L. leonurus (L.) R. Br. is a drought resistant medicinal shrub endemic to South Africa with a bi-annual flowering season (high-yielding medicinal crop). Anecdotally it is known for eliciting mild psychoactive effects akin to the smoking of Cannabis and, has a long-standing history in traditional healing practices in South Africa. Some studies have alluded to its medicinal activities however, these have largely focused on the leaves and only a few reports have dealt with the flowers [10, 11]. Furthermore, there are no reports that describe the occurrence of medicinal pCBs from the leaves or the flowers. Since pCBs have been proposed as effective TMs for the prevention and/or treatment of chronic diseases [18–20], identification of pCBs (or pCB-like compounds) in plants other than Cannabis spp. could present an attractive value proposition to complement the emergent Cannabis industry.
Based on its mild Cannabis-like effect when smoked, we conducted analyses into the flower-derived phytochemicals and the potential presence of pCB-like compounds in L. leonurus (L.) R. Br. var. albiflora (Fig. 1A). Since extensive phytochemical profiling of the leaves have never yielded any pCB-like compounds [10, 11], we suspected that such compounds may be present in flowers (akin to Cannabis). Using HPTLC, coupled to fast blue BB salt for a selective colorimetric detection of pCB compounds [14], a distinct orange spot could be observed (Rf 0.55, Fig. 1B). The fast blue method detects major neutral cannabinoids such as tetrahydrocannabinol (THC), cannabidiol (CBD), and their cannabinoid acid derivatives THCA and CBDA, among other cannabinoids with high sensitivity and selectivity [15, 16].
A single, putative pCB-like compound was isolated, and its identity determined using LC-MS/MS in both positive and negative ionization modes (Fig. 2A, B). Mass spectra from whole flower extracts and the isolated HPTLC compound were compared (Fig. 2C, D) resulting in equivalent retention times and peak masses in both negative (RT: 30.78 min; [M-H]− = 374.26) and positive (RT: 30.77 min; [M + H]+ = 376.26) ionization mode. Further investigation using the international metabolite repositories, Metabolomics Workbench, PubChem, and METLIN Metabolite and Chemical Entity Database, consistently identified this compound as 7,10,13,16-Docosatetraenoylethanolamine ([M-H]− = 374.31; [M + H]+ = 376.32; neutral m/z = 375.31), also known as Adrenoyl-ethanolamide (EA). Adrenoyl-EA is a bioactive endocannabinoid previously thought to be unique to mammals [21, 22]. However, it has recently (and for the first time) been reported to occur in methanol extracts of the Mashua plant (Tropaeolum tuberosum), extensively used in Andean folk medicine [23]. Our findings similarly demonstrate the occurrence of adrenoyl-EA in acetonitrile extracts from the flowers of L. leonurus (L.) R. Br. var. albiflora.
Adrenoyl-EA is structurally similar to the major endocannabinoid anandamide and functions as an agonist of the CB1 and TRPV1 receptor proteins, two major receptors of the human ECS [21, 24]. Agonists of these receptors are ideal candidates in a range of therapeutic targets, specifically exhibiting anti-inflammatory, neuroprotective, and anticancer activities, among others [23, 25, 26]. Within the growing market of TMs, we consider it beneficial to identify sustainable, climate resilient plant resources (high yielding medicinal crops) that accumulate pCBs or PCB-like compounds with the potential for therapeutic purposes.
A South African perspective on the development of pCB products
It is estimated that the domestic South Africa Cannabis market will reach USD $1.8 billion by 2023 however, this projection is based on export value only, excluding Cannabis-derived pCB products. The value proposition lies within the latter, given the therapeutic potential of pCBs in chronic disease treatment [23–26]. South African agriculture is typified by both large scale mechanized, and smallholder practices and Cannabis cultivation is considered viable. Although the agricultural experience and arable land in South Africa is adequate for the growth and export of Cannabis, the irrigation infrastructure and required daylight might not be sufficient to sustain the production of high quality Cannabis-derived products. One of the major predicted hurdles in establishing a sustainable Cannabis industry is the water-intensive measures required for an efficient pCB yield from Cannabis. If one could exploit a water-efficient medicinal crop capable of producing pCB-like compounds, this would serve as an ideal industry alternative into a realm of “smart-pharming” practices.
As part of a pCB-driven strategy for the development of high value TMs, we suggest that the discovery of pCBs in endemic South African plants could be highly complementary to the Cannabis industry, given that these plants are adapted to the climatic and water-scarce conditions. To our knowledge, we provide the first evidence on the presence of the pCB-like compound, adrenoyl-EA, in the flowers of L. leonurus (L.) R. Br. var. albiflora. Globally, the production of pCBs from these alternative resources could benefit the future TM market, particularly in countries where agriculture is typified by water scarcity.
Limitations
HPTLC and LC-MS/MS profiles provided secure evidence for the occurrence of the pCB-like compound adrenoyl-EA within the whole flower extract of white L. leonurus (L.) R. Br. var. albiflora. Further investigations would entail (i) quantification of the identified compound/s with LC-MS/MS using authentic standard compounds and, (ii) determining structural similarities and differences when compared to other plant-, animal- or human-derived adrenoyl-EA, with NMR. However, to support the idea of an alternative and sustainable crop to produce pCB products, it would be beneficial to isolate and identify the other compounds from the HPTLC to uncover the existence of a metabolite pathway capable of producing several different pCB or pCB-like compounds.