## 4 Discussion  
The aim of this study was to investigate the effects of _Harpagophytum procumbens_ extract (HPE) on fibroblast-like synoviocytes (FLSs) from osteoarthritis patients, with particular attention on the endocannabinoid-mediated mechanisms. Osteoarthritis (OA) is characterized by chronic inflammation, and it is currently treated with anti-inflammatory drugs, which are only able to counteract the symptoms [2; 31]. Several nutraceuticals, such as glucosamine, chondro sulfate, and curcumin, are administered with the aim of delaying the cartilage degradation, with inconsistent results [32]. Extracts from plants are also traditionally used to treat OA [16]. Among them, _H. procumbens_ DC. has been studied for its chondrocytes activities, mainly for the ability to inhibit the production of proinflammatory mediators, such as TNF\(\alpha\) and IL-1\(\beta\), and enzymes able to hydrolyze the extracellular matrix components, metalloproteinases, and elastase [19]. Iridoid glycosides have received major attention as possible bioactive compounds of _H. procumbens_ secondary metabolites [33]. Harpagsoide is the most investigated one, and it is considered a reference standard of _H. procumbens_ for titration purposes [15]. The anti-inflammatory activity of harpagsoide has been found to be mediated by the inhibition of COX-1 and COX-2 enzymes along with by a lowered cytokine and NO release [33]. It was also able to counteract inflammation in primary human osteoarthritic chondrocytes through the suppression of c-FOS/AP-1 signal and the inhibition of proinflammatory cytokine and fibrinogen factor production [23]. Moreover, harpagsoide requires hydrolysis to a bioactive metabolite to exert its anti-inflammatory activity [34]. Despite this promising evidence, the anti-inflammatory properties of harpagsoide cannot fully explain those of the entire _H. procumbens_ phytocomplex, thus, suggesting that other compounds can contribute to the activity of the plant [15; 33]. In the phytocomplex, some phenylpropanoids were reported to contribute to the _H. procumbens_ anti-inflammatory effects [15]. Some studies have highlighted that plant roots can release volatile compounds as a defense strategy to counteract pathogen and fungal infections and to mediate the interaction between plant and soil bacteria [35; 36; 37]. Accordingly, our SPME-GC-MS analysis of HPE extracts highlighted the presence of several volatile compounds, mainly recovered by \(\text{HPE}_{\text{DMSO}}\), 100% \(\forall\)/\(\text{HPE}_{\text{EOH}}\), and 50% \(\forall\)/\(\text{HPE}_{\text{EOH}}\). Among these volatile compounds, \(\beta\)-caryophyllene and eugenol were identified to be the major sesquiterpene and monoterpene present, followed by the sesquiterpenes \(\alpha\)-humulene and \(\alpha\)-copaene. Previously, 31 different volatile compounds, obtained by an heptanoic extraction, were characterized in _H. procumbens_ root, although neither sesquiterpenes nor monoterpene were detected [38]. In several preclinical models, \(\beta\)-caryophyllene, \(\alpha\)-humulene, and eugenol have been shown to possess anti-inflammatory activities, affecting proinflammatory cytokine secretion and inhibiting inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX-2) expression [39; 40]. \(\beta\)-caryophyllene acts as an agonist of the endocannabinoid CB2 receptor, which is involved in the modulation of inflammation and the immune system and inhibits the fatty acid amide hydrolase enzyme [41; 42]. Recent evidence highlighted the ability of this sesquiterpene to reduce articular and systemic inflammation in several animal models of arthritis [25; 43; 44]. Particularly, its antiarthritic effects in human articular chondrocytes were mediated by a crosstalk between CB2 and PPAR-\(\gamma\) receptors [25]. In articular joints, FLSs are involved both in supporting health chondrocytes and the immune system during inflammation [45]. In this study, we decided to use FLSs isolated from human synovial membranes as an in vitro model to study the effects of _H. procumbens_ extracts.