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The main risk factor for diabetic patients death is cardiovascular disease. Although CVD mortality rates have decreased since past decades in many countries, it still accounts for more than 40% of total mortality. Cardiovascular disease widely purposed to be associated with elevated oxidative stress (Ø±ÙØ±Ø§Ù†Ø³). Lipid peroxidation is the most commonly assessed process in oxidative stress research. There are various plasma markers of lipid peroxidation including malondialdehyde, lipid hydroperoxides, conjugated dienes, oxidation resistance assay (lag time), oxysterols and F2α-isoprostanes. Historically, the most common method for measuring lipid peroxidation has been the thiobarbituric acid reactive substances (TBARS) assay
to quantify malondialdehyde . Although increased risk of CAD has been contributed to hyperglycemia, dyslipidemia and prothrombic state, however recent studies have focused on inflammatory marker.
Markers of chronic low-grade inflammation, such as tumor necrosis factor-α (TNF-α), Interleukin-6 (IL-6), and high sensitive- C-reactive protein (hs-CRP), have been shown to predict the risk of developing type 2 diabetes and cardiovascular disease; also they are thought to be directly involved in the pathogenesis of such chronic diseases. TNF-α and IL-6 are cytokines which secreted from adipose tissue predominantly while hs-CRP is the principal downstream mediator of the acute phase response and is secreted by the liver in response to TNF-α and IL-6 .
Nutrition plays a key role in the prevention of many chronic diseases including CVD, cancers, diabetes and inflammatory diseases . Cacao is known to be rich in polyphenols, such as catechin, epicatechin, procyanidin B2 (dimer), procyanidin C1 (trimer), cinnamtannin A2 (tetramer), and other oligometric procyanidins . Studies on healthy human subjects have shown decreased levels of serum LDL-cholesterol but increased levels of HDL-cholesterol; furthermore resistance of LDL- cholesterol to oxidation following the intake of dairy cocoa powder have been reported previously [8, 9].
Some studies on proinflammatory molecules such as hs-CRP, IL-6 and TNF-α have been suggested anti-inflammatory properties for polyphenol-rich diets. In vitro studies have suggested that cocoa procyanidins and phenolic metabolites can also modify intracellular signal transduction pathways and thereby modulate the synthesis of inflammatory cytokine such as IL-6. The aim of our study is to evaluate the effect of 6 week regimen of Cacao consumption on the lipid peroxidation and profile, inflammatory markers such as TNF-α, IL-6 and hs-CRP in a randomized, double-blind clinical trial.
Material and method
Forty four subjects with type 2 diabetes participated in the study. The eligibility criteria were as follows: age between 40-70 y; total cholesterol above 240 mg/dl and triglyceride above 200 mg/dl; not to use anti-hyperlipidemia drugs; no regular consumption of supplements affecting lipid metabolism; no extreme exercise habits.
The subjects were instructed to consume cocoa powders daily for 6 weeks. The test powders was consumed as a beverage after the addition of hot water, and consumed twice each day, before noon and during the afternoon for 6 weeks. To ensure that the same foods consumed by all the subjects, we arranged home deliveries to each subject 1 week before consumption of the test powders and during the 6-weeks study period. We also gave clear guidance on the need for the subjects to maintain their normal diets for breakfast, lunch, drinks, and incidental foods. In addition, to check that the normal diets maintained, the subjects kept complete dietary records throughout the study. The subjects advised to avoid any supplements and all other cocoa products and to lead their usual life style throughout the study. Blood samples were obtained from all the volunteers before cocoa consumption (baseline) and 6 weeks after intervention. The samples were coded with random numbers and processed immediately to perform lipid profile. Blood cholesterol, triglycerides and HDL cholesterol were measured by enzymatic colorimetric kit methods and according to the package insert instructions. Low density lipoprotein cholesterol (LDL-C) calculated using Friedwald formula Ø±ÙØ±Ø§Ù†Ø³.
Malondialdehyde, end product of lipid peroxidation, levels were measured by thiobarbituric acid reactive substances (TBARS) MethodØ±ÙØ±Ø§Ù†Ø³. Inflammatory markers including hs-CRP levels, IL6 and TNF-α were measured by a sandwich ELISA methodØ§Ø³Ù… Ø´Ø±Ú©ØªÙ‡Ø§.
The data were analyzed by the SPSS package Version 13. All data reported are expressed as mean ± S.D. Statistical analysis was performed using the Student's t test. The Pearson correlation coefficient also was used for correlation estimation between malondialdehyde and inflammatory markers. P-values < 0.05 were considered significant.
The ratio of male to female patients was nearly the same. The patient's mean±SD of age is 54±5y and the mean±SD for body mass index (BMI) was 28±5kg/m2. Table 1 shows the comparison of serum lipids, lipoproteins and malondialdehyde before and after cocoa powder consumption. Cocoa powder consumption caused significant reduction (P-value=0.001) in serum cholesterol (15%) triglyceride (13%), LDL-cholesterol (11.5%), maloaldehyde (28%) and significant elevation of HDL-cholesterol (12%) (Table 1).
After 6 weeks, of cocoa powder consumption, there was a significant decrease (P-value=0.05) for serum levels of hs-CRP (−21.5%), and also non-significant decrease in serum IL-6 (−4.6%), and TNF-α (−2.6%) respectively (table 2). Also the correlation analysis reveals a positive correlation between malondialdehyde and inflammatory markers (hs-CRP , IL-6, TNF-α) which is statistically significant
The protective effect of dietary flavonoids against many diseases in particular cardiovascular disease and diabetes are supported by several studies [8, 9, 12-15].
Cocoa is a rich sources of polyphenols. The flavan-3-ol monomers, catechin,
epicatechin, and the oligomeric procyanidins are the major flavonoids in cocoa.
Recently, attention has been directed to the antioxidant potential of these flavonoids in cocoa and their potentially protective effects against the risk of cardiovascular disease. In the current study our results showed positive effects on serum lipid concentrations in type 2 diabetic patients after 6 weeks of cocoa consumption.
This study was performed in order to determine if 6 weeks consumption of cocoa produced a beneficial effect on blood lipid peroxidation. Our results have shown that consumption of cocoa increases significantly serum levels of HDL-cholesterol (12%) and decreases significantly total cholesterol (15%), triglyceride (13%) and LDL cholesterol (11.5%) in type 2 diabetic patients after 6 weeks.
Previous studies have reported that polyphenol-rich cocoa and chocolate reduce LDL-cholesterol in humans[9, 12]. In an earlier study, we were found that consumption of 26 g/d of cocoa powder for 12 wk reduces plasma LDL cholesterol by 11% in healthy men. This report supports our finding of a reduction in LDL cholesterol.
In this study, consumption of cocoa powder was increased the concentration of HDL-cholesterol compared than its baseline. Previous studies also have reported that polyphenol-rich dark chocolate and cocoa powder increases plasma HDL cholesterol [8, 9, 12-15]. Wan et al. have found that after daily consumption of 22 g of cocoa powder for 4 weeks, the concentration of HDL-cholesterol was 4% higher after consumption of a control diet . These findings suggest that absorbed polyphenolic substances in cocoa powder, such as catechins, may affect plasma HDL-cholesterol concentration. Moreover consumption of cocoa was decreased significantly the concentrations of malondialdehyde (28%), a marker of lipid peroxidation, in type 2 diabetic patients.
Previous studies have shown that consumption of cocoa decreases the formation of lipid oxidation products such as malondialdehyde, a thiobarbituric acid reactive substances (TBARS) [15, 16]. Decreased rates of lipid peroxidation in diabetic patients after cocoa administration for 6 weeks suggest that such effects may be due to flavonoids contents of cocoa.
Inflammation plays a pivotal role in cardiovascular events. Studies suggest that lifestyle and drug interventions which reduce hs-CRP levels typically delay and lower cardiovascular diseases[17, 18]. However, there are limited numbers of controlled studies that define the effects of individual dietary constituents on biomarkers of inflammation.
Mathur et al have shown not any significant change on whole-blood cytokines, IL-6, TNF-α and hs-CRP after six weeks of cocoa supplementation .
Results of this study showed that serum level of hs-CRP was decreased significantly (<0.001) while levels of IL-6 and TNF-α were decreased non-significantly after six weeks Cocoa powder consumption.
The correlation between malondialdehyde and inflammatory markers (hs-CRP, IL-6,
TNF-α) showed that there is significant positive correlations between them (r=0.0.90, r=0.76, r=0.76, p<0.05).
It is clear that the Cocoa flavonoids represent an exciting new area of nutritional research with significant implications and cardiovascular protection in diabetic patients. Further experimental studies with cocoa flavonoids are needed to define the specific mechanisms of action on the inflammatory markers. Long-term studies with large sample sizes are also warranted to determine optimal doses and long-term effects of flavonoids, including cocoa.
Acknowledgements: This work is supported by research grant from Shahid Sadoughi Yazd University of Medical Sciences and Diabetes Research Center of Yazd. We thank Yazd Central Laboratory for biochemical and serological analysis. Our special thanks go to the patients who participated in the study.