Chondroitin Sulfate in USA Dietary Supplements in Comparison to Pharma Grade Products: Analytical Fingerprint and Potential Anti-Inflammatory Effect on Human Osteoartritic Chondrocytes and Synoviocytes

Abstract

The biological activity of chondroitin sulfate (CS) and glucosamine (GlcN) food supplements (FS), sold in USA against osteoarthritis, might depend on the effective CS and GlcN contents and on the CS structural characteristics. In this paper three USA FS were compared to two pharmaceutical products (Ph). Analyses performed by HPAE-PAD, by HPCE and by SEC-TDA revealed that the CS and GlcN titers were up to -68.8% lower than the contents declared on the labels and that CS of mixed animal origin and variable molecular weights was present together with undesired keratan sulfate. Simulated gastric and intestinal digestions were performed in vitro to evaluate the real CS amount that may reach the gut as biopolymer. Chondrocytes and synoviocytes primary cells derived from human pathological joints were used to assess: cell viability, modulation of the NF-κB, quantification of cartilage oligomeric matrix protein (COMP-2), hyaluronate synthase enzyme (HAS-1), pentraxin (PTX-3) and the secreted IL-6 and IL-8 to assess inflammation. Of the three FS tested only one (US FS1) enhanced chondrocytes viability, while all of them supported synoviocytes growth. Although US FS1 proved to be less effective than Ph as it reduced NF-kB, it could not down-regulate COMP-2; HAS-1 was up-regulated but with a lower efficacy. Inflammatory cytokines were markedly reduced by Ph while a slight decrease was only found for US-FS1.

Keywords: anion exchange chromatography (HPAE-PAD); capillary electrophoresis (HPCE); food supplements (FS); human primary cells of pathological joints; inflammation biomarkers; pharmaceutical grade CS; size exclusion chromatography (SEC-TDA).

Figure 1

(a) Picture of the pharmaceutical products and US food supplement insoluble contents; (b) Determined insoluble dry weight table.

Figure 2

(a) CS, GlcN and KS contents in pharma grade samples and in US food supplements, obtained by HPAE-PAD analyses after chemical hydrolysis, reported in percentages compared to the declared values in the labels. (b) HPAE-PAD chromatograms of the US food supplements; the monosaccharide peaks are indicated by the arrows.

Figure 3

MTT assay to evaluate cell viability after 48 h of treatment. Metabolic activity performed on chondrocyte (a), and synoviocyte (b), treated with US FS in comparison to PhA and PhB pharma grade chondroitin sulfate based products. The values presented are the averages ± S.D. For chondrocytes (a) significant differences are indicated as # p < 0.05 vs. pCTR, and * p < 0.01 respect to PhA and PhB. For synoviocytes significant differences are # p < 0.01 vs. pCTR and * p < 0.01 vs. PhA, PhB and USFS1.

Figure 4

Gene expression analyses normalized respect to pathological untreated cells (pCTR) for the analyses of COMP-2 (a) and HAS-1 (b) on chondrocytes and synoviocytes, treated with US FDA approved FS in comparison to PhA and PhB samples at 6 h of incubation. The values presented are the averages ± S.D. Significant differences are indicated as * p < 0.01 vs. pCTR.

Figure 5

Western blotting analyses relative to COMP-2, NF-kB and PTX-3 on chondrocyte (a) and synoviocytes (b) treated with USA FS in comparison to PhA and PhB samples at 48 h of incubation. The expression of each protein was normalized respect to actin housekeeping protein.

Figure 6

ELISA assay for IL-6 and IL-8 on chondrocytes (a) and synoviocytes (b) treated with US FS in comparison to PhA and phB pharma grade samples at 48 h of incubation. The values presented are the averages ± S.D. Significant differences are indicated as * p < 0.01 vs. pCTR.