Phytochemical Characterization of Cannabis sativa L. Chemotype V Reveals Three New Dihydrophenanthrenoids That Favorably Reprogram Lipid Mediator Biosynthesis in Macrophages

The growing general interest surrounding Cannabis sativa L. has led to a renewal in breeding and resulted in an impressive variability of chemotypical characteristics that required the division of cannabis into different recognized chemotypes. The chemotype V has been overlooked in terms of phytochemical composition due to the almost total absence of cannabinoids, on which biomedical attention is focused. Systematic approaches addressing diverse chemotypes are, however, needed to discriminate and define phytochemical aspects beyond cannabinoids. Such thoroughly characterized chemotypes guarantee blinding in controlled studies by mimicking the sensory properties of hemp and may help to unravel the “entourage effect”. Capitalizing on the ability of cannabis to synthesize a large number of non-cannabinoid phenolic compounds, we here investigated, for the first time, the composition of the Ermo chemotype V and identified new compounds: two dihydrophenanthrenes and the methoxy-dihydrodenbinobin. All three compounds suppress pro-inflammatory leukotriene biosynthesis in activated macrophage subtypes by targeting 5-lipoxygenase, but substantially differ in their capacity to elevate the levels of specialized pro-resolving lipid mediators and their precursors in M2 macrophages. We conclude that the discovered compounds likely contribute to the anti-inflammatory properties of Cannabis sativa L. chemotype V and might promote inflammation resolution by promoting a lipid mediator class switch.


Introduction
Cannabis sativa L. was one of the first plants ever domesticated by humans, who spread its cultivation worldwide over the past 10,000 years. Due to its considerable and heterogeneous use, the plant can be considered as a multi-purpose crop, an object of constant breeding and selection [1]. The harvesting of cannabis has benefited from continuous developments and has been incredibly revived at the industrial level in several European countries such as Italy, Spain, Germany, the Netherlands and France [2]; this is also due to the progressive liberalization of non-psychoactive cultivars and their ongoing phytochemical analysis. More than 500 secondary metabolites have been identified, comprising cannabinoids, terpenoids and phenolic compounds [3]. The increasing public interest in cannabis together with the impressive variability of chemotypical characteristics inspired the division of cannabis into five recognized groups (or chemotypes) within the genus, depending on different concentrations of major cannabinoids (∆ 9 -THC 1; CBD 2; and CBG 3). The five recognized chemotypes of cannabis include: (I) the drug-type plants (narcotic) with a high content of the psychotropic ∆ 9tetrahydrocannabinol (∆ 9 -THC 1); (II) medicinal cannabis with a 1:1 content of ∆ 9 -THC 1: CBD 2; (III) industrial fiber hemp that has CBD 2 as a predominant constituent and a minimum content of ∆ 9 -THC 1 (0.2% w/w); (IV) fiber-type plants that contain cannabigerol (CBG 3) as the main cannabinoid; (V) fiber-type plants largely devoid of cannabinoids (Table 1) [4]. The latter cannabinoids are certainly spectacular secondary metabolites that have long been in the spotlight of biomedical attention. However, it is also important to discriminate and define the qualitative and quantitative aspects of other chemotypes beyond cannabinoids. Such a systematic approach allows further insights into bioactive constituents, such as cannflavins, which might synergistically contribute to the anti-inflammatory activity of cannabis [1,5]. Moreover, cannabinoid-free strains are necessary for controlled studies to mimic the sensory properties of hemp, guarantee blinding and explain the typical synergy of many botanical extracts besides the isolated cannabinoids [5]. Of all the chemotypes, type V (cannabinoid-free) has been overlooked in terms of phytochemical composition because cannabinoids are absent. Many hypotheses have arisen regarding this inability to produce cannabinoids. For example, the absence of this biosynthesis could be due to the disabling of terpeno-phenolic condensation or a total absence and dysfunctionality of glandular trichomes. The presence of a cannabinoid knock-out factor that leaves unaffected the biosynthesis of other compound classes is decisive in inactivating the pathway toward the phenolic cannabinoid precursors [6]. Anyway, cannabis is a prolific producer of metabolites and besides cannabinoids, the plant biosynthesizes other molecules, such as terpenoids responsible for the typical scent, oxylipins, amines and amides, phytosterols and a plethora of non-cannabinoid phenolic compounds [7]. Among non-cannabinoid phenols, denbinobin 4 deserves particular mention for its biological activity and for its recurrence, which is extremely rare in the plant kingdom. Denbinobin 4, a typical ingredient of several orchidaceous plants, has been identified in the IV chemotype of fiber-hemp [8]. Its anti-tumor potential has cogently been conveyed by the title of a commentary in the British Journal of Pharmacology: Escaping immune surveillance in cancer: is denbinobin the panacea? [9]. Other unique compounds of cannabis are canniprene 6 and cannflavins 7a and 8, which open a new field for chemical and biological exploration [1,10]. In particular, canniprene 6 potently inhibits the production of inflammatory eicosanoids via the 5-lipoxygenase (5-LOX) pathway [11]. It outperforms the structural analogue resveratrol in suppressing pro-inflammatory readouts in diverse cell-free test systems and shows a remarkable potential for application in skin care [12]. The prenylated lipophilic flavonoids, cannflavins 7a and 8, are potent inhibitors of inducible inflammatory enzymes such as microsomal prostaglandin E2 synthase (mPGES)-1 and act as "intelligent" suppressors of the inflammatory response [12].
Given the huge number of bioactive compounds beyond cannabinoids, we were interested in the non-cannabinoid phenolic profile present in the V chemotype, the antiinflammatory activity of individual components, and the impact of breeding on the chemical and functional fingerprint. For this reason, we here investigated, for the first time, the composition of the Ermo fiber hemp (a variety of hemp with almost no cannabinoids) provided by Canvasalus and reported on the isolation, phytochemical characterization, and biological evaluation of phenolic compounds, of which three derivatives are new. Our phytopharmacological studies employed a targeted metabololipidomics approach and focused on molecular targets within anti-inflammatory, pro-resolving and immunomodulatory lipid mediator biosynthesis pathways.

Results and Discussion
The analysis of the phytochemical profile of the Ermo chemotype V led to the isolation of trace amounts (0.14%) of cannabidiolic acid 2a and cannabigerolic acid 3a. Chromatographic purification yielded non-cannabinoid phenolic compounds, i.e., the dihydrostilbenoid canniprene 6, the C-prenylated flavonoids cannflavin A 7a, cannflavin B 8 and 5 -demethoxy-cannflavin A 7, which was first identified in Morus alba var. tatarica in 2015 [13] and only recently in Cannabis sativa [14]. The Ermo fiber-hemp provided three natural compounds 5, 9 and 10, which have never been isolated before and belong to the biosynthetic group of dihydrophenanthrenoids ( Figure 1). of inducible inflammatory enzymes such as microsomal prostaglandin E2 synthase (mPGES)-1 and act as "intelligent" suppressors of the inflammatory response [12]. Given the huge number of bioactive compounds beyond cannabinoids, we were interested in the non-cannabinoid phenolic profile present in the V chemotype, the antiinflammatory activity of individual components, and the impact of breeding on the chemical and functional fingerprint. For this reason, we here investigated, for the first time, the composition of the Ermo fiber hemp (a variety of hemp with almost no cannabinoids) provided by Canvasalus and reported on the isolation, phytochemical characterization, and biological evaluation of phenolic compounds, of which three derivatives are new. Our phytopharmacological studies employed a targeted metabololipidomics approach and focused on molecular targets within anti-inflammatory, pro-resolving and immunomodulatory lipid mediator biosynthesis pathways.
Even though a lot of work has been conducted on the phytochemical characterization of cannabis, this is the very first time the non-cannabinoid compounds 5, 9 and 10 have been isolated and characterized from this plant. The dihydrophenanthrenes 9 and 10 are particularly interesting for a possible structure-activity relationship study, since both compounds retain the same number of methoxy and hydroxyl groups but with different positions in the reciprocal scaffold. Moreover, just a few cannabis phenanthrenes have so far been described with properties that might be relevant for human health, such as the unique denbinobin 4 endowed with cell apoptosis-inducing properties that might rely on the impaired activation of the survival kinase Akt (protein kinase B) [15,16] or the interference with NF-kB signaling [17]. Given the biological relevance of denbinobin 4, the newly isolated methoxy-reduced denbinobin analogue 5 described here, is expected to be of similar biological relevance.
In human M2 macrophages, which are associated with wound healing and tissue repair [21,22] compounds 9 and 10 stimulated the production of specialized pro-resolving mediators (SPMs) (Figure 3b,f), which actively promote the termination of inflammation and return to homeostasis by inhibiting neutrophil trafficking, stimulating efferocytosis, enhancing bacterial clearance, protecting from oxidative stress, and promoting tissue regeneration [23][24][25]. Interestingly, compound 9 raised the levels of multiple SPM classes, i.e., D-series Rv (RvDs), protectins (PDs) and maresin 2 (MaR2), whereas the isomeric compound 10 selectively increased the availability of MaR2, and the quinone 5 elevated MaR2 and PDs but not RvDs levels (Figure 3b,f). It is tempting to speculate that the 3and/or 7-methoxy-groups, which are shared by 9 and 5, but not present in 10, in these isolated compounds, are required for an effective increase in PDs levels. Moreover, PDs and MaR2 were strongest upregulated by compound 5 (Figure 3b,f); although, the release of polyunsaturated fatty acids (PUFAs) was decreased (Figure 3g), which indicates that the quinone substructure is not only advantageous for 5-LOX inhibition and required for suppressing COX product formation but is also favorable for the lipid mediator class switch from pro-inflammatory LTs and prostanoids to pro-resolving SPMs.
suppressing COX product formation but is also favorable for the lipid mediator class switch from pro-inflammatory LTs and prostanoids to pro-resolving SPMs.  Mechanistically, the overall increase in SPM levels induced by 9 (Figure 3b,f) seems to arise from an enhanced formation of precursors, including 17-HDHA, 15-HEPE and 15-HETE (Figure 3b) by 15-LOX-dependent oxygenation of docosahexaenoic acid (DHA) [26,27]. Compound 5, on the other hand, slightly but significantly lowered 12/15-LOX product levels (Figure 3h), which rather excludes that 12-and 15-LOX drive the upregulation of PDs and MaR2 (Figure 3f). Of interest, soluble epoxide hydrolase (sEH) not only hydrolyzes epoxyeicosatrienoic acids (EETs) to their corresponding diols (DHETs) but also converts 13(S), 14(S)-epoxy-DHA to MaR2 [28]. Since the ratio of DHETs/EETs, which serves as an indicator of sEH activity [29], is significantly elevated by compound 5 in human M2 macrophages (Figure 3i), it is tempting to speculate that the higher sEH activity adds to the associated increase in MaR2 and potentially PDs levels (Figure 3f). Compound 10 evokes comparable but less pronounced effects (Figure 3b,f); although, sEH activity is only increased by trend (Figure 3i). Together, the newly identified dihydrophenanthrenoids promote a lipid mediator class switch from inflammation to resolution, with small structural changes deciding about the lipid mediator subgroups affected.

Plant Material
Cannabis sativa L. Ermo variety was purchased from Canvasalus Srl (Monselice, Italy). A voucher specimen (CsErmo-2022) of the vegetal material is stored in Novara laboratories. The variety is protected at Community Plant Variety Office (CPVO) with application number 20100208.

Isolation of Peripheral Blood Mononuclear Cells (PBMC) from Human Blood
Leukocyte reduction system chamber (LRSC) filters were provided by the Central Institute for Blood Transfusion and Immunological Department of Tirol Kliniken GmbH (Austria) with the informed consent of the volunteers. Only healthy blood donors between 18 and 65 years of age without medication for chronic diseases, fever, or deficiency symptoms and after physical examinations by trained medical personnel were included in the study. Human peripheral blood mononuclear cells (PBMC) were freshly isolated from LRSC filters. After dilution of the cell concentrates in PBS pH 7.4 containing 12.5 mM citrate and 14 mM glucose (130 mL, 37 • C), immune cells were separated via isopycnic density gradient centrifugation (400× g, 20 min, RT) using Histopaque ® -1077 (Sigma-Alrich, St. Louis, MO, USA). PBMC were obtained from the interphase after hypotonic lysis of erythrocytes using water (2-5 mL) and two consecutive washing steps with PBS pH 7.4 (50 mL) (270× g, 5 min, RT).

Sample Preparation and Metabololipidomic Profiling of Lipid Mediators
Lipid mediator biosynthesis in human M1/M2 macrophages (1.5 mL PBS pH 7.4 plus 1 mM CaCl 2 ) was stimulated with Staphylococcus aureus (LS1)-conditioned medium (1.0%, 3 h) after 15 min preincubation with DMSO (0.1%) or test compounds and stopped by addition of ice-cold MeOH (2.5 mL) containing deuterium-labeled internal standards (200 pg d8-5Shydroxyeicosatetraenoic acid (HETE), d4-leukotriene (LT)B 4 , d5-lipoxin (LX)A 4 , d5-resolvin (Rv)D 2 , d4-prostaglandin (PG)E 2 , and 2000 pg d8-arachidonic acid (Cayman Chemical, Ann Arbor, MI, USA)) [32]. Samples were incubated at −20 • C for at least 1 h to precipitate proteins and then centrifuged (750× g, 10 min, 4 • C). The supernatant was mixed with acidified water (7 mL, pH 3.5) and loaded onto solid phase cartridges (Sep-Pak ® Vac 6cc 500 mg/6 mL C-18 (Waters, Milford, MA, USA)), which were conditioned with MeOH (6 mL) and equilibrated with water (2 mL). Columns were washed with water (6 mL) and hexane (6 mL, 4 • C) before lipid mediators were eluted with methyl formiate (6 mL). The organic phase was evaporated to dryness using a TurboVap LV (Biotage, Uppsala, Sweden) and the remaining lipids were dissolved in MeOH/water (1:1), centrifuged twice (21,100× g, 4 • C, 5 min), and subjected to UPLC-MS/MS analysis. Chromatographic separation of lipid mediators was carried out at 55 • C on an Acquity UPLC BEH C-18 column (130Å, 1.7 µm, 2.1 × 100 mm, Waters) using an ExionLC AD UHPLC system (Sciex, Framingham, MA, USA). The gradient of mobile phase A (water/MeOH, 90/10, 0.01% acetic acid) and mobile phase B (MeOH, 0.01% acetic acid) was ramped at a flow rate of 0.35 mL/min from 35.6% to 84.4% B within 12.5 min followed by 5 min of isocratic elution at 97.8% B. Lipid mediators and fatty acids were analyzed in the negative ion mode by scheduled multiple reaction monitoring (detection window: 120 s) using a QTRAP 6500 + Mass Spectrometer (Sciex), which was equipped with an electrospray ionization source. The curtain gas was set to 40 psi, the collision gas to medium, the ion spray voltage to −4000 V, the heated capillary temperature to 500 • C, and the sheath and auxiliary gas pressure to 40 psi. Transitions selected for quantitation and the corresponding declustering potential (DP), entrance potential (EP), collision energy (CE) and collision cell exit potential (CXP) are listed in Table 4. Absolute lipid quantities refer to an external standard calibration and were normalized to a subclass-specific deuterated internal standard as well as cell numbers. Mass spectra were acquired and processed using Analyst 1.7.1 (Sciex) and Analyst 1.6.3 (Sciex), respectively.

Statistics
Data are expressed as mean ± s.e.m. and single data from n = 4-8 independent experiments. Significant outliers were removed (Grubbs'test, p < 0.05) and data were log-transformed for statistical analysis. For multiple comparisons, repeated-measures one-way ANOVA followed by Dunnett s post hoc test was performed. p values < 0.05 were considered statistically significant. Data were analyzed using Microsoft Excel (Office 365, Version: 2204, Albuquerque, New Mexico), and statistics were performed using GraphPad Prism (GraphPad Software version 9.3.1, Dotmatics, Boston, UK). IC 50 values were calculated by non-linear regression (GraphPad Prism 9.3.1).

Conclusions
The Ermo strain of Cannabis sativa belongs to the chemotype V and is almost completely devoid of cannabinoids, which makes it an excellent candidate to ensure blinding in clinical trials. We investigated, for the first time, the phytochemical profile of this fiber hemp and identified three new compounds: two dihydrophenanthrenes 9, 10 and a quinone 5 closely related to denbinobin 4. Metabololipidomics profiling showed that the three compounds target 5-lipoxygenase and suppress pro-inflammatory LT biosynthesis in activated macrophage subtypes. While each of the compounds might contribute to the resolution of inflammation, their capacity and specificity to elevate the levels of specialized pro-resolving lipid mediators and their precursors significantly differs. Taken together, our results demonstrate the potential of phytochemical and pharmacological characterization of cannabis varieties not only to explain the different biological activities of the chemotypes but also to add to our understanding of the "entourage effect" [5].
Author Contributions: Conceptualization and writing-original draft preparation F.P. and A.K., data curation, investigation and writing S.S., L.W. and A.P. investigation, G.C. formal analysis, G.G., resources. S.S and L.W. contributed with the same effort at the experimental part. All authors have read and agreed to the published version of the manuscript.