P OMEGRANATES IN C ARDIOVASCULAR D ISEASE Atherosclerosis

Several in vitro and in vivo studies using extensive animal models and human clinical trials have examined the effects of several pomegranate constituents on prevention and protection against atherosclerosis. One study investigated the effects of pomegranate juice and other polyphenolic rich fruit juices on endothelial function. In particular, the study focussed on comparing propensities to protect endothelial cells against NO and free-radical mediated destruction. Results of the antioxidant portion of the study showed that pomegranate juice contains a significantly greater antioxidant capacity at relatively lower concentrations (>1000-fold dilutions) compared to either grape or blueberry juice. This is related to the higher amounts of anthocyanin flavonoid content and total flavonoid content in pomegranate juice than the other juices.

As mentioned earlier, impaired endothelial function is an early indicator of atherosclerosis. One important study examined the effect of pomegranate juice on the proliferation of rat aortic smooth muscle cells in culture. This study showed that pomegranate juice significantly enhances the effect of NO on the cardiac endothelium at up to 2,000-fold dilutions compared to other juices.

Interestingly, pomegranate juice was unable to affect the expression of endothelial nitric oxide synthase (eNOS). The authors suggested that the antioxidant properties of pomegranate juice are likely to protect NO from oxidative insult and augment the antiproliferative action of NO on rat aortic smooth muscle cells [35-38].

The early-stage atherosclerosis has been reported to involve elevated plasma Cholesterol, increased oxidative stress and increased cholesterol esterification that contributes to the formation of foam cells, and the development and progression of the atherosclerotic plaque. Moreover, pomegranate extracts have been shown to inhibit atherogenesis in

atherosclerotic apolipoprotein-E deficient (E°) mice. Consumption of pomegranate juice to E° mice with advanced atherosclerosis for two months showed a 42% reduction in lipid peroxide content in mouse peritoneal macrophages (MPM) compared to placebo-treated mice. Similarly, the MPM lipid peroxide content in PJ-treated mice was 20% lower than in four-month-old wild-type control mice. The same study further demonstrated that MPM harvested from PJ-treated mice contained 80% lower rates of cholesterol esterification compared to placebo-treated mice. As well, the size of atherosclerotic lesions in the aorta was reduced by 17% compared to the age-matched placebo group [39-40].

Additionally, pomegranate juice and an isolated tannin fraction extracted from pomegranate juice were also given to young E° mice prior to the development of significant atherosclerosis. The results showed about 25%

reductions in plasma lipid peroxide concentrations with the isolated tannins, and 17% with the pomegranate juice. This suggests that the cardioprotective effect of pomegranates may be due to specific tannins present in the fruit [41-43].

Another study of four-month-old E° mice with significant atherosclerosis, pomegranate extract containing 51.5μg gallic acid equiv/kg/day). The study reported an eight-fold higher polyphenol concentration than pomegranate juice alone for three months. The results also showed a significant reduction in MPM oxidative status as measured by a 27% decrease in total macrophage peroxide levels, a 42% decrease in cellular lipid peroxide levels, and a 19%

decrease in peritoneal macrophage uptake of oxidized LDL [31-34].

The key question is which part of the fruit contains the most potent antiatherogenic compounds. To answer this question, one study fed atherosclerotic E° mice with six different pomegranate preparations with varying amounts of total polyphenols and gallic acid content for three months.

Antioxidant activity, atherosclerotic lesion size, MPM oxidative status, blood sugar and lipid profiles were examined. Consistent with earlier results, the study demonstrated that pomegranate flower extract most significantly reduced atherosclerotic lesion size, lipid profiles, and blood sugar levels than other extracts tested. On the contrary, two pomegranate pulp extracts demonstrated the most potent antioxidant effects. Mechanisms associated with the anti-atherogenic effects of pomegranate in this study include increased MPM uptake of oxidized LDL, decreased lipid peroxidation and decreased cholesterol levels [44-47].

The effect of pomegranate juice consumption on lipid peroxidation and the levels of plasma and HDL- and LDL-lipoproteins have also been

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previously investigated in a double-armed human trial. In the first study, 13 healthy, non-smoking men (ages 20-35) were given 50 mL pomegranate juice daily (containing 1.5 mmol total polyphenols) for two weeks. In the second study (duration ≤10 weeks), three healthy men (same age range) were given increasing doses of pomegranate juice ranging from 20-80 mL daily (0.54-2.16 mmol total polyphenols). Fasting blood samples were collected from participants pre-study, and after one and two weeks of PJ supplementation. On the contrary to previous animal studies, the human trial found no significant effect on plasma lipid profile or lipoprotein patterns. However, the results are the first to show that pomegranate juice has an inhibitory effect on lipid peroxidation in plasma and in lipoproteins in humans. An effective dose of 50 mL daily yielded a 32% decrease in plasma lipid peroxidation.

Supplementation with pomegranate juice also demonstrated up to 90%

reduction in collagen-induced platelet aggregation in human platelets ex vivo in a dose dependent manner [48-50].

Hyperlipidemia

Hyperlipidemia is a major risk factor for ischemic heart disease and impaired coronary function. There is a strong correlation between enhanced platelet activity, high LDL cholesterol levels and low platelet reactivity with low cholesterol levels. Therefore, strategies aimed at lowering blood cholesterol levels can reduce cardiovascular events.

Pomegranate flowers have been traditionally used in both the Unani and Ayurvedic systems of medicine as a natural treatment for diabetes mellitus.

Considering the historical use of pomegranates, one study examined the effects of pomegranate flower extract on cardiac lipid metabolism in 13-to 15-week old Zucker diabetic rats. These rodents were fed 500 mg/kg of pomegranate flower extract or placebo for six weeks. The parameters that were assessed were total cholesterol, triglyceride, and nonesterified free fatty acids (NEFA) prior to treatment, after 4 weeks treatment, in both rat plasma and cardiac tissue. The pomegranate flower extract was shown to activate peroxisome proliferator-activated receptor (PPAR), a well-established cardiac transcription factor associated with myocardial energy production via fatty acid uptake and oxidation. Activation of PPAR reduced cardiac uptake and circulation of lipids. The total cardiac tissue triglyceride content, and plasma total cholesterol was also reduced at the end of the study [52-58].

Another pilot study comprised of 22 type 2 diabetic patients (8 men and 14 women) investigated the cholesterol-lowering effects of 40 g concentrated pomegranate juice for eight weeks. The study found statistically significant decreases were in total cholesterol (from 202.4 mg/dL at baseline to 191.4 mg/dL at study conclusion), LDL cholesterol (124.4 mg/dL at baseline to 112.9 mg/dL at study conclusion), total/HDL cholesterol ratio (5.5 at baseline to 5.1 at study conclusion), and LDL/HDL ratio (3.4 at baseline to 3.0 at study conclusion). The authors attributed these effects to decreased absorption and increased faecal excretion of cholesterol, as well as possible effects on HMG-CoA reductase and sterol O-acyltransferase, two enzymes key to cholesterol metabolism [59-60].

Hypertension

At least 970 million people worldwide suffer from elevated blood pressure or hypertension. Hypertension represents the major cause of premature death worldwide and the problem is increasing significantly. It is a major risk factor for coronary heart disease and the single most important risk factor for stroke.

It has been estimated that over 1.56 billion adults will be diagnosed with hypertension in 2025.

Increased blood pressure is defined as a systolic blood pressure at above 140 mmHg and/or diastolic blood pressure at or above 90 mmHg. Systolic blood pressure is defined as the maximum pressure in arteries when the heart contracts. Diastolic blood pressure is defined as the minimum pressure in the arteries between the heart’s contractions. Hypertension can stress the body’s vasculature, leading to vessel weakness. Hypertension can lead to atherosclerosis and narrowing of blood vasculature making them more prone to blockage due to the formation of thrombi or fatty streaks. Extensive damage to arteries can also lead to the formation of irregular vascular projections known as aneurisms [61]

While increased blood pressure is thought to occur as part of the ageing process, several strategies have been identified aimed at reducing the risk.

These include a healthy, relatively low salt diet, physical activity and reduced calorie intake. One pilot study showed that pomegranate juice can reduce systolic blood pressure in hypertensive patients. Ten hypertensive subjects (ages 62-77; seven men and three women) were given 50 mL/ day PJ containing 1.5 mmol total polyphenols for two weeks. Two of seven patients

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were also diabetic and two were hyperlipidaemia. Seven of 10 subjects (70%) experienced a significant, five % decrease in systolic blood pressure [62-67].