The identi cation of a cannabinoid-like compound (adrenoyl-EA), in the owers of a South African medicinal plant (Leonotis leonurus)

Ethan Hunter Stellenbosch University Marietjie Stander Stellenbosch University Jens Kossmann Stellenbosch University Suparna Chakraborty University of Cape Town Department of Biological Sciences Sharon Prince University of Cape Town Department of Biological Sciences Shaun Peters Stellenbosch University Bianke Loedolff (  bianke@sun.ac.za ) Stellenbosch University Faculty of AgriSciences https://orcid.org/0000-0001-7519-0257


Introduction
Increasing interest from developed economies in the use of TMs has created the second largest global therapeutics market [1,2]. The medicinal Cannabis industry is currently one such focal area, largely because of the occurrence of phytocannabinoids (pCBs). As part of a unique class of phytochemicals that interact with the human endocannabinoid system (ECS), the use of pCBs as therapeutics holds promise in the treatment of numerous chronic diseases [3][4][5][6][7][8]. However, many countries still abide by strict regulatory laws pertaining to the commercial growth of Cannabis and, consequently research into pharmacologically relevant products has until recently, been relatively protracted. Since the discovery of pCBs, similar compounds (pCB-like) have been reported in several plant species, beyond Cannabis, that are commonly used in TM practices [9]. The growing interest in the use of TMs enabled an opportunity for the discovery and evaluation of new compounds from medicinal and nonmedicinal plants for the development of natural therapeutics.
The use of the 'wild cannabis' plant, L. leonurus (L.) R. Br., endemic to South Africa, is commonly reported in TM to treat numerous ailments including eczema, headaches, hypertension, and chest infections [10,11]. The leaves (when smoked) have been described to elicit similar effects to Cannabis, including mild psychoactive symptoms, as well as the capacity to alleviate anxiety and induce calming effects [10,12].
Despite the amount of research available on the leaves, little to no information exists on the medicinal properties of the owers of this plant. Furthermore, the occurrence of pCBs or pCB-like compounds has never been reported. In this research note, we report on the identi cation of adrenoyl-EA, a pCB-like compound, in the owers of the white ower variety of L. leonurus (L.) R. Br. var. albi ora.

Materials And Methods
Plant material L. leonurus (L.) R. Br. var. albi ora seeds (white ower variety) were obtained from a commercial seed supplier (Seeds for Africa, South Africa) and grown on a residential property at the Bottom Road Sanctuary, Zeekoevlei, Western Cape Province, South Africa (GPS coordinates: -34.057951, 18.499391). Flower samples were harvested at mature stage ( Fig. 1, photo courtesy of Dr B Loedolff) and immediately stored at -20°C.

Whole ower metabolite extraction
Flowers were lyophilized (20 h; 100 mTorr, -60°C) and ground to a ne powder using a pestle and mortar. Metabolites were extracted from ower material (50 mg) as previously described [13,14], evaporated, and the resin reconstituted in 200 µL ddH 2 O, calibrating the extracts to 0.50 mg/µL. Phytocannabinoid screening and isolation using HPTLC Phytocannabinoid screening was performed on 10 × 20 cm glass-back plates pre-coated with 0.25 mm silicagel 60 (REF 811213, Macherey-Nagel, Germany). Flower derived-extracts (0.50 mg/µL) were applied to the plates in 2 µL increments and allowed to dry completely before placement into a glass chromatography chamber (Camag, Switzerland), pre-conditioned with the mobile phase, chloroform:methanol (9:1; 30 min). Subsequently, the chromatogram was developed in the dark using fast blue B reagent (0.1% w/v; dissolved in 1 mM NaOH). Based on R f ranges (comparative to the upper R f values representing the less polar range of pCBs from Cannabis, under the same chromatographic conditions [15]) and change in colour (in the range of orange-red [16]), a single compound of interest was scraped from the preparative HPTLC plate and resuspended in 50% (v/v) methanol, prior to tandem mass spectrometry (LC-MS/MS) analyses.

Tandem mass spectrometry (LC-MS/MS) analyses
LC-MS/MS analyses were performed, as previously described [14], with a Waters Synapt G2 quadrupole time-of-ight mass spectrometer (Waters Corporation, Milford, MA, USA) equipped with a Waters Acquity UPLC. Samples were separated on a Waters UPLC BEH C18 column (2.1 × 100 mm; 1.7 µm) at a ow rate of 0.25 ml/min at 55 C. Solvent A consisted of 0.1% (v/v) formic acid in water and solvent B was 0.1% (v/v) formic acid in acetonitrile. The mobile phase gradient was initiated at 100% solvent A for 1 min and linearly reduced to 28% solvent A over 22 min. Subsequently, the mobile phase was changed to 40% solvent B over 8 min followed by a 1 min wash step in 100% solvent B before the column was reequilibrated to the initial conditions for 4 min. Electrospray ionization was applied, and samples were analysed in a negative mode run and a positive mode run. Data was acquired in MS E mode, which consists of a high collision energy scan range of m/z 125-1500 and a low collision energy scan from m/z 40-1500. The photo diode array detector was set to scan from 220-600 nm. The capillary voltage was set at 3.5 kV and the collision energy either 6 V (low collision energy scan from) or 30-60 V (high collision energy scan), the cone voltage was 15 V, the source temperature 120 C and the desolvation temperature was 275 C. The desolvation and cone gas (nitrogen) ows were 650 L/h and 50 L/h, respectively. Sodium formate was used for calibration and leucine encephalin was infused in the background as lock mass for accurate mass determinations. Metabolites were monitored using their deprotonated quasi-molecular ions.
Compounds were tentatively identi ed using the Metabolomics workbench [17]. The database was searched, using the m/z mass obtained from total ion chromatograms, with parameters set to the 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 owers [10,11]. Furthermore, there are no reports that describe the occurrence of medicinal pCBs from the leaves or the owers. Since pCBs have been proposed as effective TMs for the prevention and/or treatment of chronic diseases [18][19][20], identi cation 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 ower-derived phytochemicals and the potential presence of pCB-like compounds in L. leonurus (L.) R. Br. var. albi ora (Fig. 1A). Since extensive phytochemical pro ling of the leaves have never yielded any pCB-like compounds [10,11], we suspected that such compounds may be present in owers (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 (R f 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 ower extracts and the isolated HPTLC compound were compared (Fig. 2C, D)  , 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 rst time) been reported to occur in methanol extracts of the Mashua plant (Tropaeolum tuberosum), extensively used in Andean folk medicine [23]. Our ndings similarly demonstrate the occurrence of adrenoyl-EA in acetonitrile extracts from the owers of L. leonurus (L.) R. Br. var. albi ora.
Adrenoyl-EA is structurally similar to the major endocannabinoid anandamide and functions as an agonist of the CB 1 and TRPV 1 receptor proteins, two major receptors of the human ECS [21,24]. Agonists of these receptors are ideal candidates in a range of therapeutic targets, speci cally exhibiting antiin ammatory, neuroprotective, and anticancer activities, among others [23,25,26]. Within the growing market of TMs, we consider it bene cial 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][24][25][26]. South African agriculture is typi ed 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 su cient to sustain the production of high quality Cannabis-derived products. One of the major predicted hurdles in establishing a sustainable Cannabis industry is the waterintensive measures required for an e cient pCB yield from Cannabis. If one could exploit a water-e cient 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 rst evidence on the presence of the pCB-like compound, adrenoyl-EA, in the owers of L. leonurus (L.) R. Br. var. albi ora. Globally, the production of pCBs from these alternative resources could bene t the future TM market, particularly in countries where agriculture is typi ed by water scarcity.

Declarations
Ethics approval and consent to participate Not applicable.

Consent for publication
Not applicable.

Availability of data and materials
Any data, including the full LC-MS/MS datasets, used and/or analysed during the current study are available from the corresponding author (bianke@sun.ac.za) on reasonable request.

Competing interests
Not applicable.

Funding
Not applicable.
Author's contributions HPTLC and LC-MS/MS pro les provided secure evidence for the occurrence of the pCB-like compound adrenoyl-EA within the whole ower extract of white L. leonurus (L.) R. Br. var. albi ora. Further investigations would entail (i) quanti cation of the identi ed 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 bene cial 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.  leonurus (L.) R. Br. var. albi ora white ower phenotypic appearance (Photo courtesy of Dr B Loedolff) and (B) its corresponding acetonitrile (75%) extract, HPTLC phytochemical pro le. Whole ower metabolite extractions were prepared using various extraction solvents (full HPTLC plate visual is available in Supplementary Figure, acetonitrile cropped area is indicated with red dotted block) and, the acetonitrile (75%) extracts were selected for downstream analyses. The phytochemical pro le was developed fully (mobile phase; chloroform:methanol 9:1, stationary phase; silica60Å) before reagent visualization (0.1% Fast blue B salt dissolved in 1 mM NaOH) was applied to reveal potential pCB candidates, based solely on an orange-red colour development. Compound x (Rf value: 0.55) represents a colorimetric-speci c pCB-like candidate compound and, was selected for further LC-MS/MS analyses.