Pharmacological effects of a C-phycocyanin-based multicomponent nutraceutical in an in-vitro canine chondrocyte model of osteoarthritis

Abstract

Multicomponent nutraceuticals are becoming increasingly popular treatments or adjunctive therapies for osteoarthritis in veterinary medicine despite lack of evidence of efficacy for many products. The objective of this study was to evaluate the anti-inflammatory and antioxidant activities of a commercially available C-phycocyanin-based nutraceutical and select constituent ingredients in an in-vitro model of canine osteoarthritis. Normal canine articular chondrocytes were used in an in-vitro model of osteoarthritis. Inflammatory conditions were induced using interleukin-1β. The nutraceutical preparation as a whole, its individual constituents, as well as carprofen were evaluated at concentrations of 0 to 250 μg/mL for reduction of the following inflammatory mediators and indicators of catabolism of the extracellular matrix: prostaglandin E₂ (PGE₂), tumor necrosis factor-α (TFN-α), interleukin-6 (IL-6), metalloproteinase-3 (MMP-3), nitric oxide, and sulfated glycosaminoglycans (sGAGs). Validated, commercially available assay kits were used for quantitation of inflammatory mediators. The antioxidant capacities, as well as cyclooxygenase-1 (COX-1), cyclooxygenase-2 (COX-2), and lipoxygenase (LOX) inhibitory activities of the whole nutraceutical preparation and select constituents, were also assessed using validated commercially available assay kits. The antioxidant capacity of the nutraceutical and constituents was concentration-dependent. The nutraceutical and constituents appear to display anti-inflammatory activity primarily through the inhibition of COX-2. The nutraceutical displayed similar strength to carprofen in reducing TNF-α, IL-6, MMP-3, nitric oxide, and sGAGs at select concentration ranges. The C-phycocyanin (CPC)-based nutraceutical and constituents may be able to mediate 3 primary pathogenic mechanisms of osteoarthritis: inflammation, chondral degeneration, and oxidative stress in vitro. The nutraceutical may be clinically useful in veterinary medicine and its efficacy should be further investigated in vivo.

Introduction

Osteoarthritis (OA) poses significant therapeutic problems in humans, dogs, horses, and other companion animals. Non-steroidal anti-inflammatory drugs (NSAIDs) remain the most common treatment across species for attenuation of clinical signs of OA (1–3). Long-term use of NSAIDs is associated with adverse effects, including gastrointestinal toxicity, renal toxicity, negative effects on chondrocytes and cartilage-matrix formation, and possible delay in bone healing (1,4–12). Due to the possible adverse effects of NSAIDs, there is an interest in both human and veterinary medicine in identifying natural products, nutraceuticals, and supplements that may provide safer alternatives to NSAIDs for managing OA while maintaining efficacy.

Currently, joint health products, such as glucosamine hydrochloride and chondroitin sulfate, represent the largest category of nutraceutical and natural supplements for veterinary medicine (13,14). In humans, the use of nutraceuticals and natural health supplements is increasing (15–18) and pet owners who purchase and use natural products themselves are also likely to purchase them for their animals as well (19) as owners see it as part of responsible pet ownership (13). Presently, multicomponent formulations are a trend in nutraceutical and natural product supplements, with the idea that several compounds may interact with multiple targets to evoke interdependent pharmacological activities to achieve optimal effects (20).

One of such a group of multicomponent joint health veterinary nutraceuticals on the market is a canine soft chew formulation, which is part of the Phycox product line produced by Dechra Pharmaceuticals (Northwich, England). The products are marketed as potent cyclooxygenase-2 (COX-2) inhibitors that support joint mobility and healthy bone structure (21). One ingredient of note in the product line is C-phycocyanin (CPC), a biliprotein containing a biliverdin-like chromophore, which is a constituent of the Phycox active ingredient, a proprietary blue-green algae extract (ingredients listed in Table I). C-phycocyanin (CPC) is reported to be a selective COX-2 inhibitor (22) and potent antioxidant (23). The nutraceutical also contains glucosamine hydrochloride, methyl-sulfonylmethane (MSM), citrus bioflavonoids, grape seed extract, and turmeric, all of which have been reported to possess anti-inflammatory and/or antioxidant properties in vitro and, in some cases, in vivo (24–31).

Table I.

Phycox soft chew formula for canines (21) (Phycox active ingredient is a proprietary blue-green algae extract that contains c-phycocyanin)

Active ingredients per soft chew
Glucosamine hydrochloride*†‡°	450 mg
Methylsulfonylmethane*†‡°	400 mg
Creatine monohydrate*‡°	250 mg
Alpha-linolenic acid	200 mg
Proprietary blend of citrus bioflavonoids*†‡°, calcium phosphate, manganese sulfate, ascorbic acid (vitamin C)*°, zinc sulfate, alpha lipoic acid*°, and grape seed extract*†‡°	132 mg
Turmeric*†‡°	50 mg
Phycox active*†‡°	30 mg
Eicosapentaenoic acid (EPA)	9 mg
Docosahexaenoic acid (DHA)	6 mg
Boron	100 μg
Selenium	10 μg
Alpha tocopheryl acetate (vitamin E)	25 IU
Inactive ingredients
Flaxseed oil, hydrolyzed vegetable protein, magnesium stearate, marine lipid concentrates, natural liver flavor, and sucrose

^*Tested in the in-vitro canine chondrocyte osteoarthritis model.

^†Tested as a COX inhibitor.

^‡Tested as a LOX inhibitor.

^°Tested for antioxidant capacity.

It is important to evaluate the efficacy of nutraceuticals and supplements used in veterinary medicine as they are not regulated in North America like traditional pharmaceuticals and therefore are not required to undergo rigorous safety and efficacy studies. The efficacy of the nutraceutical to mediate joint inflammation has not been evaluated in the literature.

The objectives of the present study are to measure the anti-inflammatory effects of the nutraceutical and select constituents compared to carprofen in an in-vitro canine chondrocyte (CnC) model of OA. This is done by quantitating inflammatory mediators and the catabolism of the extracellular matrix (cytokines, prostaglandins, and other molecular markers) and assessing the antioxidant capacity, COX inhibition, and lipoxygenase (LOX) inhibition of the nutraceutical and select constituents.

Materials and methods

Chemicals and reagents

Phycox canine chewable whole tablets and individual components, which consist of glucosamine hydrochloride, MSM, creatine monohydrate, alpha-lipoic acid, ascorbic acid, grape seed extract, turmeric, citrus bioflavonoids, and Phycox active ingredient (a proprietary blue-green algae extract) were provided by Dechra Pharmaceuticals (Northwich, England). C-phycocyanin (CPC) was generously provided by Cerule (Klamath Falls, Oregon, USA). Carprofen was purchased from Sigma-Aldrich (St. Louis, Missouri, USA).

Cell culture

Canine chondrocytes (CnCs) isolated from normal canine articular cartilage (Cell Applications, San Diego, California, USA) were maintained in Dulbecco’s Modified Eagle’s Medium/Ham’s Nutrient Mixture F-12, without phenol red supplementation, with 20% heat-inactivated fetal bovine serum (FBS) and penicillin-streptomycin (10 mg/L), and incubated at 37°C in a 5% carbon dioxide (CO₂) atmosphere. The cell sub-culture and cell number procedures were followed as described previously (32).

In-vitro osteoarthritis model

An OA model for CnCs was followed that was previously used to study the anti-inflammatory effects of canine dietary supplements and other natural products relevant to human and animal health (32,33). Briefly, CnCs were counted and seeded on 24-well plates. The seeded cells were incubated at 37°C in a 5% CO₂ atmosphere for 72 h. On the day of the experiment, stock solutions of the previously mentioned compounds and preparations of interest were made [10% dimethyl sulfoxide (DMSO) and 90% cell culture medium]. Stock solutions were diluted to yield final concentrations of 0.1, 1.0, 5.0, 10, 50, 100, and 250 μg/mL. All solutions were sterile-filtered through 0.2-μm filters. At the start of the experiment, the cells were serum-starved for 2 h. After aspiration of the serum-free media from the wells, cells were treated with 500 μL of the pro-inflammatory cytokine, interleukin-1β (IL-1β) (10 ng/mL in serum-free medium) to induce inflammatory conditions and incubated at 37°C in 5% CO₂ atmosphere for 2 h. Cells were treated with the compounds and preparations of interest (0.1 to 250 μg/mL). Additional cells were treated with either DMSO diluted in medium to reflect total DMSO concentrations in treatment groups (0.1%) or only medium. The final concentration of FBS in the wells was 20%. Treated and control cells were incubated at 37°C in 5% CO₂ atmosphere for 72 h, after which the cell plates were removed from the incubator. Media were then collected and stored at −80°C until further analysis for the following 6 key mediators and indicators of extracellular matrix catabolism released during inflammatory events: prostaglandin E₂ (PGE₂), tumor necrosis factor-α (TNFα), interleukin-6 (IL-6), metalloproteinase-3 (MMP-3), nitric oxide, and sulfated glycosaminoglycans (sGAGs).

Determination of inflammatory biomarkers

Validated, commercially available assay kits were used to quantify concentrations of biomarkers of interest in the collected in-vitro OA model media. Prostaglandin E₂ (PGE₂), TNF-α, and IL-6 concentrations were measured by enzyme immunoassays from Cayman Chemical (Ann Arbor, Michigan, USA), catalog numbers 514010, 589201, and 583361, respectively. Metalloproteinase-3 (MMP-3) was determined using an enzyme-linked immunosorbent assay (ELISA) from RayBiotech (Norcross, Georgia, USA), catalog number ELH-MMP-3-001. The nitric oxide assay kit used was from Active Motif (Carlsbad, California, USA), catalog number 40020. Concentrations of sGAG were quantified using Alcian Blue dye as supplied in the assay kit from Kamiya Biomedical (Seattle, Washington, USA), catalog number BP-004. All assays were carried out in quadruplet. More information about each assay is available in the instructions provided with each kit.

Cell viability

Cytotoxicity of the compounds and preparation of interest at the experimental concentrations used in the in-vitro OA model were assessed using the resazurin fluorescent dye method following previously described methodology (32,33) and was carried out in quadruplet. Cell viability was indirectly measured as conversion of resazurin to resorufin. Briefly, the non-fluorescent resazurin dye compound is metabolized into the fluorescent compound, resorufin, by intact and viable cells. The emission of fluorescence is quantified using a plate reader, which allows the number of viable cells to be assessed based on fluorescent intensity. The median lethal dose values, or doses to cause 50% cell death (LC₅₀), for each compound of interest were determined by using pharmacodynamic modeling with Phoenix WinNonlin 6.3 software (Certara, St. Louis, Missouri, USA).

Cyclooxygenase inhibitory activity

The inhibition of COX-1 and -2 by the compounds and preparations of interest were measured with a validated commercial assay kit using an ELISA from Cayman Chemical (catalog number 560131). The assay was carried out in quadruplet. More information about the assay protocol is provided in the instructions for the kit. To determine COX selectivity of the compounds and preparations, COX ratios were determined using the half maximal inhibitory concentration values (IC₅₀) for COX-1 and -2 for each compound and preparation by pharmacodynamic modeling with Phoenix WinNonlin 6.3 software. The COX-2 IC₅₀ value for a compound was divided by the COX-1 IC₅₀ value to yield the COX ratio.

Lipoxygenase inhibitory activity

The inhibition of LOX by the compounds and preparations of interest was measured using a commercial validated colorimetric assay kit from Cayman Chemical (catalog number 7607000). The assay was carried out in triplicate. More information about the assay protocol is provided in the instructions for the kit.

Determination of antioxidant capacity

The antioxidant capacities of the nutraceutical and select constituents were measured through the commonly used ABTS [2,2′-azino-di-(3-ethylbenzthiazoline sulfonate)] method (34) using a commercially available and validated assay from Cayman Chemical (catalog number 709091). The assay was carried out in quadruplet. More information about the assay protocol is provided in the instructions for the kit.

Statistical analysis

All data are expressed as the mean ± standard error of the mean (SEM) or mean ± standard deviation (SD). When possible, the data were analyzed for statistical significance using GraphPad Prism 6 statistical software (GraphPad Software, La Jolla, California, USA). Student’s t-test was used for unpaired samples. Comparisons between the control and treatment groups were made using repeated-measures analysis of variance (ANOVA) with Fisher’s least significant difference test.

Significance was set at P < 0.05 for all statistical testing.

Results

Cell viability

Calculated across the tested concentration range (0.1 to 250 μg/mL), not enough cell death occurred to calculate LC₅₀ values for all compounds administered in the in-vitro OA model. Thus, cell death did not exceed 50% even at 250 μg/mL and the LC₅₀ concentrations for all compounds and preparations were greater than 250 μg/mL (data not shown). This indicates that inflammatory mediator results were not affected due to cell toxicity from high concentrations of the compounds of interest.

Prostaglandin E₂

Incubation of CnCs with IL-1β increased the concentration of PGE₂ (56%) compared to the untreated control cells (Figure 1A and Supplementary Table I). The treatment of CnCs with the nutraceutical (0.1 and 1 μg/mL) resulted in a significant decrease in PGE₂ concentrations from the positive control (48% to 84%) similar to baseline. Treatment with the nutraceutical at higher concentrations, however, resulted in significantly increased PGE₂ concentrations from the positive control (14% to 32%). Carprofen-treated cells at all concentrations yielded significantly decreased PGE₂ from the positive control (96% to 99%), as did cells treated with many of the nutraceutical constituents. The nutraceutical and constituents across all concentration ranges did not result in PGE₂ levels significantly lower than those produced by carprofen at specific concentration ranges.

Figure 1.

Select results from Supplementary Table 1 (please contact author for table) for cells treated with carprofen, nutraceutical, proprietary blue-green algae extract, and C-phycocyanin (CPC) (n = 4, mean ± SEM) for production of: A — prostaglandin E₂ (PGE₂), B — tumor necrosis factor-α (TNF-α), C — interleukin-6 (IL-6), D — metalloproteinase-3 (MMP-3), E — nitrite, F — nitrate, and G — sulfated glycosaminoglycans (sGAGs).

* Significantly lower concentrations of inflammatory biomarker than positive control (IL-1β) cells (P < 0.05).

^† Significantly lower concentrations of inflammatory biomarker than carprofen-treated cells at the same concentration (P < 0.05).

Tumor necrosis factor-α

Untreated control CnCs yielded a concentration of 0.362 ± 0.140 pg/mL of TNF-α (Figure 1B and Supplementary Table I). When CnCs were treated with IL-1β, the TNF-α concentration rose by 64% to 0.954 ± 0.147 pg/mL. All concentrations of carprofen, the nutraceutical, and its constituents failed to significantly reduce TNF-α concentrations from those of the positive control. Treatment with all concentration of the nutraceutical and constituents did not result in a statistical difference in TNF-α concentrations from CnCs treated with carprofen at specific concentration ranges, with the exception of α-lipoic acid at 100 μg/mL and creatine at 0.1 and 50 to 250 μg/mL, which resulted in statistically greater concentrations of TNF-α than carprofen at the same concentrations.

Interleukin-6

Untreated control CnCs yielded a concentration of 1.48 ± 0.206 pg/mL of IL-6 (Figure 1C and Supplementary Table 1). When CnCs were treated with IL-1β, the IL-6 concentration rose 88% to 2.78 ± 0.229 pg/mL. All concentrations of carprofen, the nutraceutical, and most of the constituents failed to significantly reduce IL-6 concentrations from those of the positive control cells treated with IL-1β. Treatment with carprofen at 100 and 250 μg/mL resulted in significantly greater concentrations of IL-6 than the positive control. Canine chondrocytes (CnCs) treated with CPC, citrus bioflavonoids, turmeric, grape seed extract, and ascorbic acid at 10 μg/mL resulted in significant decreases of IL-6 compared to IL-6 in CnCs dosed at 10 μg/mL of carprofen. The remainder of nutraceutical and constituent concentrations resulted in no significant difference in IL-6 concentrations compared to those produced by carprofen at the same concentrations.

Metalloproteinase-3

Untreated control CnCs yielded a concentration of 0.717 ± 0.008 ng/mL of MMP-3 (Figure 1D and Supplementary Table I). When CnCs were treated with IL-1β, the MMP-3 concentration rose by 3.5% to 0.742 ± 0.006 pg/mL. Treatment with carprofen at all concentrations showed no significant change in MMP-3 concentrations compared to the positive control IL-1β cells. Treatment with the nutraceutical also did not result in significant changes in MMP-3 concentrations compared to the positive control, with the exception of concentrations of 1.0 and 250 μg/mL, which resulted in significant increases of MMP-3. The blue-green algae extract at 10, 50, and 250 μg/mL resulted in a significant decrease in MMP-3 from the positive control. However, cells treated with 1.0 μg/mL of the blue-green algae extract resulted in a significant increase in MMP-3 from the positive control. All concentrations of CPC and citrus bioflavonoids resulted in a significant decrease in MMP-3 from positive control cells treated with IL-1β, as did cells dosed with turmeric at concentrations from 1.0 to 100 μg/mL. Treatment with all experimental concentrations of CPC and citrus bioflavonoids resulted in a statistically significant decrease in MMP-3 concentrations compared to treatment with specific concentrations of carprofen. At 1.0 and 250 μg/mL, however, the nutraceutical resulted in a significant increase in MMP-3 compared to cells treated with the same concentrations of carprofen, as did cells treated with blue-green algae extract at 1.0 μg/mL.

Nitric oxide

Nitrite

The CnC control produced an average nitrite concentration of 8.41 ± 0.000 μM (Figure 1E and Supplementary Table 1). When treated with IL-1β, nitrite concentrations increased 5% to 8.82 ± 0.096. Canine chondrocytes (CnCs) treated with all concentrations of carprofen, the nutraceutical, and most constituents did not result in a significant change in nitrite compared to the positive control. The nutraceutical and most of the constituents tested at all concentrations did not significantly differ in nitrite compared to CnCs treated with the same concentrations of carprofen.

Nitrate

The control produced a nitrate concentration of 5.49 ± 2.91 μM (Figure 1F and Supplementary Table I). When treated with IL-1β, the nitrate concentrations increased 22% to 6.71 ± 3.48. Neither carprofen nor the nutraceutical at all concentrations produced a significant change in nitrate from the positive control. The majority of nutraceutical constituents also did not significantly alter the nitrate concentrations from the positive control. There was no statistical difference between nitrite concentrations in samples treated with the nutraceutical or most constituents and carprofen.

Sulfated glycosaminoglycans

The untreated control samples contained an average sGAG concentration of 7.77 ± 1.69 μg/mL (Figure 1G and Supplementary Table I). When cells were treated with IL-1β, sGAG concentration increased by 25% to 9.68 ± 1.12 μg/mL. Carprofen significantly decreased sGAG concentrations in CnCs when treated with 1.0 to 50 and 250 μg/mL concentrations as did the nutraceutical at concentrations of 1.0 and 5.0 to 250 μg/mL. Many nutraceutical constituents at various concentrations also significantly decreased sGAG concentrations in CnCs compared to the positive control. There was no statistical difference between sGAG concentrations in the samples treated with carprofen and the nutraceutical at all concentrations.

Cyclooxygenase inhibition

The COX ratios (Figure 2) for carprofen, nutraceutical, and select constituents were calculated by dividing the determined IC₅₀ of COX-2 inhibition by the IC₅₀ of COX-1 for each substance. A ratio of 1 indicates the compound is a non-preferential COX inhibitor. A ratio of less than 1 indicates preferential COX-2 inhibition and a ratio of greater than 1 indicates selectivity for COX-1 inhibition. The majority of nutraceutical constituents displayed selectivity for COX-2 inhibition. C-phycocyanin (CPC), citrus bioflavonoids, turmeric, and glucosamine hydrochloride all had COX ratios of less than 1. The blue-green algae extract and MSM both had COX ratios of 1, which indicates that they are non-preferential COX inhibitors. Grape seed extract had a COX ratio slightly over 1, which indicates a mild preference for the inhibition of COX-1.

Figure 2.

Cyclooxygenase (COX) ratios for carprofen, nutraceutical, and select nutraceutical constituents [proprietary blue-green algae extract, C-phycocyanin (CPC), citrus bioflavonoids, turmeric, grape seed extract, glucosamine hydrochloride, and methylsulfonylmethane (MSM)] (n = 4, mean ± SEM). A ratio of 1:1 indicated equal inhibition of both COX iso-forms, a ratio < 1 indicates preferential COX-2 inhibition, and a ratio > 1 indicates preferential COX-1 inhibition.

* Significantly different COX ratio than that of carprofen (P < 0.05).

Lipoxygenase inhibition

Initial LOX activity was determined to be 9.56 ± 4.01 nM/min/mL (Figure 3). All concentrations of carprofen, nutraceutical, and constituents failed to significantly affect LOX activity compared to initial activity. Carprofen, the nutraceutical, and most constituents were not found to be statistically different from each other at all concentrations.

Figure 3.

Lipoxygenase (LOX) inhibitory activity by carprofen, nutraceutical, and select nutraceutical constituents [proprietary blue-green algae extract, C-phycocyanin (CPC), citrus bioflavonoids, turmeric, grape seed extract, glucosamine hydrochloride, methylsulfonylmethane (MSM), and creatine)] (n = 3, mean ± SEM).

* Significantly different than initial activity (P < 0.05).

^† Significantly different than carprofen at the same concentration (P < 0.05).

Antioxidant capacity

The antioxidant capacity of the nutraceutical and select constituents is expressed as mM Trolox equivalents (Figure 4). While the nutraceutical exhibited a positive dose-response relationship, only at 100 μg/mL does the nutraceutical display antioxidant capacity significantly different from baseline. The blue-green algae extract, CPC, α-lipoic acid, MSM, and creatine failed to display antioxidant capacity that was significantly different from baseline at all concentrations. The citrus bioflavonoids at 1.0 and 50 to 100 μg/mL, turmeric at 5.0 to 100 μg/mL, grape seed extract at 5.0 to 100 μg/mL, ascorbic acid at 50 to 100 μg/mL, and glucosamine hydrochloride at 10 to 50 μg/mL displayed antioxidant capacity significantly different from baseline. Ascorbic acid and grape seed extract displayed the highest observable activity of all the constituents examined.

Figure 4.

Antioxidant activity by the nutraceutical and select constituents [proprietary blue-green algae extract, C-phycocyanin (CPC), citrus bioflavonoids, turmeric, grape seed extract, α-lipoic acid, ascorbic acid, glucosamine hydrochloride, methylsulfonylmethane (MSM), and creatine] (n = 4, mean ± SEM).

* Significantly different than baseline activity (P < 0.05).

Discussion

The nutraceutical was not found to be statistically different from carprofen in its ability to inhibit TNF-α, IL-6, MMP-3, nitrate, nitrite, and sGAG production in IL-1β stimulated CnCs across the concentration ranges tested. However, the nutraceutical and carprofen failed to significantly decrease concentrations of the following inflammatory mediators at lower concentrations (0.1 to 10 μg/mL): TNF-α, IL-6, MMP-3, nitrate, and nitrite. Many of the nutraceutical constituents also demonstrated the ability to reduce inflammatory mediators after an inflammatory insult. These observations largely agree with the reports of anti-inflammatory activities in the literature (33,35–38). The data also demonstrated that the nutraceutical inhibits COX from providing its anti-inflammatory effects and may provide some antioxidant activity.

The pharmacological endpoints selected to evaluate the efficacy of the nutraceutical and select constituents against inflammation induced by IL-1β were chosen as all are elevated in chondrocytes by IL-1β and present at increased concentrations in synovial fluid of osteoarthritic joints (37,39). The in-vitro activity of the nutraceutical and select constituents suggests that they may have some activity in the pathogenesis of OA through inflammation (TNF-α, IL-6, PGE₂, and COX-2), chondral degeneration (MMP-3 and sGAG), and oxidative stress (nitric oxide and antioxidant capacity). Carprofen was chosen for comparison of the pharmacological activities of the nutraceutical as it is a commonly prescribed veterinary NSAID used clinically for inflammation related to OA and postoperative pain. The concentration range examined (0.1 to 250 μg/mL) was chosen to provide a wide concentration-response range of both biologically relevant and super-physiological concentrations. However, it is unlikely that concentrations over 10 μg/mL of the constituents would be observed in vivo in the synovium, which is a target site in OA (33).

The relationship between inflammatory cytokines, oxidative stress, and chondral degeneration in the pathogenesis of OA is dynamic and interrelated, which may explain the concentration-response curves of many of the compounds of interest in this study. The nutraceutical is a multicomponent nutraceutical and differences in pharmacological activities were observed between the selected active constituents and the whole preparation. With the exception of PGE₂ production and 1.0 μg/mL and 250 μg/mL concentrations for reduction of MMP-3, the nutraceutical was not observed to have significantly different pharmacological activities from carprofen in this study, although lack of statistical difference does not imply sameness. The nutraceutical was able to significantly reduce (P < 0.05) the concentrations of PGE₂ and sGAG from the positive control at biologically relevant concentrations.

Results for COX and LOX revealed that the nutraceutical is a poor LOX inhibitor and strong COX inhibitor with preferential COX-2 inhibition, which suggests that the nutraceutical may provide anti-inflammatory activity in vivo. Many of the nutraceutical constituents also displayed selectivity for COX-2 inhibition and proved to be poor LOX inhibitors. The nutraceutical appeared to display concentration-dependent antioxidant activity. Grape seed extract and ascorbic acid were observed to be the constituents with the greatest antioxidant potency.

Based on the results of the present study, there is support for continued study of the nutraceutical and constituents as anti- inflammatory agents. At certain concentration ranges, the nutraceutical and constituents inhibit IL-1β induced PGE₂, TNF-α, IL-6, nitric oxide, MMP-3, and sGAG production, which could lead to reduced proteoglycan catabolism after an inflammatory insult. The nutraceutical and constituents may be able to mediate 3 major pathogenic mechanisms of OA: inflammation (PGE₂, TNF-α, IL-6, and COX-2), chondral degeneration (MMP-3 and sGAG), and oxidative stress (nitric oxide and antioxidant capacity) (40). The nutraceutical may be able to treat the clinical signs of OA in dogs and other species. If used as an adjunct therapy to NSAIDs, prudent clinical assessments should be undertaken due to the COX potency that the nutraceutical displayed in-vitro. In light of these findings, an in-vivo efficacy study of the nutraceutical in osteoarthritic dogs is warranted. A multi-center in-vivo efficacy study has now been undertaken by our laboratories based on these initial in-vitro findings of anti-inflammatory and antioxidant activities.