japanese R. campanigera and R. chinensis, and in Costa Rican R. laxiramea (Cullmann & Becker, 1999). PET was also found in the Aotearoa/New Zealand (A/NZ) endemic liverwort Radula marginata Taylor ex Gottsche, Lindenb. & Nees, together with presumed biosynthetic precursor perrottetinenic acid (perrottetinene acid (PETA), Fig. 1) (Toyota et al., 2002). PETA 2 is analogous to THCA 4, the biosynthetic product in Cannabis that is thermally decarboxylated to give psychoactive THC (Reason et al., 2022). These Radula bibenzyl analogs of trans-THC and trans-THCA are also noteworthy for their inverted stereoconfiguration at C4 (Fig. 1), which may affect the biological potency of the molecule (Chicca et al., 2018).

Deep sequencing, de novo assembly and annotation of a R. marginata transcriptome resulted in the identification of candidate precursor genes for the PET biosynthetic pathway (Hussain et al., 2018). The authors also putatively identified bibenzyl-4-gerolic acid (BB4GA 9, Fig. 1), a potential biosynthetic precursor of PET analogous to cannabigerolic acid (CBGA 11, Fig. 1), in a R. marginata extract.

Further biological activities have been reported for chemically synthesized cis- and trans-PET (Stott et al., 2021). This patent also described syntheses of cis and trans isomers of perrottetinene diol (PTD 5, Fig. 1) analogous to Cannabis cannabidiol (CBD 7, Fig. 1), complementing the syntheses of Crombie et al. (1988), who hypothesized the occurrence of these compounds in nature.

Known by some Māori as Wairuakohu (Caddie, 2024), R. marginata is endemic to A/NZ, growing as an epiphyte on bark or leaves, or on rocks (Fig. 2). It is found in shaded area of native forest across Te Ika-a-Māui/North Island and the north of Te Wai Pounamu/South Island (Hodgson, 1944–1945). The original PETA report (Toyota et al., 2002) was from a single R. marginata collection and therefore did not report on potential variability within the species. Intraspecific variation of specialized metabolites in vascular plants is quite common, with cannabis chemotypes the most instructive model (Toth et al., 2020). However, studies on intraspecific variation of metabolites in liverworts are scarce. Blatt-Janmaat et al. (2023) reported untargeted metabolomic analyses of multiple collections of epiphytic R. complanata from Europe and Canada, although there was no mention of the presence of bibenzyls.

Fig. 2

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Wild population of Radula marginata at collection site S1 (Fig. 3) (a) and light microscopy images of R. marginata (b–d). Ventral views of a whole branch (b). Lobe medial cells (c) and margin cells (d), where variation in oil body morphology and cell wall thickness can be seen. Bars: (a) 1 cm; (b) 500 μm; (c) 100 μm; (d) 50 μm.

This study is a comprehensive investigation into the bibenzyl cannabinoid profile of multiple R. marginata collections across various sites and seasons, uncovering distinct chemotypes that persisted under controlled and in vitro conditions. Our work on this taonga (culturally significant) species was carried out in collaboration with kaitiaki Māori (indigenous guardians), with a focus on exploring potential therapeutics from native flora. The discoveries reported here complete the parallels of R. marginata bibenzyl cannabinoids with the main Cannabis cannabinoids.