# Guineensine: Isolation, Synthesis, and Biological Activity
## 4 Biological Activities  
### Inhibition of Endogenous Cannabinoids and Anti-Inflammatory Activity  
The endocannabinoid system (ECS) is a cell signalling network that encompasses chemical signalling molecules called endocannabinoids (ECs), receptors and enzymes, involved in the synthesis and degradation of ECS. These include monoacylglycerolglycerol lipase (MAGL) and fatty acid amide hydrolase (FAAH). Several types of endocannabinoids receptors were described; nevertheless, the most discussed are CB1 and CB2. They are mostly expressed in the immune tissues as far as CB2 and the central nervous system concerning CB1 receptor [71]. Upon stimulation by endogenous cannabinoids, many signalling pathways are activated, giving rise to different pharmacological effects responsible for maintaining homeostasis and for regulating pathophysiology of inflammation and pain. The best discussed ECs are arachidylethanolamine or anandamine (AEA) and arachidylglycerol (2-AG), biosynthetic products of arachidonic acid. Both compounds are hydrophobic, and it was discussed that their penetration through the cell membrane occurs via membrane transporter proteins [72; 73; 74]. Specifically, for anandamine, a FAAH-like anandamine transporter [FLAT] was described [75]. According to this study, FLAT is expressed in brain and liver cells of rats and has low binding activity for AEA. The overexpression of the protein resulted in an accumulation of AEA to the extracellular space, without hindering other molecules entering the cell membrane. However, whether FLAT is finally a protein carrier for AEA was doubted by [PERSON] et al. [76]. Authors found no protein expression to the peripheral nervous system of mice models, while similar results were also obtained after Western blot analysis. ECs' activity is short due to their degradation because of the presence of hydrolytic enzymes or by their reuptake by the postsynaptic neuron. Since several disease conditions are attributed to low levels of ECs, strategies to increase their concentration in synaptic cells were examined. Some inhibitors of EC reuptake were proposed, among which naturalproducts feature prominently on the list [48; 77]. In particular, \(N\)-isobutylamide guineensine was reported to induce cannabinoidic effects related to pain [48; 78]. However, central nervous system side effects, after the direct activation of cannabinoid receptors, occur often. Therefore, targeting degradation enzymes like FAAH and MAGL, inhibiting EC reuptake or alternatively, transport carriers like FLAT, are approaches under study. Such an approach was followed by [PERSON] et al. [79] who suggested guineensine as a candidate compound to target FLAT and control neuropathic pain. Authors compared the protein bound efficacy of guineensine and retrofracamide A, an amide isolated from _Pipper_ spp., against FLAT. The results were compared to a control compound, namely FAAH-1 inhibitor (9Z)-1-(5-pyridin-2-yl-1,3_A_-oxadiazol-2-yl)octadec-9-en-1-one. The length of the hydrocarbon chain, number of carbon atoms, bond distance, and electropolocial state were parameters to consider for the inhibition of FLAT. The bioactivity of guineensine was comparable to the control compound and better than that of retrofracamide A. Guineensine formed hydrophobic bonds with FLAT in contrast to retrofracamide A and the control, which formed both hydrogen and hydrophobic bonds with the protein, a result attributed to the length of their hydrocarbon skeleton. In addition, the stability of the formed complexes (ligand compound and FLAT protein) was investigated with molecular docking simulation. Binding affinity of the two tested compounds was lower than that of the control. However, guineensine caused stronger vibrations to FLAT than the control compound and this was suggested as the possible mechanism of action against FLAT inhibition. In the study of [PERSON] et al. [48], extracts of _P. nigrum_ L. and _P. longum_ L. were tested for their capacity to inhibit AEA and 2-AG reuptake.