Curcumin nanomicelle versus curcumin improves lipid profile, stress oxidative factors and inflammatory markers in patients undergoing coronary elective angioplasty; A Randomized Clinical Trial

Background: Curcumin exhibited antioxidant and anti-inflammatory effects. The aim of this study, assess and compare curcumin and nano- curcumin effects on lipid profile, oxidative stress index and inflammatory factors of heart patients. Methods: This Randomized, Double-Blind, Placebo-Controlled Clinical Trial conducted on 90 patients undergoing coronary elective angioplasty. Patients were randomly divided into 3 groups. The first group received a 500 mg capsule of curcumin daily. The second group received an 80 mg capsule of nano- curcumin daily. The placebo group also received capsules similar to curcumin for 8 weeks. Lipid profile, stress oxidative factors and inflammatory markers measured in baseline and end of the investigation. Results: At the end of study, statistically significant changes was seen in the total cholesterol (TC), triglyceride (TG) and low density lipoprotein (LDL) in the intervention groups to the control group (p<0.05). These changes in the nano-curcumin group were greater than the curcumin group. Curcumin and nano-curcumin supplementation also caused a statistically significant improvement in plasma levels of total antioxidant capacity (TAC), malondialdehyde (MDA), Superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), high-sensitivity C-reactive protein (hs-CRP), Interleukin 1 beta (IL-1β) and tumor necrosis factor alpha (TNF-α) in comparison to the placebo (p<0.05). Conclusion: Complementary therapy of cardiovascular patients with curcumin and nano-curcumin supplements, could improve lipid profile, stress oxidative index and, inflammatory factors. The effects of curcumin on nano formula may be better for cardiac patients due to high bioavailability. However, further investigation is


Introduction
One of the mainly factors resulted in death is the cardiovascular disease (CVD) in the world wide [1]. According to the World Health Organization (WHO) report prevalence of CVD increased, that projected combined death toll of 24 million by 2030 [2]. Epidemiologic studies have reported the several risk factors for this category of diseases. In the pathophysiology of atherosclerosis, impaired plasma levels of lipoproteins including high cholesterol, high LDL cholesterol, high serum TG, low HDL cholesterol, and lipid metabolism disorders are more than other factors affecting atherosclerosis [3]. Also, increased inflammation and oxidative stress are considered to be major risk factors for atherosclerosis and cardiovascular disease.
Among the main causes of cardiovascular disease, inflammation has a pivotal role in all steps atherothrombosis. Inflammatory cytokines, including IL-1, TNF-α, and CRP induce the expression of cellular adhesion molecules, which mediate adhesion of leukocytes to the vascular endothelium. CRP can also induce monocytes to express tissue factor, a glycoprotein that plays an important role in coagulation [4,5].
Furthermore, Reactive Oxygen Species (ROS) at physiological levels are now known as a signaling molecules to regulate a wide range of actions in the cardiovascular system and to contribute to the maintenance of cardiovascular homeostasis [6].
Many scientific investigations have to be done on the role, mechanism and sources of ROS in heart cells. Much studies are required to determine ROS inhibitors for clinical applications [7]. Decreasing in blood circulation to the heart and straitened arteries are the main features of the disease [8]. With the advancement of surgical techniques in last years, some techniques such as percutaneous coronary intervention (PCI) and coronary artery bypass grafting (CABG) have been introduced for boosting in coronary heart disease (CHD) patients conditions. Nevertheless, on the reports of the several studies, reperfusion injury and microvascular blocking may occur after PCI. Also in subjects who experience new remedy such as stenting, resulted in no-reflow development. Endothelial layer integrity by eliminating risk factors associated with it by lipid lowering agents, free radical scavengers, and antioxidants can be effective in restoring endothelial function and reducing disease progression [9,10].
Curcumin is a polyphenols compound separated from turmeric. It is found that curcumin has many pharmacological effects including antioxidant, antiinflammation, eliminating free radicals, anti-tumor, lipid regulation and anticoagulation [11]. Clinical trials showed no severe toxic or side effect. It is also reported that after application of curcumin in coronary heart disease, the lipid level is effectively controlled and the incidence of cardiovascular event is reduced [12].
Various methods are used to increase the availability of curcumin in tissue or plasma. The results show that the use of nano-carriers with curcumin increases the effectiveness of curcumin in preventing diseases and increases 22-fold access to curcumin, and this action and the use of curcumin in the nano formulation can be an important and reliable [13]. Curcumin nano-micelle which is a registered curcumin product (SinaCurcumin®) was prepared for oral use. Considering the fact that there is no study about nano-type turmeric effect in patients undergoing coronary angioplasty, we decided to study and compare curcumin and nano-curcumin effects on lipid profile, inflammatory factors and oxidative stress index of heart patients.

Design
All patients met the inclusion criteria were randomly divided into 3 groups: curcumin, nano-curcumin, and placebo groups. The first group received a 500 mg capsule of curcumin daily. The second group received an 80 mg capsule of nanocurcumin daily. The placebo group also received capsules similar to curcumin. The study lasted 60 days. Randomization number allocated for qualified subject that was formed by a computer software. Next a table of randomization was created by the method of random permuted blocks. Each qualified patient received a randomization number, which was determined by a computer generated schedule. Then a randomization table was generated by the method of random permuted blocks. The investigator and patients were blinded to the treatment condition. Nanomicelle formulation of curcumin is a novel formulation of curcumin in world that produced by Exir Nano Sina company. Patient's demographic data, including age, gender, medical history and medications intake were collected. Anthropometric indices including: height and weight were measured then body mass index (BMI) was calculated in kilograms per meters squared. In order to assess the diet of patients in terms of energy intake, macronutrients, types of fatty acids, some vitamins and

Laboratory Methods
At baseline and at the end of the investigation, after 12 hours of fasting, 10 cc of venous blood samples were collected and after serum isolation, they were kept at -80 ° C until assay. Blood samples without EDTA centrifuged (Beckman Avanti J-25, USA) at a rate of 3000 rpm for 10 minutes in order to separation of serum.
Total cholesterol levels were determined by the enzymatic spectrophotometric method using an auto-analyzer (Abbott, model Alcyon 300, USA) with Pars-Azmoon Kit (Tehran, Iran). Triglyceride and HDL were determined by the enzyme colorimetric method using an automatic analyzer (Abbott, Model Alcyon 300, and USA) with Pars-Azmoon Kit. LDL-C was calculated by Friedwald formula; LDL-C (mg/dl) = TC-(HDL-C + TG/5).
Serum MDA levels were measured by tiobarbituric acid method. Colorimetric method was used for analyzing serum TAC (Randox Laboratories Ltd, UK). This method has completely been explained by Khosrowbeygi et al [14]. Also glutathione peroxidase enzyme was measured by spectrophotometric and superoxide dismutase enzyme was performed with Randox lab, UK.
Turbidimetric immunoassay was used for measuring of hs-CRP levels (BioSystems Co, Barcelona, Spain). Also enzyme-linked immunosorbent assay (ELISA) (DIAsource Co, Belgium) was used for determining serum levels of TNF-α. The amount of IL-1β was measured by using the Bendermed quantitative detection kit made in Austria, with precision of picograms per milliliter by ELISA method according to the manufacturer's instructions in the laboratory.

Sample size calculation
To determine the sample size, we used the MDA factor before and after the intervention, which was used in Yunes Panahi et al. study [15]. Thus, if the mean and standard deviation of the MDA before and after the supplementation was 19.84 ± 3.60 and 16 ± 2.88, for each group of 25 people was calculated. Considering the drop in participants during the study, 30 people were considered for each group.
Statistical Analysis SPSS software v.20 was utilized as a data analyst. Due to the normal distribution of variables, which were checked by Kolmogorov-Smirnov test. Also paired t-test is used to compare before and after supplementation in each group and one way ANOVA was applied to compare groups. The percentage change of each variable was also calculated by the formula [(E-B)/ B* 100], where E is the end of treatment values and B is the baseline values. All data presented as mean ± SD and statistically signification was defined as P < 0.05.

Results
One hundred patients were qualified to participate in this study at the beginning.
Ninety two patients completed the intervention. One patient from the curcumin group withdrew from the study because of personal reasons. One patient from the placebo group dropped out due to personal reasons. Their data were excluded from the final statistical analysis ( Fig. 1)

Demographic and baseline characteristics
The patient's demographic and baseline characteristics are presented in Table 1.
According to Table 1 there are no significant differences in terms of age, weight, body mass index (BMI), ejection fraction and Hemoglobin. Coronary risk factors for coronary artery disease were evenly distributed between the groups. The stented target vessels were similarly among the groups. Table 1 Comparison of characteristics between groups in the baseline. The mean of energy and macronutrient intake at baseline of the study were summarized in Table 2. As shown there were no statistically significant difference between the groups in terms of average daily intake the energy, protein, fat, saturated fatty acids, unsaturated fatty acids and some micronutrients (P > 0.05).

Lipid profile
There were no statistically significant differences in lipid profile between groups at baseline of the study (P > 0.05, Table 3). There were no significant changes in the placebo group in the lipid profile, but total cholesterol, triglyceride and LDL in the intervention groups after the completion of the study significantly decreased.
According to Table 3, these changes in the nano-curcumin group were greater than the curcumin group. Also the HDL levels was increased in intervention groups, but these changes were not significant. Table 3 Comparison of changes in levels of lipid profiles before and after intervention in study groups

Antioxidant Indices
The mean of antioxidant indices are shown in groups before and after study in Table   4. In the intervention groups, the mean of TAC, SOD and GSH-Px were increased (p ≤ 0.05) that were statistically significant, but there was no significant change in placebo group. Plasma MDA was also affected by curcumin and nano-curcumin supplementation. This factor significantly decreased in the intervention groups (P < 0.05). As shown in Table 4, these changes in the nano-curcumin group were greater than the curcumin group.

Inflammatory factors
The effect of supplementations on inflammatory biomarkers in cardiovascular patients have been summarized in Table 5. After 8 week, inflammatory biomarkers levels were affected by curcumin and nano-curcumin. This factors significantly decreased in the intervention groups (P < 0.05). As shown in Table 5, these changes in the nano-curcumin group were greater than the curcumin group.

Discussion
CVD are the main critical healthiness complication of the worldwide and their outcomes have been reported as an out of every three deaths in the United States [16]. While remarkable scientific attempts has been straighten to identifying bimolecular functions regulating the beginning and developing of disease, much remains controversial [17]. One of the important and useful remedy option for subjects with acute coronary syndrome is the PCI that can be main way for improving in patients with coronary artery anomalies. However some complications such as myocardial injury due to PCI were reported that causing to impecunious prognosis [18]. According to the current investigations antioxidant and antiinflammatory properties of polyphenols have been detected [19]. Turmeric has a main polyphenol ingredient called curcumin that is obtainable as an over-thecounter (OTC) supplement widespread of world [20]. This randomized, controlled profiles [24]. In order to justify the results of this study, in addition to the low sample size, supplementation of curcumin has no effect in healthy subjects with normal lipid profile. for about 50% of daily cholesterol excretion [28].
The another possible mechanism of curcumin in improving dyslipidemia improves cholesterol catabolism by enhancing the activity of the cholesterol 7 hepatic hydroxylase enzyme, which inhibits the synthesis of cholesterol by inhibiting the HMGCOA reductase enzyme [29]. Enhancing of the expression of hepatic CYP7A1 gene by curcumin consumption, resulted in reducing the blood cholesterol levels and approved the hypocholesterolemic properties of curcumin [30]. Expression of AMP-activated protein kinase and peroxisome proliferator-activated receptor boosted by curcumin that affected energy metabolism of subcutaneous adipocyte tissue in mice that treated with curcumin-supplemented high fat diet [31].
Interestingly, although curcumin shows antioxidant properties by supplying hydrogen radicals and scavenging free radicals, there are a growing number of evidences that it can act as pro-oxidant under certain condition by generating ROS [32]. Results of our study indicated that curcumin supplementation improved status of oxidative stress. In this study intake of curcumin and nano-curcumin increased the mean of TAC, SOD and GSH-Px, also plasma MDA significantly decreased in the intervention groups. Results of Banaeifar et al. study demonstrated that Eight weeks consumption of curcumin caused a significant increase on the level of SOD and GSH-Px enzymes in the rats [33]. Also the results of Quiles et al. showed that curcumin reduces oxidative stress and lipid peroxidation and attenuates aortic fatty streak development in rabbits that treated with atherogenicity diets [34]. According to the Alizadeh F and et al. scientific report, 10 weeks of curcumin nanomicelle supplementation resulted in a statistically significant improvement in plasma levels of TAC, MDA, CRP and TNF-α in comparison to the placebo in asthenoteratospermia patients [35].
Immoderate ROS generation has a main role in the beginning, developing and clinical outcomes in CVD. Although many studies have been done and considerable development and advances occurred in field of free radical biology and cardiovascular treatment, but main mechanisms of CVD and outcomes of pathophysiologically increased ROS in cardiovascular tissue not fully understood [7]. Anti-oxidant properties of curcumin have been greatly researched. This polyphenol can inhibit ROS degradation and also reduce inflammatory factors through down regulation of pro-inflammatory cytokines and transcription factors [29]. Scientific research has also paid great attention to the immunomodulatory role of curcumin.
In this regard, activity of immune system cells such as T-cells, B-cells, macrophages, neutrophils, natural killer (NK) cells, and dendritic cells are regulated with curcumin. Also, curcumin, at low doses, may boost antibody responses. These molecular affirmations propose that its detailed beneficial effects could be because of direct anti-oxidative and anti-inflammatory properties, additionally in part to its capacity to control the immune systems [26,20].
Curcumin inhibits the production of active oxygen species. It linked to thyroxin reductase and turns it to nicotine amide dinucleotide phosphate oxidase (NADPH oxidase) which prevents from ROS formation. Also it increases glutathione gene expression and through iron binding it can do its antioxidant role [36]. Results of this study indicated that inflammatory biomarkers levels were significantly decreased in the intervention groups. Curcumin has suppressive effect on production of the inflammatory cytokine TNF-α, which in turn leads to inhibition of

Availability of data and material
The data analyzed during the current study are available from the corresponding author on reasonable request.

Ethics approval and consent to participate
We confirm that any aspect of the work covered in this manuscript that has involved

Consent for publication
Not applicable.  The authors would like to thank the all of the participants who completed the study protocol.

Authors' contributions
BH, HG, SKH, and HKH: designed the project; MK and HKH: developed the study; BH, HH, and SKH: developed the cardiovascular measures; MK and HKH: contributed to the study design and developed the statistical approach; HG: contributed to the study design; BH, MK, and HH: conducted the trial and collected study data; HKH: prepared the manuscript; and all authors: reviewed manuscript drafts and read and approved the final version.

Funding
This work was financially supported by supported by a Grant (Number: NRC-9620) from Vice-Chancellor for Research Affairs of Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran.

Availability of data and material
The data analyzed during the current study are available from the corresponding author on reasonable request.

Ethics approval and consent to participate
We confirm that any aspect of the work covered in this manuscript that has involved either