*6.1. Cardiovascular Studies*

A series of experiments have been conducted to assess the efficacy of DDA in protecting the myocardium using the myocardial ischemia-reperfusion model in rats, rabbits and dogs [35,36,53,54]. Prior treatment for four-days with intravenous DDA using doses of 25, 50 and 75 mg/kg body weight in Sprague-Dawley rats significantly reduced myocardial infarct size [35]. The degree of cardiac protection correlated with the dose of DDA administered. In a study in rabbits using a myocardial ischaemia-reperfusion model prior intravenous treatment with 50 mg/kg/day of DDA for four days resulted in a significant decrease in the size of the myocardial infarction and an improvement in myocardial salvage [52]. Animals treated with DDA had an attenuation of inflammation and complement activation suggesting there was a reduction in tissue inflammation [52]. In another study using a dog model intravenous DDA was administered daily for four-days prior to occlusion of the left anterior descending coronary artery or two hours prior to coronary artery occlusion [54]. After an hour of coronary occlusion and three hours of reperfusion there was a significant reduction in myocardial infarct size in the dogs treated with DDA. In the four-day treatment group, two out of three dogs had complete cardiac protection [54]. In a rat study, the effects of seven days of pre-treatment with oral DDA, 125 and 500 mg/kg/day on the concentrations of free astaxanthin in myocardial tissue [36]. The astaxanthin concentration in the myocardium was 400 nM after oral DDA at a dose of 125 mg/kg/day for seven-days and it was 1634 nM after 500 mg/kg/day. There was also a reduction of multiple lipid peroxidation products. The doses of DDA used in these experiments were quite high and at this stage it is not known whether such doses would be safe to use in humans. 

The effects of astaxanthin on blood pressure (BP) were assessed in spontaneously hypertensive rats (SHR). There was a significant reduction in BP after 14-days of oral astaxanthin administration whereas this did not occur in normotensive Wistar Kyoto rats [71]. Astaxanthin administered orally for five-weeks in stroke prone SHR also resulted in a significant BP reduction [71]. Oral astaxanthin also enhanced nitric oxide induced vascular relaxation in the rat aortas [71] In experiments in SHR, oral astaxanthin significantly decreased nitric oxide end products indicating that it may be exerting its BP effects via this pathway [72]. Studies using the SHR aorta and coronary arteries demonstrated that astaxanthin reduced the wall/lumen ratio in coronary arteries and decreased elastin bands in the aorta [72]. This suggests that astaxanthin may beneficially mediate atherosclerotic CVD processes. 

Recently, a series of two experiments were reported in the one article, one using the synthetic astaxanthin (CDX-085) and the other using free astaxanthin [55]. CDX-085 administered orally to C57BL/6 mice resulted in the presence of free astaxanthin in the plasma, heart, liver and platelets. Mice that were fed astaxanthin had significantly increased basal arterial blood flow and a delay in occlusive thrombosis after endothelial injury. Also, in an *in vitro* study, human umbilical vein endothelial cells and platelets isolated from Wistar-Kyoto rats that were treated with free astaxanthin has significantly increased nitric oxide release and a decrease in peroxynitrite levels [55]. The authors concluded the results support the potential  of astaxanthin as a potential therapy to prevent thrombosis associated with cardiovascular disease. 

Astaxanthin administered to C57BL/6 mice resulted in a reduction in exercise-induced increases in the oxidative stress biomarkers 8-hydroxy-2ȝ-deoxyguanosine and 4-hydroxy-2- nonenal-modified protein in both cardiac and gastrocnemius muscle [63]. Increases in myeloperoxidase and creatinine kinase activity in cardiac and gastrocnemius muscle were also reduced by astaxanthin. After three-weeks of astaxanthin supplementation there was evidence of accumulation of astaxanthin in gastrocnemius and cardiac muscle. Astaxanthin given to female BALB/c mice for eight-weeks resulted in a dose dependent increase in plasma astaxanthin but no significant changes in blood glutathione or change in lymphocyte mitochondrial membrane potential and cardiac contractility index measured on echocardiography. The mice that were fed 0.08% astaxanthin in the diet had higher cardiac mitochondrial membrane potential and contractility index compared with control animals [74]. This suggests dietary astaxanthin provides cardiac protection. Astaxanthin administered for four weeks to eight week old ICR mice resulted in increased exercised induced fat utilization and prevention of increased hexanoyl-lysine modification of carnitine palmitoyltransferase I (CTP I) [73]. In a canine carotid artery thrombosis model, administration of DDA resulted in a dose-dependent reduction in carotid artery re-thrombosis and a reduction of re-thrombosis after thrombolysis but there was no effect on hemostasis [34]. 

## *6.2. Diabetes Studies*

Diabetes mellitus and its associated nephropathy is a common cause of chronic kidney disease and is complicated by accelerated atherosclerotic cardiovascular disease [75]. In studies involving diabetic db/db mice, supplementation with astaxanthin produced a reduction in the levels of blood glucose [60]. In the kidney there was significantly decreased relative mesangial area in the animals supplemented with astaxanthin. Also proteinuria and urinary excretion of 8-OHdG were attenuated. Mice supplemented with astaxanthin had less glomerular 8-OHdG immunoreactive cells [60]. Hyperglycemia induced reactive oxygen species production, activation of transcription factors, and cytokine expression and production by normal human mesangial cells was suppressed significantly by astaxanthin [66]. 

## **7. Astaxanthin Studies in Humans**

Although no cardiovascular outcomes studies using astaxanthin have been reported in humans there have been clinical studies that have investigated the effects of astaxanthin in human health and other diseases (Table 2). The majority of these have been conducted in healthy participants who volunteered to assess astaxanthin dose, bioavailability, safety and oxidative stress, which are all potentially relevant to the cardiovascular system. Studies have also been conducted in other medical conditions such as reflux oesophagitis, where measurements of oxidative stress and/or inflammation have been included. 

*Mar. Drugs* **2011**, *9*, 447–465 
