Tariq A. Akhtar

A key factor that regulates melanogenesis is the activity of tyrosinase, and inhibitors of this enzyme are used as skin-whitening and lightening agents in the skincare and cosmetics industries. The methanolic extract from male inflorescences of Artocarpus altilis (Parkinson) Fosberg (breadfruit) was investigated for its bioactivities which are important for the skincare industry, including anti-tyrosinase activity. Bioassay-guided fractionation led to the isolation of a geranylated dihydrochalcone, 2-Geranyl-2’,3,4,4’-tetrahydroxydihydrochalcone (hereinafter referred to as Artochalcone), present at 1.64% of the inflorescence dry weight. Its structure was determined by a combination of 1D and 2D-NMR and HR-ESI-MS analysis. Artochalcone was found to inhibit tyrosinase with an IC₅₀ value of 28.4 μM, making it sixteen times more effective than the leading commercial skin-whitening agent, arbutin. In addition, Artochalcone displayed significant antioxidant activity determined by 2,2-diphenyl-1-picrylhydrazyl (IC₅₀ = 20.4 μM) and nitric oxide scavenging assays (IC₅₀ = 20.7 μM).

Despite the potential of Cannabis bibenzyls to remedy acute and chronic inflammation, their relative scarcity, in planta, has hindered applications for them in mainstream therapeutic efforts. Here, we describe the biocatalytic synthesis of cannabistilbene I (1), a prototypical Cannabis bibenzyl, and demonstrate its utility as an anti-inflammatory agent. A Cannabis O-methyltransferase (CsOMT1) was first identified that catalyzes the 3-hydroxymethylation of dihydroresveratrol (2) to produce pinobistilbene (3). Structural characterization of CsOMT1 revealed that the substrate-binding pocket requires the ethyl bridge on 2 to twist with a dihedral angle of −110°, thereby explaining why less flexible aromatics such as stilbenes serve as poor enzymatic substrates. Next, a prenyltransferase (CloQ) from the Gram-positive bacterium Streptomyces was shown to prenylate the 3′-position of the B-ring on 3 into 1. Using these two enzymes, a cell-free method was then developed to synthesize 1 and the compound was shown to inhibit both microsomal prostaglandin E2 synthase-1 and 5-lipoxygenase enzyme activity, in vitro, more effectively than the leading commercially available inhibitors. Together, these results establish a platform for producing cannabistilbene I (1) that circumvents the challenges of traditional “chemical synthesis”, and which is amenable to produce similar value-added compounds that are not easily accessible from nature.

Background: Cannabis sativa L. accumulates a wide array of specialized compounds, many of which are non-psychotropic and show significant promise in medical and therapeutic applications. One such group of C. sativa compounds is prenylated flavonoids, which have emerged as potential treatments for chronic pain and inflammation. Accordingly, the aim of this study was to isolate, identify, and synthesize prenylated flavonoids from C. sativa and test their efficacy as anti-inflammatory agents. Methods: An enriched polyphenol extract from C. sativa was fractionated using flash chromatography and high-performance liquid chromatography to isolate prenylated flavonoids. Liquid chromatography–mass spectrometry (LC–MS) and nuclear magnetic resonance (NMR) spectroscopy were employed to determine their structures. Phylogenomic and classical biochemical approaches were combined to identify the enzyme involved in the biosynthesis of the isolated compounds. Finally, these prenylated flavonoids were tested to determine their inhibitory properties against microsomal prostaglandin E synthase-1 (mPGES-1) activity. Results: Two prenylated flavonoids were isolated from the aerial parts of the C. sativa plant using classical chromatographic procedures and identified as 6-prenylapigenin (6-PA) and 6-geranylapigenin (6-GA). A C. sativa prenyltransferase (CsPT3) from the UbiA superfamily was identified to complete the final prenylation step in 6-PA and 6-GA biosynthesis from the widespread plant flavonoid known as apigenin. The inhibitory potentials of 6-PA and 6-GA against mPGES-1 activity were approximately as effective as, or better than, that of a leading commercially available inhibitor, MK-886. Molecular docking simulations confirmed strong binding affinities of 6-PA and 6-GA to mPGES-1 compared to its natural substrate. Conclusions: 6-PA and 6-GA are prenylated derivatives of the widespread plant flavonoid known as apigenin. These non-psychotropic flavonoids accumulate in C. sativa and exhibit potent inhibition of mPGES-1, a chief mediator in the pro-inflammatory pathway. Identification of the final step in 6-PA and 6-GA biosynthesis, together with their now-established anti-inflammatory activity, presents propitious biotechnological avenues for these therapeutically relevant C. sativa compounds.

There has been growing interest in natural products as potential therapeutics for the core and comorbid symptoms of autism spectrum disorders. Almost all the studies on autism have focused on the therapeutic benefits of cannabis and its associated cannabinoids. In this study the potential therapeutic efficacy of cannflavin B, a related, yet non-psychoactive component of the Cannabis sativa plant, was evaluated. Using prenatal valproic acid (VPA) exposure in rats, a model that has been widely used to study aspects of autism, we showed that cannflavin B was anxiolytic in the female VPA rats, and normalized sociality in VPA animals of both sexes. When neuronal oscillatory activity was examined, in female VPA rats cannflavin B normalized alterations in low frequency power within the cingulate cortex (Cg), and theta-gamma cross frequency coupling between the dorsal hippocampus (dHIP) and the prefrontal cortex (PFC). In male VPA animals, cannflavin B induced frequency-specific alterations in power within the PFC, Cg, and dHIP and ameliorated the VPA-induced suppression of oscillatory coherence between all three regions. In each brain region, cannflavin B also attenuated the sex-specific VPA-induced elevations in microglia. In vitro , cannflavin B normalized VPA-induced elevations in cortical and HIP neuronal activity and promoted more organized cortical firing. These findings demonstrate cannflavin B normalizes behavioural and neuronal systems function alterations induced by prenatal VPA in rats. The present study highlights the importance of alternative cannabis compounds in autism and other disorders.

SUMMARY: Zea mays (maize) makes phytoalexins such as sesquiterpenoid zealexins, to combat invading pathogens. Zealexins are produced from farnesyl diphosphate in microgram per gram fresh weight quantities. As farnesyl diphosphate is also a precursor for many compounds essential for plant growth, the question arises as to how Z. mays produces high levels of zealexins without negatively affecting vital plant systems. To examine if specific pools of farnesyl diphosphate are made for zealexin synthesis we made CRISPR/Cas9 knockouts of each of the three farnesyl diphosphate synthases (FPS) in Z. mays and examined the resultant impacts on different farnesyl diphosphate‐derived metabolites. We found that FPS3 (GRMZM2G098569) produced most of the farnesyl diphosphate for zealexins, while FPS1 (GRMZM2G168681) made most of the farnesyl diphosphate for the vital respiratory co‐factor ubiquinone. Indeed, fps1 mutants had strong developmental phenotypes such as reduced stature and development of chlorosis. The replication and evolution of the fps gene family in Z. mays enabled it to produce dedicated FPSs for developmentally related ubiquinone production (FPS1) or defense‐related zealexin production (FPS3). This partitioning of farnesyl diphosphate production between growth and defense could contribute to the ability of Z. mays to produce high levels of phytoalexins without negatively impacting its growth.

Cannflavins A and B are flavonoids that accumulate in the Cannabis sativa plant. These specialized metabolites are uniquely prenylated and highly lipophilic, which, a priori , may permit their interaction with membrane-bound enzymes and receptors. Although previous studies found that cannflavins can produce anti-inflammatory responses by inhibiting the biosynthesis of pro- inflammatory mediators, the full extent of their cellular influence remains to be understood. Here, we studied these flavonoids in relation to the Tropomyosin receptor kinase B (TrkB), a receptor tyrosine kinase that is activated by the growth factor brain-derived neurotrophic factor (BDNF). Using mouse primary cortical neurons, we first collected evidence that cannflavins prevent the accumulation of Activity-regulated cytoskeleton-associated (Arc, also known as Arg3.1) protein upon TrkB stimulation by exogenous BDNF in these cells. Consistent with this effect, we also observed a reduced activation of TrkB and downstream signaling effectors that mediate Arc mRNA transcription when BDNF was co-applied with the cannflavins. Of note, we also performed a high-throughput screen that demonstrated a lack of agonist action of cannflavins towards 320 different G protein-coupled receptors, a result that specifically limit the possibility of a TrkB transinactivation scenario via G protein signaling to explain our results with dissociated neurons. Finally, we used Neuro2a cells overexpressing TrkB to show that cannflavins can block the growth of neurites and increased survival rate produced by the higher abundance of the receptor in this model. Taken together, our study offers a new path to understand the reported effects of cannflavins and other closely related compounds in different cellular contexts.

SUMMARY: This study focuses on the biosynthesis of a suite of specialized metabolites from Cannabis that are known as the ‘bibenzyls’. In planta, bibenzyls accumulate in response to fungal infection and various other biotic stressors; however, it is their widely recognized anti‐inflammatory properties in various animal cell models that have garnered recent therapeutic interest. We propose that these compounds are synthesized via a branch point from the core phenylpropanoid pathway in Cannabis, in a three‐step sequence. First, various hydroxycinnamic acids are esterified to acyl‐coenzyme A (CoA) by a member of the 4‐coumarate‐CoA ligase family (Cs4CL4). Next, these CoA esters are reduced by two double‐bond reductases (CsDBR2 and CsDBR3) that form their corresponding dihydro‐CoA derivatives from preferred substrates. Finally, the bibenzyl backbone is completed by a polyketide synthase that specifically condenses malonyl‐CoA with these dihydro‐hydroxycinnamoyl‐CoA derivatives to form two bibenzyl scaffolds: dihydropiceatannol and dihydroresveratrol. Structural determination of this ‘bibenzyl synthase’ enzyme (CsBBS2) indicates that a narrowing of the hydrophobic pocket surrounding the active site evolved to sterically favor the non‐canonical and more flexible dihydro‐hydroxycinnamoyl‐CoA substrates in comparison with their oxidized relatives. Accordingly, three point mutations that were introduced into CsBBS2 proved sufficient to restore some enzymatic activity with an oxidized substrate, in vitro. Together, the identification of this set of Cannabis enzymes provides a valuable contribution to the growing ‘parts prospecting’ inventory that supports the rational metabolic engineering of natural product therapeutics.

Mitragyna speciosa (“kratom”), employed as a traditional medicine to improve mood and relieve pain, has shown increased use in Europe and North America. Here, the dose-dependent effects of a purified alkaloid kratom extract on neuronal oscillatory systems function, analgesia, and antidepressant-like behaviour were evaluated and kratom-induced changes in ΔFosB expression determined. Male rats were administered a low or high dose of kratom (containing 0.5 or 1 mg/kg of mitragynine, respectively) for seven days. Acute or repeated low dose kratom suppressed ventral tegmental area (VTA) theta oscillatory power whereas acute or repeated high dose kratom increased delta power, and reduced theta power, in the nucleus accumbens (NAc), prefrontal cortex (PFC), cingulate cortex (Cg) and VTA. The repeated administration of low dose kratom additionally elevated delta power in PFC, decreased theta power in NAc and PFC, and suppressed beta and low gamma power in Cg. Suppressed high gamma power in NAc and PFC was seen selectively following repeated high dose kratom. Both doses of kratom elevated NAc-PFC, VTA-NAc, and VTA-Cg coherence. Low dose kratom had antidepressant-like properties whereas both doses produced analgesia. No kratom-induced changes in ΔFosB expression were evident. These results support a role for kratom as having both antidepressant and analgesic properties that are accompanied by specific changes in neuronal circuit function. However, the absence of drug-induced changes in ΔFosB expression suggest that the drug may circumvent this cellular signaling pathway, a pathway known for its significant role in addiction.