The Marine ‐ derived , Multi ‐ mineral formula , AquaPT Reduces TNF ‐ a Levels in Osteoarthritis Patients

Carola T Murphy2, Conal Martin2, Andrea M Doolan2,4, Michael G Molloy3, Timothy G Dinan2, Denise M O Gorman5 and Ken Nally1,2* 1School of Biochemistry and Cell Biology, University College Cork (UCC), Cork, Ireland 2Alimentary Pharmabiotic Centre, UCC, Cork, Ireland 3Department of Rheumatology, Cork University Hospital, Cork, Ireland 4Atlantia Food CRO, UCC, Cork, Ireland 5Marigot Limited, Strand Farm, Currabinny, Carrigaline, Co. Cork, Ireland Journal of Nutritional Health & Food Science Open Access Short Communication


Background
Osteoarthritis (OA) results from the breakdown of cartilage in joints over time, leading to joint pain and damage.It is the leading cause of chronic disability in the United States and affects about eight million people in the United Kingdom.Symptoms and effects include joint stiffness, swelling and pain, loss or restriction of joint mobility and joint deformity.Treatment for OA generally involves a combination of exercise, lifestyle modification and analgesics.Current anti-inflammatory treatments, while providing some relief from symptoms, are suboptimal and the side effects of these treatments, in particular the COX-2 specific

Treatment
For 6 consecutive weeks, the subjects with OA received either Aquamin (Food and Drug Administration (FDA) Generally recognized as safe (GRAS) 000028) (Marigot Ltd, Cork, Ireland) or Aquamin supplemented with 2% Enzogenol (Enzo Nutraceuticals, New Zealand), a pine bark extract, and 8% Sunphenon (Taiyo Kagaku Co. Japan), a green tea extract (AquaPT).The daily dose of 4 capsules of Aquamin provided 800mg calcium, (the European Union (EU) recommended dietary allowance (RDA) for calcium) and 74 mg of Magnesium (EU RDA 375mg).The daily dose of 4 capsules of AquaPT provided 720mg calcium, 200mg green tea (polyphenols) and 50mg pine bark extract.Study participation involved 3 study visits over a period of 8 weeks.

Study measurements and statistical analyses
At the first visit, subjects were screened for symptoms and effects of OA, such as joint swelling, joint tenderness and decreased range of motion in joints, visible joint damage (i.e bony growths) and crepitus.Once deemed eligible, upon review of a number of inclusion and exclusion criteria, subjects returned for a second visit (WK 0) and received a six-week supply of Aquamin or AquaPT along with food diaries.They were required to come off all NSAIDs, steroids, probiotics and vitamin supplements for 2 weeks prior to study entry and for the 6 week duration of the study and returned for their final visit at week 6 (WK 6).
At each visit, WOMAC scores for pain, stiffness and difficulty in performing daily activities were assessed.In addition, the subjects were queried about changes in their medications and any adverse events were recorded.One subject from the OA + Aquamin group was removed from the study due to taking medications prohibited in the study criteria.These effects were not treatment-related.
On WK 0 and WK 6, blood from subjects was collected in EDTA vacutainers, centrifuged @ 1200 g for 10 min and the serum was aspirated and stored at -80°C.Serum TNF-α levels were measured using a Meso Scale Discovery (MSD) assay kit and the electrochemiluminescent multiplex system Sector 2400 imager (Meso Scale Discovery, Gaithersburg, MD, USA), as per the manufacturer's instructions.All statistical tests were performed using commercially available statistic software (GraphPad Software, San Diego, CA, USA).Data are represented by mean ± standard error of the mean (SEM).P values of < 0.05 were considered significant.

Results and Discussion
The data presented here demonstrate that treatment with the green tea and pine bark extract-supplemented formulation of Aquamin (AquaPT) significantly reduced basal serum levels of the pro-inflammatory cytokine TNF-α in patients with moderate to severe OA (P < 0.05) (Figure 1).Serum TNF-α levels in those that received Aquamin on its own were decreased by 23.6%, but this reduction, whilst trending towards significance was not significant (Figure 1).Both in vitro and in vivo data from OA studies support the role of TNF-α in disrupting the normal biochemical metabolism of synovial joint tissues [9].In addition, evidence from a canine model of OA suggests that both TNF-α and its receptors play a role in early development of the disease [10].Two studies have demonstrated the anti-inflammatory effects of Aquamin in vitro [5,6].However, the present study is the first to demonstrate such an effect in human subjects with OA.Of note, the levels of TNF-α detectable in the sera of OA patients were overall very low.This phenomenon has been previously reported in a study comparing rheumatoid arthritis and OA, where cytokines were only detectable in the synovial fluid of OA patients [11].Despite trending towards significance, no significant differences in WOMAC scores were evident post treatment, seven of the eleven OA subjects that received Aquamin and seven of the twelve that received AquaPT had improved WOMAC composites scores for pain, stiffness and activity post treatment (Figure 2).More specifically, out of the eleven subjects that received Aquamin; eight felt less pain, six felt less stiff and six felt an improvement in performing daily activities (Figure 2).Similarly, out of the twelve subjects that received AquaPT; nine felt less pain, six felt less stiff and eight felt an improvement in performing daily activities (Figure 2).These results are in agreement with previous reports that Aquamin reduces the symptoms of OA in humans [4] and reduces TNF-α levels in vitro [5].Additionally, this is the first time that both of these parameters have been investigated in a single study.The potential limitations of this study include the relatively small sample size and short duration of treatment (6 weeks), however similar effects in terms of WOMAC scores have been observed in previous studies with Aquamin conducted over a 12-week period.However, it is important to note the difficulty in recruiting OA sufferers willing to come off all analgesics for a pro-longed period of time.The 'active ingredient' for Aquamin is not known since a number of the minerals present in the complex may be having anti-inflammatory effects.The prominent molecule calcium (daily dosage providing 98-100% RDA) ameliorated pain symptoms in OA patients [12].Furthermore with regards AquaPT, Suphenon prevented disease in a rat model of colorectal cancer [7], while Enzogenol reduced a number of risk factors associated with cardiovascular disease [8].

Conclusion
This study provides initial evidence for the potential antiinflammatory effects of AquaPT in vivo and confirms previous findings showing trends of improved pain, stiffness and activity scores with increased supply of minerals from Aquamin in patients with moderate to severe OA.These data suggest that supplementation of the seaweed-derived formula Aquamin with green tea and pine bark extract enhances the nutraceutical's beneficial and anti-inflammatory effect in OA.Finding alternative treatments for OA are merited due to the undesirable effects associated with current therapies for the disease.Our findings warrant further mechanistic studies to identify the specific immunomodulatory effects of AquaPT and further appropriately controlled clinical studies with larger subject groups conducted over longer time periods to support the interesting observations made in this study.

Figure 1 :
Figure 1: Effect of six week treatments with Aquamin or AquaPT on serum TNF-alpha levels in patients with OA.Data are expressed as mean ± the standard error of the mean for 11-12 patients per group.The significance of differences between week 6 and week 0 was determined by two way anova with repeated measures and post hoc analysis (*p<0.05).

Figure 2 :
Figure 2: Effect of six week treatments with Aquamin or AquaPT on WOMAC scores in patients with OA.Reduction in WOMAC scores trended towards significance in patients with OA following six weeks treatment with AquaCal and AquaPT .Data are expressed as mean ± the standard error of the mean for 11-12 patients per group.