Antiarthritic Effects of a Root Extract from Harpagophytum procumbens DC: Novel Insights into the Molecular Mechanisms and Possible Bioactive Phytochemicals

Abstract

Harpagophytum procumbens (Burch.) DC. ex Meisn. is a traditional remedy for osteoarticular diseases, including osteoarthritis (OA), although the bioactive constituents and mechanisms involved are yet to be clarified. In the present study, an aqueous H. procumbens root extract (HPE; containing 1.2% harpagoside) was characterized for its effects on synoviocytes from OA patients and phytochemical composition in polyphenols, and volatile compounds were detected. HPE powder was dissolved in different solvents, including deionized water (HPE_H2O), DMSO (HPE_DMSO), 100% v/v ethanol (HPE_EtOH100), and 50% v/v ethanol (HPE_EtOH50). The highest polyphenol levels were found in HPE_DMSO and HPE_EtOH50, whereas different volatile compounds, mainly β-caryophyllene and eugenol, were detected in all the extracts except for HPE_H2O. HPE_H2O and HPE_DMSO were able to enhance CB2 receptor expression and to downregulate PI-PLC β2 in synovial membranes; moreover, all the extracts inhibited FAAH activity. The present results highlight for the first time a multitarget modulation of the endocannabinoid system by HPE, likely ascribable to its hydrosoluble compounds, along with the presence of volatile compounds in H. procumbens root. Although hydrosoluble compounds seem to be mainly responsible for endocannabinoid modulation by HPE, a possible contribution of volatile compounds can be suggested, strengthening the hypothesis that the entire phytocomplex can contribute to the H. procumbens healing properties.

Keywords: FAAH; cannabinoid receptors; eugenol; nutraceuticals; osteoarthritis; phospholipases; polyphenols; volatile compounds; β-caryophyllene.

Figure 1

Immunohistochemical analysis of synovial membranes from OA (osteoarthritis) and non-OA patients. Left panel: Slices were stained with anti-CB1 and anti-CB2 receptor antibodies. Right panel: Slices were stained with anti-PI-PLC β2 and anti-PI-PLC β3 antibodies. Slides were counterstained with hematoxylin and mounted with permanent mounting media. This figure shows representative images of different experiments (n = 5 non-OA and n = 6 OA).

Figure 2

Characterization of human primary synoviocytes (FLSs) and analysis of cell viability. (A) Human primary FLSs, isolated by synovial membranes and cultured in vitro, were stained with anti-vimentin primary antibody and with Alexa Fluor 488 (green) secondary antibody. (B) Cell viability was assessed by the MTS colorimetric method, and FLSs were treated with three concentrations, 1, 0.5, and 0.1 mg/mL of Harpagophytum procumbens root extract (HPE) dissolved in deionized water (HPE_H2O), DMSO (HPE_DMSO), 100% v/v EtOH (HPE_EtOH100), and 50% v/v EtOH (HPE_EtOH50), for 24, 48, and 72 h. Cell viability of treated samples was normalized to the untreated cells, which is reported as 100% and represented by a horizontal line.

Figure 3

Effects of all HPE extracts on CB1 and CB2 mRNA expression level in human primary FLSs. After 24 h treatment with 0.1 mg/mL of Harpagophytum procumbens root extract (HPE) dissolved in deionized water (HPE_H2O), DMSO (HPE_DMSO), 100% v/v EtOH (HPE_EtOH100), and 50% v/v EtOH (HPE_EtOH50), cells were harvested and mRNA was extracted and analyzed by RT-PCR. CB1 and CB2 receptor mRNA levels were reported as relative mRNA expression level with respect to 18S mRNA (2^−ΔΔCt method). Results are expressed as mean ± S.E.M. of data obtained by three different experiments. Statistical significance was * p < 0.05; ** p < 0.01.

Figure 4

Effects of all HPE extracts on CB2 receptor and PI-PLC β2 protein production. Upper panel: Cells were treated with 0.1 mg/mL of Harpagophytum procumbens root extract (HPE) dissolved in deionized water (HPE_H2O), DMSO (HPE_DMSO), 100% v/v EtOH (HPE_EtOH100), and 50% v/v EtOH (HPE_EtOH50), for 24 h and then, analyzed by immunofluorescence using anti-CB2 and anti-PI-PLC β2 primary antibodies and Alexa Fluor 488 (green, CB2) and Alexa Fluor 568 (red, PI-PLC β2) secondary antibodies, respectively. Nuclei were stained with DAPI (original magnification 40×). Lower panel: The pixel intensities in the region of interest were obtained by ImageJ. * p < 0.05.

Figure 5

Effects of Harpagophytum procumbens root extract (HPE) on fatty acid anandamide hydrolase (FAAH) expression and enzymatic activity. (A) Cells were treated with 0.1 mg/mL of Harpagophytum procumbens root extract (HPE) dissolved in deionized water (HPE_H2O), DMSO (HPE_DMSO), 100% v/v EtOH (HPE_EtOH100), and 50% v/v EtOH (HPE_EtOH50), for 24 h. Cells were then harvested, and mRNA was extracted and analyzed by RT-PCR. FAAH mRNA levels were reported as relative mRNA expression level with respect to 18S mRNA (2^−ΔΔCt method). Results are expressed as mean ± S.E.M. of data obtained by three different experiments. (B) Concentration–response curves showing the inhibitory effects on FAAH from HPE_DMSO, 100% v/v HPE_EtOH100, 50% v/v HPE_EtOH50, and HPE_H2O. Data are the mean ± SE of at least three independent experiments with two replicates for each experiment (n = 6).