The cannabinoid (CB) and opioid receptors are seven-transmembrane domain G-protein coupled receptors (GPCRs) known to modulate various cellular, neuronal, and cardiovascular functions [1]. Cannabinoid receptor type 1 (CB1R) antagonists/inverse agonists have the potential for treating obesity [2], obesity-related cardiometabolic disorders [3], and drug/substance abuse [4]; however, there is no such drug currently available on the market. CB1R is abundant in the central nervous system (CNS) and in peripheral tissues [5,6]. The cannabinoid receptor type 2 (CB2R) is a target for the discovery of several categories of therapeutics, such as for neuro-inflammation, cardiometabolic disorder, renal ischemia-reperfusion injury, and other diseases/disorders, including multiple sclerosis and arthritis [7,8,9,10]. Opioid receptors include three subtypes, δ- (delta), κ- (kappa), and µ- (mu), and agonism of these receptors regulates pain inhibitory pathways of the CNS [11]. Research studies have shown that both CB and opioid receptors share several pharmacological properties and act synergistically in analgesic effects at lower doses with fewer side effects [12]. Medicinal plants and their constituents have been used extensively as supplements to treat various neurological disorders, including pain, anxiety, convulsions, epilepsy, hysteria, and inflammation [13].

In the United States, Magnolia grandiflora L. (Magnoliaceae), commonly known as Southern magnolia, is a plant of medicinal significance that is often grown as an ornamental tree [14]. Its flowers and seeds are used in various traditional herbal remedies, including fever, rheumatism, and inflammation [15,16]. From the same genus, the bark of M. officinalis plays an important role in traditional Chinese and Japanese herbal medicine for the treatment of anxiety, sleep-related disorders, and allergic diseases [16]. In addition, magnolia-based products have been used for smoking cessation therapy and as aphrodisiacs, anti-depressants, and sedatives due to their hypothesized cannabimimetic and GABA-ergic-like effects [17]. Extensive research has been carried out on magnolia extracts and their major constituents to study their anti-cancer [18], anti-anxiety [19], anti-depressant [16,20], cardiovascular [21], and anti-inflammatory [22,23] activities. These pharmacological effects were proposed [16] to be primarily mediated by the presence of the neolignans 4-O-methylhonokiol (1), magnolol (2), honokiol (3), and 2′s hydrogenated derivative, tetrahydromagnolol (4) (Figure 1). The presence of these neolignans in the other species of the genus Magnolia, including M. officinalis, M. obovata, and M. virginiana, has led to the use of supplements prepared from these extracts as anti-depressants, analgesics, aphrodisiacs, and appetite suppressants or stimulants [17]. Earlier studies [24] have indicated that 1 and 2 could potentiate γ-aminobutyric acid A (GABA_A) receptors and could act as positive allosteric modulators (PAMs); however, these neolignans have shown no direct interaction with the norepinephrine transporter [25]. In 2004, Kong et al., evaluated the MAO inhibitory potential of 2 and 3 using rat brain mitochondria; however, no significant rat brain MAO inhibitory activity was noted [26]. Remarkably, M. officinalis extract, its constituents, and their synthetic analogs have been reported as CB2 agonists and inverse agonists [27,28,29,30] and similarly as CB-related orphan receptor GPR55 inhibitors [27]; however, the safety and toxicological properties of 2, 3, and bark extracts of M. officinalis and M. obovate have been reported as safe for consumption [31].

In a continuation to our research efforts to establish potential leads towards human cannabinoids and opioid receptors, M. grandiflora seed extract showed significant binding affinities to CB and opioid receptors, and therefore a detailed investigation of its active constituents and their structure-activity relationships (SAR) has been conducted using in silico studies that utilize the CB2 X-ray crystal structure. A bioassay-guided isolation of the hexane extract, which showed significant displacement of radioligand in cannabinoid and opioid receptors (64% CB1, 74% CB2, δ 93%, κ 61%, and µ 85%), yielded 4-O-methylhonokiol (1), magnolol (2), and honokiol (3) with selective CB2 activity (K_i 0.29, 1.40, and 1.94 μM, respectively). The structures of these neolignans are closely related to the structures of the potent CB1 agonist Δ⁹-tetrahydrocannabinol (Δ⁹-THC) and the CB1/CB2 non-selective agonist CP55,940 (Figure 1). In addition to CB and opioid activity, we report the first systematic in silico molecular docking studies of neolignans (1–4) and their structurally-related previously reported analogs (4a, 5, 5a, 6, 6a, 6b) [29], using the active-state X-ray crystal structures of CB1R and CB2R to gain an understanding of the various protein–ligand interaction patterns, their putative binding modes, and the observed SAR of these ligands. A detailed understanding of the SAR of the isolated metabolites, as well as the additive effect between compounds 2 and 3, has been reported for the first time, as supported via the in vitro data and the in silico docking studies that take advantage of the first CB2 X-ray crystal structure, which was released in 2020 [32].

The n-hexane extract of M. grandiflora showed significant radioligand displacement of CB1, CB2, δ, κ, and µ opioid receptors (Table 1). The extract was subjected to centrifugal preparative thin-layer chromatography (CPTLC; for details, see Experimental Section) to afford 4-O-methylhonokiol (1), magnolol (2), and honokiol (3), together with marked fatty acids. The structures of the isolated compounds were identified based on 1D- and 2D- nuclear magnetic resonance (NMR) and electrospray ionization mass spectrometry (ESI-MS). Compounds 1–3, as well as 4 (tetrahydromagnolol, purchased from Sigma-Aldrich), were evaluated using in vitro binding assays with CB and opioid receptors (Table 1 and Table 2 and Figure 1, Figure 2 and Figure 3). The preliminary results determined at a concentration of 10 µM revealed strong displacement by 1 towards both CB1R and CB2R (i.e., 91.3% and 82.2%, respectively). The mixture of 2 + 3 (1:1) showed similar displacement of radioligand (99.8% and 91.0%, respectively), compared to either compound 2 or 3 alone (Table 1). Based on the secondary assay results, compounds 1–3 displayed strong selective affinity towards CB2R compared to CB1R, with K_i values of 1 towards CB2R as being 0.29 ± 0.022 μM, which is significantly better than those of 2 and 3 (K_i 1.44 ± 0.138 and 1.94 ± 0.162 µM, respectively), while tetrahydromagnolol (4) showed similar displacement to that of 2 (Figure 2 and Figure 3).

The hexane extract displayed significant radioligand displacement at all three opioid receptors: δ (93%), κ (60.8%), and µ (84.5%); however, all the isolated neolignans (1–3) were devoid of binding affinity at δ and κ. Nevertheless, the isolated neolignans showed high micromolar binding affinity at the µ receptor. Indeed, a 1:1 mixture of 2 and 3 showed better additive percentage displacement (%) of radioligand at δ (93.3%), κ (59.2%), and µ (74.0%) receptors, compared to 1–3 alone, but was similar to those of the hexane extract (Table 1), which provides an explanation for the strong displacement activity of the n-hexane extract. Additionally, 2 and 3 showed a low affinity for µ opioid receptors during IC₅₀ and K_i determinations (Figure 4).

CB binding affinities of compounds 1–4 have been previously reported [27,28,29], and are in accordance with the results obtained for CB2R in our laboratory. Furthermore, compounds 1–4 were reported to possess binding affinities (K_i) between 2–9 μM for CB1R [27,28,29]. However, in our lab, this observation was only true for 4-O-methylhonokiol (1); the remaining three compounds possessed binding affinities (K_i) between 14–20 μM (Table 2). Rempel et al. (2013) have reported magnolol (2), and tetrahydromagnolol (4) as partial CB2R agonists (K_i 1.44 and 0.41 μM, respectively), and honokiol (3) was considered as a CB2R antagonist or inverse agonist (K_i 5.61 μM) [27]. Schuehly et al. (2011) reported intriguing nonspecific heteroactive behavior of 4-O-methylhonokiol (1) at the CB2R as an inverse agonist at G_i/o and as a full agonist regarding intracellular Ca²⁺ [Ca²⁺]_i. In contrast, Fuchs et al. (2013) reported 1 as an agonist at both CB receptor subtypes in forskolin-induced cAMP (3’,5’-cyclic adenosine monophosphate) accumulation assays [29]. Our CB binding affinity data of 1–4 are in close agreement with those reported previously [27,28,29].

Bioassay-guided isolation revealed that neolignans 1–3 from M. grandiflora seeds and an analog of 2 (4) displayed various degrees of displacement affinities against cannabinoid and opioid receptors. These neolignans were previously reported as CB2 agonists [29]. The observed in vitro displacement binding affinities and previously published functional data provided further evidence that neolignan 1 acts as a promising CB2 agonist. An in-depth understanding of the SAR of the isolated compounds as well as the additive effect between compounds 2 and 3 has been reported for the first time, supported via the in vitro data and the in silico docking studies that take advantage of the first CB2 X-ray crystal structure, which was released in 2020. Methylation of 2 at the p-position and the replacement of the hydroxyl group with a methoxy substituent increase the displacement activity towards CB1 as shown in Table 1. Similarly, an equal (1:1) mixture of 2 and 3 showed additive displacement activity towards the tested receptors as compared to either 2 or 3 alone, which in turn provides an explanation for the strong displacement activity of the n-hexane extract. The in silico docking studies of these neolignans (1–4) and their related previously reported analogs (4a, 5, 5a, 6, 6a, and 6b) [29] with the active states X-ray crystal structures of CB1R and CB2R revealed the putative binding mechanism, selectivity, and interaction profile of the protein–ligand complex. The docking and binding free-energy results indicate that one of the hydroxyl moieties of the molecules in the present study formed strong H-bonding through Ser383 and Ser285 with CB1R and CB2R, respectively. The impact of the methoxy group on the affinity towards CB1R and CB2R was explained in terms of binding free-energy data and the orientation of the alkyl chain within the CB1R. The additional strong hydrophobic interactions between the methoxy moiety of 5a and 6a, and Phe200 and Cys386 of CB1R, as well as the orientation of the pentyl and propyl chains, are interchanged across 5 and 5a, which could explain the increased affinity towards CB1R compared to the CB2R. We believe that insights gained from this study could provide a platform for medicinal chemists working in this important area of cannabinoid research to utilize a new scaffold for the design of new analogs from neolignans. This, in turn, can lead to the identification of new synthetic compounds with improved affinity, functional activity, and/or selectivity for the CB receptors.

Our findings suggest the potential utility of Magnolia neolignans and their derivatives for the development of new CB agonists for use as analgesic or anti-inflammatory lead compounds. Furthermore, the combined effect of potent cannabinoid (CB) and weak μ opioid binding affinity of a mixture of magnolol and honokiol analogs offers a new window for the treatment of opioid dependence and opioid withdrawal symptoms; however, further in vivo studies should be implemented to help deepen the understanding of the mechanisms of action of these new lead natural compounds.