Phytochemical characterization and biological activity of a commercial Harpagophytum procumbens (Burch.) DC. ex Meisn. extract

Background: Harpagophytum procumbens (Burch.) DC. ex Meisn. is a medicinal plant used worldwide as a remedy for joint pain associated with osteoarthritis and rheumatic ailments. Iridoid glucosides are considered the bioactive constituents, although further unknown phytochemicals seem to be involved in its bioactivities. In the present study, a commercially available extract from H. procumbens root, characterized to contain 1% harpagoside, has been evaluated for its phytochemical composition and biological activities, in order to identify novel bioactive compounds; particularly, its ability to modulate different targets of endocannabinoid system, including CB2 receptor and FAAH enzyme, was studied. Methods: In order to better recover the bioactive phytochemicals, the crude extract was dissolved in different solvents, including dimethyl sulfoxide (DMSO), ethanol (EtOH) 100% v/v, EtOH 50% v/v and deionized water, and any insoluble residue was removed by centrifugation. Total polyphenols, tannins and flavonoids were spectrophotometrically determined, while the volatile compounds were identified by GC-MS analysis. A preliminary cytotoxicity assay in human primary synoviocytes was performed; then, nontoxic concentrations of the extracts were assessed for the ability to affect endocannabinoid system. The effects of the treatments on protein and gene expression of the cannabinoid receptors were determined by immunofluorescence and quantitative-real time-PCR. Furthermore, the ability of the extracts to inhibit the fatty acid amide hydrolase (FAAH) was evaluated using a fluorescence-based kit. Results: DMSO fully dissolved the crude extract, whereas insoluble residues were produced with the other solvents; moreover, the solvent allowed to recover the highest levels of total polyphenols, tannins and flavonoids, followed by EtOH 100% v/v, EtOH 50% v/v and H2O. GC-MS highlighted the presence of different terpenoids, among which β-caryophyllene and eugenol. The extracts induced a significant increase in the CB2 mRNA expression and protein levels in synoviocytes. Moreover, they inhibited FAAH activity, although with a different potency. Under the same experimental conditions, harpagoside lacked FAAH inhibition, despite a strong effect of the positive control JZL 195. On the basis of this evidence, a modulation of the endocannabinoid system seems to be involved in the bioactivities of the tested extracts. Moreover, our results highlight the presence of different terpenoids along with polyphenols in H. procumbens samples. Among the identified constituents, β-caryophyllene is known to induce CB2-mediated antinflammatory effects, thus suggesting its possible contribution to phytocomplex bioactivity. Conclusions: The present results highlight, for the first time, the involvement of an endocannabinoid system modulation in the antinflammatory and analgesic effects of H. procumbens and improve the mechanistic knowledge in support of its use in arthritic diseases