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Hesperidin Supplementation Modulates Inflammatory Responses Following Myocardial Infarction a
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F. Haidari PhD , H. Heybar DMD , M. T. Jalali PhD , K. Ahmadi Engali MD PhD , B. Helli & E. Shirbeigi Msc
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Nutrition and Metabolic Diseases Research Center, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, IRAN b
School of Medicine, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, IRAN
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Hyperlipidemia Research Center, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, IRAN d
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Faculty of Public Health, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, IRAN Published online: 11 Mar 2015.
To cite this article: F. Haidari PhD, H. Heybar DMD, M. T. Jalali PhD, K. Ahmadi Engali MD PhD, B. Helli & E. Shirbeigi Msc (2015): Hesperidin Supplementation Modulates Inflammatory Responses Following Myocardial Infarction, Journal of the American College of Nutrition, DOI: 10.1080/07315724.2014.891269 To link to this article: http://dx.doi.org/10.1080/07315724.2014.891269
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Original Research
Hesperidin Supplementation Modulates Inflammatory Responses Following Myocardial Infarction F. Haidari, PhD, H. Heybar, DMD, M. T. Jalali, PhD, K. Ahmadi Engali, MD, PhD, B. Helli, E. Shirbeigi, Msc Nutrition and Metabolic Diseases Research Center (F.H., B.H., E.S.), School of Medicine (H.H.), Hyperlipidemia Research Center (M.T.J.), Faculty of Public Health (K.A.E.), Ahvaz Jundishapur University of Medical Sciences, Ahvaz, IRAN
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Key words: flavonoids, hesperidin, inflammation, lipid profile, myocardial infarction Objective: A growing number of studies have suggested a crucial role for a variety of inflammatory mediators in myocardial infarction. Recently, several flavonoids have been shown to have cardioprotective and anti-inflammatory properties. Therefore, the aim of this study was to investigate the effect of hesperidin—a common constituent of citrus fruits—on the serum levels of inflammatory markers and adipocytocines in patients with myocardial infarction. Methods: Seventy-five patients with myocardial infarction were participated in this randomized, doubleblind controlled clinical trial and were assigned to 2 intervention and control groups. Subjects consumed 600 mg/d pure hesperidin supplement and placebo in the intervention and control groups, respectively, for 4 weeks. Serum concentrations of inflammatory markers and adipocytocines were measured at baseline and at the end of the intervention. Results: Consumption of 600 mg/day hesperidin significantly decreased the serum levels of E-selectin and increased adiponectin and high-density lipoprotein cholesterol (HDL-C) concentrations in patients with myocardial infarction. The improvement in other inflammatory markers, such as interleukin (IL)-6, highsensitivity C-reactive protein (hs-CRP), leptin, and other lipid profile was also observed at the end of the intervention, compared to the baseline values, but the difference between the hesperidin and placebo groups was not statistically significant (p > 0.05). Conclusion: Hesperidin supplementation could compensate for decreased levels of adiponectin and HDL-C and increased levels of E-selectin in patients with myocardial infarction. These results support the concept that certain flavonoids in the diet can be associated with significant health benefits, including heart health.
INTRODUCTION
vascular biology, and energy hemoestasis and it has been shown that adiponectin is also associated with MI [4]. According to some studies, adiponectin levels decrease in patients with MI and a low adiponectin level is associated with an increase in the level of inflammatory markers such as IL-6 and CRP [5,6]. Leptin is an endocrine hormone secreted from white adipose tissue and is believed to be involved in MI through various mechanisms [7]. Flavonoids are nonnutrient polyphenolic compounds that occur in edible plants and possess a wide range of antioxidants and anti-inflammatory effects [8,9]. Recently, several flavonoids have been recommended as chemopreventive agents or
Myocardial infarction (MI) is a major cause of death and disability worldwide that can result in serious psychological and functional changes in patients [1]. It is considered an inflammatory disease due to the high levels of some inflammatory markers such as acute phase proteins, cytokines, and intracellular adhesion molecules that have been detected in patients with MI [2]. It is speculated that interleukin (IL)-6, high-sensitivity C-reactive protein (hs-CRP), and E-selectin play key roles in the progression of MI [2,3]. Adiponectin appears to play an important role in glucose and lipid metabolism,
Address correspondence to: Esmat Shirbeigi, Nutrition and Metabolic Diseases Research Center, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, IRAN. Email: [email protected]
Journal of the American College of Nutrition, Vol. 0, No. 0, 1–7 (2015) Ó American College of Nutrition Published by Taylor & Francis Group, LLC 1
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Hesperidin Supplementation nutritional supplements [8]. Hesperidin (30 ,5,7-trihydroxy-40 methoxy-flavanone-7-rhamnglucoside) belongs to the flavanone subclass of flavonoids and is mainly found in citrus fruits such as oranges [9]. In recent years, there has been a significant interest in the anti-oxidative, anti-inflammatory, and antihypercholesterolemic properties of the citrus flavanone hesperidin in oxidative stress–related diseases [10]. We previously demonstrated that hesperetin, the aglycone form of hesperidin, at a dose of 5 mg/kg reduced hepatic xanthine oxidoreductase activity, the key enzyme in the catabolism of purines, and consequently decreased the serum levels of uric acid and malondialdehyde and increased the total antioxidant capacity in hyperuricemic rats [11]. Supplementation of orange juice and hesperetin could also restore paraoxonase activity and partially improve the lipid profile in hyperuricemic rats [12]. In another study, Sahu et al. revealed that hesperidin suppressed the generation of reactive oxygen species and pro-inflammatory cytokines such as tumor necrosis factor (TNF)-a, lipid peroxidation, and apoptosis/necrosis in cisplatin-induced acute renal injury [13]. Because MI is a multifactorial disease and chronic inflammation plays a major role in the pathogenesis of the disease [2], the present study aimed to determine the effects of hesperidin supplementation on the serum levels of inflammatory markers (E-selectin, IL-6, hs-CRP) and adipocytocines (adiponectin and leptin) in patients with MI.
Study Design A randomized, double-blind, placebo-controlled clinical trial was conducted to investigate the effects of 600 mg pure hesperidin supplement on the serum levels of inflammatory markers and adipocytocines in patients with MI. Seventy-five patients with MI were divided into 2 groups by randomized block allocation (intervention: n D 38, control: n D 37). For the subjects in the intervention group, one capsule of 600 mg pure hesperidin was provided for consumption in the morning with breakfast every day for 4 weeks. Similarly, the control group received one placebo capsule of 600 mg starch. The hesperidin and placebo supplements were produced by Herbal Extracts Plus Company (Croydan, PA, USA) and the School of Pharmacy, Ahvaz Jundishapur University of Medical Sciences, respectively. Pharmacological treatment with aspirin, statins, beta-blockers, inhibitors of angiotensin II, nitrates, and diuretics was similar in the 2 groups. All subjects were instructed to continue their habitual diets and were taking no vitamin/mineral supplements or flavonoid-rich beverages during the study. Anthropometric indices, dietary intakes, blood pressure, and physical activity were evaluated before and after the intervention. Dietary intakes were also measured in the middle of the intervention. To assess physical activity, we used the International Physical Activity Questionnaire (IPAQ). This questionnaire evaluates participants’ activity in 5 major domains (work, transportation, housework, sport, and leisure-time physical activity). Data from the IPAQ were converted to metabolic equivalent of task-minutes/wk using existing guidelines [14].
MATERIAL AND METHODS Biochemical Analysis Subjects In this study, 75 patients with MI at Ahvaz Golestan Hospital participated. MI was defined as a combination of 2 of 3 characteristics: typical symptoms (i.e., chest discomfort), enzyme rise, and a typical echocardiograph pattern involving the development of Q waves (echocardiograph abnormalities). If the diagnosis of MI was confirmed by a cardiologist, patients were asked to participate in this study. Inclusion criteria were patients with MI, age range 40–65 years, body mass index (BMI) between 18.5 and 30, and patient satisfaction. Exclusion criteria included chronic disease (such as hepatic disorders, renal failure, and diabetes), gastrointestinal disorders, major psychiatric disorder, taking antidepressants, absolute vegetarian diet, consumption of antioxidants and vitamin supplements at least 6 months before sampling, high consumption of flavonoid-rich beverages (including green tea and coffee), smoking, allergies to any vitamin supplements, lack of patient follow-up, and lack of consent. The study was approved by and performed under the guidelines of the Research Ethics Committee of Ahvaz Jundishapur University of Medical Sciences, Iran (ETH-569). Written consent was obtained from all subjects.
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Fasting blood samples were collected for biochemical analysis. Blood samples centrifuged at 3000 g for 5 min. To prepare serum, blood samples were stored at ¡80 C until analysis. Serum concentrations of E-selectin, hs-CRP, IL-6, leptin, and adiponectin were measured using a commercially available enzyme-linked immunosorbent assay method (Orgenuim Laboratories Business Unit, Finland for IL-6, leptin, and adiponectin; Labor Diagnostika Nord, Germany for hs-CRP; Boster Biological Technology, China for E-selectin). All assays were performed according to the manufacturer’s instructions.
Statistical Analysis All statistical analyses were performed with the Statistical Package for Social Sciences (SPSS Inc., Chicago, IL) version 18 for Windows. First, normal distribution of all variables was checked with the Kolmogorov-Smirnov test. We compared the means of variables of each group with each other group using both independent sample t test and analysis of covariance in the adjusted models, which were adjusted for age, sex, energy,
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Hesperidin Supplementation physical activity, and weight. The end values of each variable were also compared with the baseline values using paired sample t tests. The percentage change of each variable was also calculated by the formula [(E ¡ B)/B £ 100], where E is the end of treatment value and B is the baseline value. The independent sample t test and analysis of covariance in the adjusted models were also used for comparing the percentage change of variables between 2 groups. The differences with p values < 0.05 were considered significant. In this study, Nutritionist IV software (First Databank, San Bruno, CA), which was modified for Iranian foods, was used to estimate patients’ dietary intakes.
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RESULTS In this study, 75 participants, 55.49 § 5.98 years old, were included the study. Patients did not report any serious adverse effects during the study related to hesperidin or placebo consumption. Sixty-eight percent (n D 26) and 72% (n D 27) of the study population in the hesperidin and placebo groups were males, respectively. Some demographic and anthropometric characteristics of the participants at baseline and postintervention are shown in Table 1. There were no significant differences at baseline and at the end of the study (anthropometric indices, blood pressure, and physical activity) between the 2 groups (p > 0.05). However, weight, BMI, waist circumference, head circumference, and
IPAQ significantly decreased within groups at the end of study when compared to the baseline values (p < 0.05). Dietary intake of subjects in different times of study was assessed by 3-day diet records (Table 2). Regarding dietary intake analysis, although energy and nutrient intakes of patients increased during the study (from time 1 to time 3) in both the hesperidin and placebo groups, there were no significant differences between the 2 groups at the beginning, middle, and end of the intervention. These results remained insignificant after adjusting for confounders. The effect of hesperidin supplementation on the serum levels of inflammatory markers (E-selectin, IL-6, hs-CRP), adipocytocines (adiponectin and leptin), and lipid profile in patients with myocardial infarction are shown in Table 3. As can be seen, no significant differences in baseline characteristics of participants were found between the 2 groups. However, hesperidin consumption, compared to placebo, significantly increased adiponectin and HDL-C levels and decreased Eselectin concentrations at the end of the study (p < 0.05). These effects also remained significant after adjusting for age, sex, energy, and physical activity. However, other inflammatory factors and lipid profile were not significantly changed in the hesperidin group compared to the control group (p > 0.05). Statistical analyses (within groups) also showed that the serum levels of leptin, E-selectin, IL-6, hs-CRP, triglycerides
Table 1. Demographic and Anthropometric Characteristics of the Study Participantsa Variables Weight (kg)
BMI (kg/m2)
WC (cm)
HC (cm)
WHR
SBP (mmHg)
DBP (mmHg)
IPAQ (metabolic equivalent of task-min/wk)
Baseline End P3 Baseline End P3 Baseline End P3 Baseline End P3 Baseline End P3 Baseline End P3 Baseline End P3 Baseline End P3
Hesperidin N D 38
Placebo N D 37
P1
P2
76.15 § 8.82 73.07 § 8.82 0.001 25.97 § 2.87 24.92 § 2.89 0.000 97.50 § 7.61 95.16 § 7.15 0.000 94.34 § 5.95 92.5 § 5.52 0.000 1.03 § 0.03 1.01 § 0.03 0.537 135.55 § 15.99 131.89 § 15.73 0.145 84.10 § 10.49 85.81 § 8.30 0.421 2392 § 708 2145 § 663 0.000
76.63 § 8.85 73.85 § 9.12 0.000 26.82 § 2.61 25.83 § 2.66 0.000 96.56 § 8.33 94.02 § 7.93 0.000 95.08 § 7.38 93.59 § 7.04 0.003 1.01 § 0.03 1.00 § 0.05 0.024 136.62 § 18.99 132.72 § 15.74 0.431 86.05 § 9.14 84.54 § 8.38 0.513 2432 § 678 2091 § 753 0.000
0.814 0.710 — 0.158 0.163 — 0.614 0.516 — 0.634 0.489 — 0.067 0.102 — 0.793 0.819 — 0.395 0.510 — 0.803 0.744 —
0.522 0.796 — 0.050 0.123 — 0.621 0.470 — 0.531 0.472 — 0.034 0.078 — 0.885 0.927 — 0.556 0.512 — 0.590 0.855 —
BMI D body mass index, WC D waist circumference, HC D hip circumference, WHR D waist-to-hip ratio, SBP D systolic blood pressure, DBP D diastolic blood pressure, IPAQ D International Physical Activity Questionnaire. a Data are expressed as mean § SD. P1 are results from independent sample t tests; P2 are results from analysis of covariance in the adjusted models (adjusted for age, sex, energy, and physical activity); P3 are results from paired sample t tests.
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Hesperidin Supplementation Table 2. Dietary Intakes of the Study Population during the Interventiona
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Variables Energy (kcal) Time 1 Time 2 Time 3 P3 Protein (g) Time 1 Time 2 Time 3 P3 Carbohydrate (g) Time 1 Time 2 Time 3 P3 Fat (g) Time 1 Time 2 Time 3 P3 SFA (g) Time 1 Time 2 Time 3 P3 MUFA (g) Time 1 Time 2 Time 3 P3 PUFA (g) Time 1 Time 2 Time 3 P3 Cholesterol (mg) Time 1 Time 2 Time 3 P3 Fiber (g) Time 1 Time 2 Time 3 P3
Hesperidin N D 38 Placebo N D 37
P1
P2
1221 § 372 1559 § 525 1906 § 485 0.000
1164 § 204 1472 § 375 1892 § 392 0.000
52.23 § 24.33 60.02 § 26.80 73.40 § 40.50 0.000
42.27 § 19.18 0.053 0.669 50.44 § 18.97 0.079 0.069 76.94 § 39.95 0.704 0.715 0.000
153.97 § 54.72 210.60 § 52.70 263.92 § 52.95 0.000
148.00 § 64.06 0.665 0.538 198.17 § 73.77 0.403 0.369 258.33 § 62.68 0.678 0.720 0.000
36.60 § 19.98 49.78 § 24.31 64.12 § 23.93 0.000
42.24 § 28.59 0.325 0.336 55.82 § 31.61 0.356 0.400 66.89 § 28.92 0.652 0.700 0.000
10.18 § 6.63 13.88 § 6.90 20.11 § 12.00 0.000
9.91 § 7.38 14.71 § 8.02 21.46 § 12.93 0.000
0.870 0.630 0.642 —
7.84 § 4.54 11.75 § 6.01 19.22 § 6.55 0.000
7.05 § 5.28 12.39 § 7.14 19.13 § 6.96 0.000
0.490 0.326 0.677 0.844 0.954 0.812
8.23 § 9.87 12.90 § 12.25 8.23 § 9.87 0.000
11.02 § 10.84 0.248 0.356 16.10 § 13.79 0.293 0.408 29.37 § 12.31 0.306 0.358 0.000
131.68 § 66.59 207.84 § 96.64 230.16 § 69.57 0.000
114.54 § 52.39 0.220 0.245 172.97 § 66.43 0.073 0.080 208.98 § 55.99 0.151 0.141 0.000
2.02 § 1.46 3.23 § 2.08 7.05 § 5.01 0.000
2.37 § 2.19 3.77 § 2.89 7.56 § 5.42 0.000
0.416 0.253 0.414 0.277 0.892 0.764
0.804 0.692 0.691 —
0.414 0.435 0.350 0.382 0.673 0.621
Time 1 D at the baseline, Time 2 D middle of the intervention, Time 3 D end of the intervention, SFA D saturated fatty acid, MUFA D mono-unsaturated fatty acid, PUFA D polyunsaturated fatty acid. a Data are expressed as mean § SD. P1 are results from independent sample t tests; P2 are results from analysis of covariance in the adjusted models (adjusted for age, sex, energy, and physical activity); P3 are results from repeated measures analysis of variance.
(TG), total cholesterol (TC), and LDL-C significantly decreased and the mean concentration of adiponectin and HDL-C significantly increased postintervention in both groups (p < 0.05). Percentage changes in inflammatory markers and lipid profile are shown in Table 4. Results showed significant differences in percentage changes of adiponectin, HDL-C, and low-
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density lipoprotein (LDL)-C between the 2 groups (p D 0.040, p D 0.001, p D 0.014, respectively). However, after adjustment for age, sex, energy, and physical activity, this significant effect did not remain for adiponectin (p D 0.081). There were no significant differences in percentage changes of leptin, E-selectin, IL-6, hs-CRP, TG, and TC between the 2 groups (p > 0.05).
DISCUSSION In this clinical trial, we have shown for the first time the effect of daily consumption of pure hesperidin (600 mg) on inflammatory markers, lipid profile, and blood pressure in patients with MI. The main findings of this study was that consumption of a hesperidin supplement, the major orange flavonoid constituent, for 4 weeks significantly decreased E-selectin and elevated adiponectin and HDL-C levels in patients with MI. An improvement in other inflammatory markers, such as IL-6, hs-CRP, leptin, and other lipids, was also observed at the end of the intervention, compared to baseline values, but the difference between the hesperidin and placebo groups was not statistically significant (p > 0.05). Although the beneficial effects of hesperidin on lipid profile have been shown in several animal models [15–18], studies regarding the anti-inflammatory effects of hesperidin are very limited and its effect in humans, especially in patients with MI, remains unclear. The results from a crossover study that was conducted in healthy subjects have shown that consumption of orange juice or hesperidin for 4 weeks did not affect plasma concentrations of CRP and IL-6 in healthy subjects [9]. Contrary to this finding, one study reported that oral consumption of hesperidin (500 mg once daily for 3 weeks) reduced E-selectin and hs-CRP in subjects with metabolic syndrome [19]. Adhesion of leukocytes to endothelial cells is the initial event in the acute inflammatory response, which is thought to play an important role in myocardial reperfusion injury. E-selectin is a cell-surface glycoprotein that belongs to the selectin family, and it appears to be the initial adhesion molecule for leukocytes during the inflammatory process [20]. Results of a previous study suggested that an increase in plasma E-selectin level may reflect enhanced endothelial cell activation in patients with MI [21]. It is transiently expressed in vascular endothelial cells in response to pro-inflammatory cytokines (such as TNF-a and IL-1) and its serum levels decreased slowly during the chronic phase of MI [3]. Therefore, it seems that the decreased levels of E-selectin in the intervention group in this study might be due to the antioxidant properties and inhibitory effects of hesperidin on generation of TNF-a [10,13]. Several studies demonstrated the decreased levels of adiponectin in patients with MI [4,5], but the effects of flavonoids
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Hesperidin Supplementation Table 3. Effects of Hesperidin Supplementation on Serum Levels of Inflammatory Markers and Lipid Profile in Patients with Myocardial Infarctiona Variables Adiponectin (mg/L)
E-selectin (ng/ml)
IL-6 (pg/ml)
hs-CRP (mg/L)
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Leptin (ng/ml)
TC (mg/dl)
TG (mg/dl)
HDL-C (mg/dl)
LDL-C (mg/dl)
Baseline End P3 Baseline End P3 Baseline End P3 Baseline End P3 Baseline End P3 Baseline End P3 Baseline End P3 Baseline End P3 Baseline End P3
Hesperidin N D 38
Placebo N D 37
P1
P2
6.71 § 1.05 8.10 § 1.24 0.000 41.32 § 5.27 33.21 § 3.85 0.000 54.76 § 7.38 16.85 § 4.31 0.000 5.99 § 1.55 1.77 § 0.84 0.000 13.95 § 4.32 6.52 § 2.91 0.000 207.6 § 58.9 186.3 § 51.3 0.003 148.8 § 38.6 139.9 § 31.7 0.096 34.7 § 7.2 42.9 § 9.8 0.000 143.0 § 61.1 115.4 § 56.0 0.000
6.82 § 1.07 7.57 § 0.93 0.000 40.21 § 7.59 36.26 § 7.34 0.041 52.45 § 8.02 17.27 § 6.00 0.000 6.37 § 1.55 1.82 § 0.85 0.000 13.53 § 3.90 7.23 § 3.49 0.000 203.5 § 49.1 193.4 § 48.5 0.041 151.7 § 41.3 140.6 § 35.2 0.014 35.3 § 8.7 37.7 § 10.8 0.037 137.8 § 52.5 127.5 § 51.6 0.000
0.646 0.043 — 0.463 0.027 — 0.200 0.728 — 0.290 0.837 — 0.662 0.342 — 0.749 0.541 — 0.759 0.930 — 0.776 0.034 — 0.698 0.333 —
0.896 0.049 — 0.632 0.036 — 0.126 0.658 — 0.328 0.925 — 0.665 0.383 — 0.733 0.517 — 0.953 0.992 — 0.931 0.031 — 0.732 0.309 —
IL-6 D interleukin-6, TC D total cholesterol, TG D triglycerides, HDL-C D high-density lipoprotein cholesterol, LDL-C D low-density lipoprotein cholesterol. a Data are expressed as mean § SD. P1 are results from independent sample t tests; P2 are results from analysis of covariance in the adjusted models (adjusted for age, sex, energy, and physical activity); P3 are results from paired sample t test.
on adiponectin levels in these patients are limited and rare. The present study also indicated a significant increase in adiponectin level in the intervention group compared with the control group after 4 weeks. Adiponectin is regarded as a protective adipokine associated with a lower risk of MI [22]. Therefore, the increased levels of adiponectin in the hesperidin group compared to the placebo group in this study could be explained in part by the anti-inflammatory, anti-oxidative, and enzyme inhibitory effects of hesperidin [12]. In the present study, the serum level of HDL-C in the hesperidin group was significantly increased compared to the control group; however, there was no significant difference in serum levels of other lipid profile between the hesperidin and placebo groups. Previous studies have shown conflicting results regarding the effect of hesperidin on lipid profile [23–26]. Our results concur with those of Yasim and colleagues, who showed that diosmin– hesperidin administration to rats fed a high-cholesterol diet significantly increased HDL-C levels but did not significantly affect other lipid parameters [16]. In another study, Kurowska et al. showed that consumption of orange juice (750 ml once a day for 4 weeks) increased HDL-C levels in hypercholesterolemic individuals [23]. Kim et al. also reported that 12 weeks’ administration of hesperetin and its metabolite, ferulic acid, in
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hypercholesterolemic hamsters increased the HDL-C/total cholesterol ratio and paraoxonase levels [27]. The correlation between paraxonase activity and protective role of HDL-C in oxidative stress and atherosclerosis pathogenesis has been suggested in
Table 4. Percentage Changes in Inflammatory Markers and Lipid Profile in Patients with MIa
Variables Adiponectin (mg/L) E-selectin (ng/ml) IL-6 (pg/ml) hs-CRP (mg/L) Leptin (ng/ml) TC (mg/dl) TG (mg/dl) HDL-C (mg/dl) LDL-C (mg/dl)
Hesperidin N D 38 23.32 ¡18.26 ¡68.66 ¡70.93 ¡50.33 ¡8.31 ¡3.54 24.92 ¡16.97
Placebo N D 37
P1
P2
§ 27.55 12.39 § 15.52 0.040 0.081 § 14.72 ¡4.73 § 34.88 0.055 0.077 § 9.17 ¡66.84 § 10.67 0.151 0.061 § 9.85 ¡72.09 § 9.96 0.182 0.250 § 27.90 ¡45.04 § 28.46 0.270 0.298 § 18.87 ¡5.19 § 1.02 0.091 0.110 § 21.25 ¡5.90 § 15.89 0.750 0.788 § 21.80 8.44 § 21.24 0.001 0.000 § 29.87 ¡8.23 § 7.81 0.014 0.017
IL-6 D interleukin-6, hs-CRP D high-sensitivity C-reactive protein, TC D total cholesterol, TG D triglycerides, HDL-C D high-density lipoprotein cholesterol, LDL-C D low-density lipoprotein cholesterol. a Data are expressed as mean § SD. P1 are results from independent sample t tests; P2 are results from analysis of covariance in the adjusted models (adjusted for age, sex, energy, weight change, and physical activity).
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Hesperidin Supplementation several studies [12]. Therefore, the significantly higher levels of HDL-C in the hesperidin group compared to the placebo control group in our study might be partly explained by the antioxidant properties of hesperidin. Contrary to these findings, some animal and clinical trials demonstrated that hesperidin or orange juice administration could decrease total cholesterol [15,24] and triglyceride levels [25]. The mechanisms by which hesperidin can affect the lipid profile are largely unknown, but previous studies have shown that in a human liver cell line (HepG2), principal flavanones from oranges and grapefruit, hesperetin and naringenin, both reduced the medium content of Apo lipoprotein B, the main protein component of LDL [15,24]. However, in the present study, comparison of groups (before and after intervention) showed that the serum levels of all inflammatory biomarkers and lipid profile significantly improved at the end of study in both groups (p < 0.05). This result might be masked by the effect of hesperidin on some inflammatory markers, adipocytocines, and lipid profile. In other words, one possible reason for the lack of a significant effect of hesperidin on leptin, IL-6, hs-CRP, TG, TC, and LDL-C in our study may be that the concurrent drug therapy (such as statins and aspirin) [28] and lifestyle modifications (such as weight loss) have powerful effects on modulating these factors after MI, similar to the way in which the same effect of hesperidin is masked. In our study, no significant changes in systolic or diastolic blood pressure were observed during the intervention. Our results are consistent with data reported by Wang and colleagues, who showed that consumption of hesperidin in rats fed a high-cholesterol diet did not cause a significant change in blood pressure [15]. On the other hand, these finding contrast with a study that demonstrated that consumption of orange juice as well as hesperidin for 4 weeks in healthy subjects resulted in a significantly lower diastolic blood pressure compared to that measured after consumption of placebo [9]. The possible mechanisms by which flavonoid-rich foods lower blood pressure may involve a chronic increase in the production of NO by vascular endothelium [29]. Other mechanisms, such as an inhibitory effect on angiotensin-converting enzyme, could also be responsible for the blood pressure–lowering effects of flavanones [30]. In conclusion, the present study showed that the intake of 600 mg/day hesperidin could compensate decreased levels of adiponectin and HDL-C and increased levels of E-selectin in patients with MI. The effect of hesperidin on blood pressure and other inflammatory factors and lipid profile in individuals with MI, however, was not significant in this study. The natural process of the disease and acute changes in these parameters following MI might be responsible for masking the effect of hesperidin. Further investigation to explore the effect of a higher dosage of hesperidin and to define its clinical efficacy would be highly desirable. Furthermore, the search for safe anti-inflammatory and anti-oxidative foods must continue.
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REFERENCES 1. Tehrani DM, Seto AH: Third universal definition of myocardial infarction: update, caveats, differential diagnoses. Cleve Clin J Med 80:777–786, 2013. 2. Ghuge GD, Zine R, Mogrekar MR: High sensitivity C-reactive protein, paraxonase 1 and high density lipoprotein cholesterol in myocardial infarction. Int J Biomed Adv Res 3:637–639, 2012. 3. Suefuji H, Ogawa H, Yasue H, Sakamoto T, Miyao Y, Kaikita K, Soejima H, Misumi K, Miyamoto S, Kataoka K: Increased plasma level of soluble E-selectin in acute myocardial infarction. Am Heart J 140:243–248, 2000. 4. Shojaie M, Sotoodah A, Shafaie G: Is Adiponectin associated with acute myocardial infarction in Iranian non obese patients? Lipids Health Dis 28:8–17, 2009. 5. Ambroziak M, Bartoszewicz Z, Budaj A: Total and high molecular weight adiponectin are decreased in young patients with myocardial infarction. J Am Coll Cardiol 59:E530–E530, 2012. 6. Gustafson B: Adipose tissue, inflammation and atherosclerosis. J Atheroscler Thromb 17:332–341, 2010. 7. Akram S, Ahmed Z, Fayyaz I, Mehmood S, Ghani M, Choudhary AM, Shah I: Serum leptin levels in patients with coronary artery disease. J Ayub Med Coll Abbottabad 23:13–14, 2011. 8. Kim SW, Kim CE, Kim MH: Flavonoids inhibit high glucoseinduced up-regulation of ICAM-1 via the p38 MAPK pathway in human vein endothelial cells. Biochem Biophys Res Commun 415:602–607, 2011. 9. Morand C, Dubray C, Milenkovic D, Lioger D, Martin JF, Scalbert A, Mazur A: Hesperidin contributes to the vascular protective effects of orange juice: a randomized crossover study in healthy volunteers. Am J Clin Nutr 93:73–80, 2011. 10. Raza SS, Khan MM, Ahmad A, Ashafaq M, Khuwaja G, Tabassum R, Javed H, Siddiqui MS, Safhi MM, Isalam F: Hesperidin ameliorates functional and histological outcome and reduces neuroinflammation in experimental stroke. Brain Res 1420:93–105, 2011. 11. Haidari F, Keshavarz SA, Rashidi MR, Mohammad Shahi M: Orange juice and hesperetin supplementation to hyperuricemic rats alter oxidative stress markers and xanthine oxidoreductase activity. J Clin Biochem Nutr 45:285–291, 2009. 12. Haidari F, Rashidi MR, Mohammad Shahi M: Effects of orange juice and hesperetin on serum paraxonase activity and lipid profile in hyperuricemic rats. BioImpacts 2:39–45, 2012. 13. Sahu BD, Kuncha M, Sindhura GJ, Sistla R: Hesperidin attenuates cisplatin-induced acute renal injury by decreasing oxidative stress, inflammation and DNA damage. Phytomedicine 20:453–460, 2013. 14. Wolin KY, Heil DP, Askew S, Matthews CE, Bennett GG: Validation of the International Physical Activity Questionnaire–Short among blacks. J Phys Act Health 5:746–760, 2008. 15. Wang X, Hasegawa J, Kitamura Y, Wang Z, Matsuda A, Shinoda W, Miura N, Kimura K: Effects of hesperidin on the progression of hypercholesterolemia and fatty liver induced by high-cholesterol diet in rats. J Pharmacol Sci 117:129–138, 2011. 16. Yasim A, Ozbag D, Kilinc M, Ciralik H, Toru I: The effects of diosmin–hesperidin combination treatment on the lipid profile and oxidative–antioxidative system in high-cholesterol diet-fed rats. T€urk G€og€us Kalp Damar Cer Derg 19:55–61, 2011.
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Hesperidin Supplementation 17. Akiyama S, Katsumata S, Suzuki K, Ishimi Y, Wu J, Uehara M: Dietary hesperidin exerts hypoglycemic and hypolipidemic effects in streptozotocin-induced marginal type 1 diabetic rats. J Clin Biochem Nutr 46:87–92, 2010. 18. Akiyama S, Katsumata S, Suzuki K, Nakaya Y, Ishimi Y, Uehara M: Hypoglycemic and hypolipidemic effects of hesperidin and cyclodextrin-clathrated hesperetin in Goto-Kakizaki rats with type 2 diabetes. Biosci Biotechnol Biochem 73:2779–2782, 2009. 19. Rizza S, Muniyappa R, Iantorno M, Kim JA, Chen H, Pullikotil P, Senese N, Tesauro M, Lauro D, Cardillo C, Quon MJ: Citrus polyphenol hesperidin stimulates production of nitric oxide in endothelial cells while improving endothelial function and reducing inflammatory markers in patients with metabolic syndrome. J Clin Endocrinol Metab 96:E782–E792, 2011. 20. Ala A, Dhillon AP, Hodgson HJ: Role of cell adhesion molecules in leukocyte recruitment in the liver and gut. Int J Exp Pathol 84:1–16, 2003. 21. Suefuji H, Ogawa H, Yasue H, Sakamoto T, Miyao Y, Kaikita K, Soejima H, Misumi K, Miyamoto S, Kataoka K: Increased plasma level of soluble E-selectin in acute myocardial infarction. Am Heart J 140:243–248, 2000. 22. Piestrzeniewicz K, Luczak K, Komorowski J, Maciejewski M, Piechowiak M, Jankiewicz-Wika J, Goch JH: Obesity and adiponectin in acute myocardial infarction. Cardiol J 14:29–36, 2007. 23. Kurowska EM, Spence JD, Jordan J, Wetmore S, Freeman DJ, Piche LA, Serratore P: HDL-cholesterol-raising effect of orange juice in subjects with hypercholesterolemia. Am J Clin Nutr 72:1095–1100, 2000.
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24. Cesar TB, Aptekmann NP, Araujo MP, Vinagre CC, Maranh~ao RC: Orange juice decreases low-density lipoprotein cholesterol in hypercholesterolemic subjects and improves lipid transfer to highdensity lipoprotein in normal and hypercholesterolemic subjects. Nutr Res 30:689–694, 2010. 25. Miwa Y, Yamada M, Sunayama T, Mitsuzumi H, Tsuzaki Y, Chaen H, Mishima Y, Kibata M: Effects of glucosyl hesperidin on serum lipids in hyperlipidemic subjects: preferential reduction in elevated serum triglyceride level. J Nutr Sci Vitaminol (Tokyo) 50:211–218, 2004. 26. Demonty I, Lin Y, Zebregs YE, Vermeer MA, van der Knaap HC, J€akel M, Trautwein EA: The citrus flavonoids hesperidin and naringin do not affect serum cholesterol in moderately hypercholesterolemic men and women. J Nutr 140:1615–1620, 2010. 27. Kim HJ, Jeon SM, Lee MK, Cho YY, Kwon EY, Lee JH, Choi MS: Comparison of hesperetin and its metabolites for cholesterollowering and antioxidative efficacy in hypercholesterolemic hamsters. J Med Food 13:808–814, 2010. 28. Antonopoulos AS, Margaritis M, Lee R, Channon K, Antoniades C: Statins as anti-inflammatory agents in atherogenesis: molecular mechanisms and lessons from the recent clinical trials. Curr Pharm Des 18:1519–1530, 2012. 29. Grassi D, Desideri G, Croce G, Tiberti S, Aggio A, Ferri C: Flavonoids, vascular function and cardiovascular protection. Curr Pharm Des 15:1072–1084, 2009. 30. Actis-Goretta L, Ottaviani JI, Fraga CG: Inhibition of angiotensin converting enzyme activity by flavanol-rich foods. J Agr Food Chem 54:229–234, 2006. Received December 23, 2013; revision accepted February 1, 2014.
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Journal of the American College of Nutrition Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/uacn20
Hesperidin Supplementation Modulates Inflammatory Responses Following Myocardial Infarction a
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F. Haidari PhD , H. Heybar DMD , M. T. Jalali PhD , K. Ahmadi Engali MD PhD , B. Helli & E. Shirbeigi Msc
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Nutrition and Metabolic Diseases Research Center, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, IRAN b
School of Medicine, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, IRAN
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Hyperlipidemia Research Center, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, IRAN d
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Faculty of Public Health, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, IRAN Published online: 11 Mar 2015.
To cite this article: F. Haidari PhD, H. Heybar DMD, M. T. Jalali PhD, K. Ahmadi Engali MD PhD, B. Helli & E. Shirbeigi Msc (2015): Hesperidin Supplementation Modulates Inflammatory Responses Following Myocardial Infarction, Journal of the American College of Nutrition, DOI: 10.1080/07315724.2014.891269 To link to this article: http://dx.doi.org/10.1080/07315724.2014.891269
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Original Research
Hesperidin Supplementation Modulates Inflammatory Responses Following Myocardial Infarction F. Haidari, PhD, H. Heybar, DMD, M. T. Jalali, PhD, K. Ahmadi Engali, MD, PhD, B. Helli, E. Shirbeigi, Msc Nutrition and Metabolic Diseases Research Center (F.H., B.H., E.S.), School of Medicine (H.H.), Hyperlipidemia Research Center (M.T.J.), Faculty of Public Health (K.A.E.), Ahvaz Jundishapur University of Medical Sciences, Ahvaz, IRAN
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Key words: flavonoids, hesperidin, inflammation, lipid profile, myocardial infarction Objective: A growing number of studies have suggested a crucial role for a variety of inflammatory mediators in myocardial infarction. Recently, several flavonoids have been shown to have cardioprotective and anti-inflammatory properties. Therefore, the aim of this study was to investigate the effect of hesperidin—a common constituent of citrus fruits—on the serum levels of inflammatory markers and adipocytocines in patients with myocardial infarction. Methods: Seventy-five patients with myocardial infarction were participated in this randomized, doubleblind controlled clinical trial and were assigned to 2 intervention and control groups. Subjects consumed 600 mg/d pure hesperidin supplement and placebo in the intervention and control groups, respectively, for 4 weeks. Serum concentrations of inflammatory markers and adipocytocines were measured at baseline and at the end of the intervention. Results: Consumption of 600 mg/day hesperidin significantly decreased the serum levels of E-selectin and increased adiponectin and high-density lipoprotein cholesterol (HDL-C) concentrations in patients with myocardial infarction. The improvement in other inflammatory markers, such as interleukin (IL)-6, highsensitivity C-reactive protein (hs-CRP), leptin, and other lipid profile was also observed at the end of the intervention, compared to the baseline values, but the difference between the hesperidin and placebo groups was not statistically significant (p > 0.05). Conclusion: Hesperidin supplementation could compensate for decreased levels of adiponectin and HDL-C and increased levels of E-selectin in patients with myocardial infarction. These results support the concept that certain flavonoids in the diet can be associated with significant health benefits, including heart health.
INTRODUCTION
vascular biology, and energy hemoestasis and it has been shown that adiponectin is also associated with MI [4]. According to some studies, adiponectin levels decrease in patients with MI and a low adiponectin level is associated with an increase in the level of inflammatory markers such as IL-6 and CRP [5,6]. Leptin is an endocrine hormone secreted from white adipose tissue and is believed to be involved in MI through various mechanisms [7]. Flavonoids are nonnutrient polyphenolic compounds that occur in edible plants and possess a wide range of antioxidants and anti-inflammatory effects [8,9]. Recently, several flavonoids have been recommended as chemopreventive agents or
Myocardial infarction (MI) is a major cause of death and disability worldwide that can result in serious psychological and functional changes in patients [1]. It is considered an inflammatory disease due to the high levels of some inflammatory markers such as acute phase proteins, cytokines, and intracellular adhesion molecules that have been detected in patients with MI [2]. It is speculated that interleukin (IL)-6, high-sensitivity C-reactive protein (hs-CRP), and E-selectin play key roles in the progression of MI [2,3]. Adiponectin appears to play an important role in glucose and lipid metabolism,
Address correspondence to: Esmat Shirbeigi, Nutrition and Metabolic Diseases Research Center, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, IRAN. Email: [email protected]
Journal of the American College of Nutrition, Vol. 0, No. 0, 1–7 (2015) Ó American College of Nutrition Published by Taylor & Francis Group, LLC 1
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Hesperidin Supplementation nutritional supplements [8]. Hesperidin (30 ,5,7-trihydroxy-40 methoxy-flavanone-7-rhamnglucoside) belongs to the flavanone subclass of flavonoids and is mainly found in citrus fruits such as oranges [9]. In recent years, there has been a significant interest in the anti-oxidative, anti-inflammatory, and antihypercholesterolemic properties of the citrus flavanone hesperidin in oxidative stress–related diseases [10]. We previously demonstrated that hesperetin, the aglycone form of hesperidin, at a dose of 5 mg/kg reduced hepatic xanthine oxidoreductase activity, the key enzyme in the catabolism of purines, and consequently decreased the serum levels of uric acid and malondialdehyde and increased the total antioxidant capacity in hyperuricemic rats [11]. Supplementation of orange juice and hesperetin could also restore paraoxonase activity and partially improve the lipid profile in hyperuricemic rats [12]. In another study, Sahu et al. revealed that hesperidin suppressed the generation of reactive oxygen species and pro-inflammatory cytokines such as tumor necrosis factor (TNF)-a, lipid peroxidation, and apoptosis/necrosis in cisplatin-induced acute renal injury [13]. Because MI is a multifactorial disease and chronic inflammation plays a major role in the pathogenesis of the disease [2], the present study aimed to determine the effects of hesperidin supplementation on the serum levels of inflammatory markers (E-selectin, IL-6, hs-CRP) and adipocytocines (adiponectin and leptin) in patients with MI.
Study Design A randomized, double-blind, placebo-controlled clinical trial was conducted to investigate the effects of 600 mg pure hesperidin supplement on the serum levels of inflammatory markers and adipocytocines in patients with MI. Seventy-five patients with MI were divided into 2 groups by randomized block allocation (intervention: n D 38, control: n D 37). For the subjects in the intervention group, one capsule of 600 mg pure hesperidin was provided for consumption in the morning with breakfast every day for 4 weeks. Similarly, the control group received one placebo capsule of 600 mg starch. The hesperidin and placebo supplements were produced by Herbal Extracts Plus Company (Croydan, PA, USA) and the School of Pharmacy, Ahvaz Jundishapur University of Medical Sciences, respectively. Pharmacological treatment with aspirin, statins, beta-blockers, inhibitors of angiotensin II, nitrates, and diuretics was similar in the 2 groups. All subjects were instructed to continue their habitual diets and were taking no vitamin/mineral supplements or flavonoid-rich beverages during the study. Anthropometric indices, dietary intakes, blood pressure, and physical activity were evaluated before and after the intervention. Dietary intakes were also measured in the middle of the intervention. To assess physical activity, we used the International Physical Activity Questionnaire (IPAQ). This questionnaire evaluates participants’ activity in 5 major domains (work, transportation, housework, sport, and leisure-time physical activity). Data from the IPAQ were converted to metabolic equivalent of task-minutes/wk using existing guidelines [14].
MATERIAL AND METHODS Biochemical Analysis Subjects In this study, 75 patients with MI at Ahvaz Golestan Hospital participated. MI was defined as a combination of 2 of 3 characteristics: typical symptoms (i.e., chest discomfort), enzyme rise, and a typical echocardiograph pattern involving the development of Q waves (echocardiograph abnormalities). If the diagnosis of MI was confirmed by a cardiologist, patients were asked to participate in this study. Inclusion criteria were patients with MI, age range 40–65 years, body mass index (BMI) between 18.5 and 30, and patient satisfaction. Exclusion criteria included chronic disease (such as hepatic disorders, renal failure, and diabetes), gastrointestinal disorders, major psychiatric disorder, taking antidepressants, absolute vegetarian diet, consumption of antioxidants and vitamin supplements at least 6 months before sampling, high consumption of flavonoid-rich beverages (including green tea and coffee), smoking, allergies to any vitamin supplements, lack of patient follow-up, and lack of consent. The study was approved by and performed under the guidelines of the Research Ethics Committee of Ahvaz Jundishapur University of Medical Sciences, Iran (ETH-569). Written consent was obtained from all subjects.
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Fasting blood samples were collected for biochemical analysis. Blood samples centrifuged at 3000 g for 5 min. To prepare serum, blood samples were stored at ¡80 C until analysis. Serum concentrations of E-selectin, hs-CRP, IL-6, leptin, and adiponectin were measured using a commercially available enzyme-linked immunosorbent assay method (Orgenuim Laboratories Business Unit, Finland for IL-6, leptin, and adiponectin; Labor Diagnostika Nord, Germany for hs-CRP; Boster Biological Technology, China for E-selectin). All assays were performed according to the manufacturer’s instructions.
Statistical Analysis All statistical analyses were performed with the Statistical Package for Social Sciences (SPSS Inc., Chicago, IL) version 18 for Windows. First, normal distribution of all variables was checked with the Kolmogorov-Smirnov test. We compared the means of variables of each group with each other group using both independent sample t test and analysis of covariance in the adjusted models, which were adjusted for age, sex, energy,
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Hesperidin Supplementation physical activity, and weight. The end values of each variable were also compared with the baseline values using paired sample t tests. The percentage change of each variable was also calculated by the formula [(E ¡ B)/B £ 100], where E is the end of treatment value and B is the baseline value. The independent sample t test and analysis of covariance in the adjusted models were also used for comparing the percentage change of variables between 2 groups. The differences with p values < 0.05 were considered significant. In this study, Nutritionist IV software (First Databank, San Bruno, CA), which was modified for Iranian foods, was used to estimate patients’ dietary intakes.
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RESULTS In this study, 75 participants, 55.49 § 5.98 years old, were included the study. Patients did not report any serious adverse effects during the study related to hesperidin or placebo consumption. Sixty-eight percent (n D 26) and 72% (n D 27) of the study population in the hesperidin and placebo groups were males, respectively. Some demographic and anthropometric characteristics of the participants at baseline and postintervention are shown in Table 1. There were no significant differences at baseline and at the end of the study (anthropometric indices, blood pressure, and physical activity) between the 2 groups (p > 0.05). However, weight, BMI, waist circumference, head circumference, and
IPAQ significantly decreased within groups at the end of study when compared to the baseline values (p < 0.05). Dietary intake of subjects in different times of study was assessed by 3-day diet records (Table 2). Regarding dietary intake analysis, although energy and nutrient intakes of patients increased during the study (from time 1 to time 3) in both the hesperidin and placebo groups, there were no significant differences between the 2 groups at the beginning, middle, and end of the intervention. These results remained insignificant after adjusting for confounders. The effect of hesperidin supplementation on the serum levels of inflammatory markers (E-selectin, IL-6, hs-CRP), adipocytocines (adiponectin and leptin), and lipid profile in patients with myocardial infarction are shown in Table 3. As can be seen, no significant differences in baseline characteristics of participants were found between the 2 groups. However, hesperidin consumption, compared to placebo, significantly increased adiponectin and HDL-C levels and decreased Eselectin concentrations at the end of the study (p < 0.05). These effects also remained significant after adjusting for age, sex, energy, and physical activity. However, other inflammatory factors and lipid profile were not significantly changed in the hesperidin group compared to the control group (p > 0.05). Statistical analyses (within groups) also showed that the serum levels of leptin, E-selectin, IL-6, hs-CRP, triglycerides
Table 1. Demographic and Anthropometric Characteristics of the Study Participantsa Variables Weight (kg)
BMI (kg/m2)
WC (cm)
HC (cm)
WHR
SBP (mmHg)
DBP (mmHg)
IPAQ (metabolic equivalent of task-min/wk)
Baseline End P3 Baseline End P3 Baseline End P3 Baseline End P3 Baseline End P3 Baseline End P3 Baseline End P3 Baseline End P3
Hesperidin N D 38
Placebo N D 37
P1
P2
76.15 § 8.82 73.07 § 8.82 0.001 25.97 § 2.87 24.92 § 2.89 0.000 97.50 § 7.61 95.16 § 7.15 0.000 94.34 § 5.95 92.5 § 5.52 0.000 1.03 § 0.03 1.01 § 0.03 0.537 135.55 § 15.99 131.89 § 15.73 0.145 84.10 § 10.49 85.81 § 8.30 0.421 2392 § 708 2145 § 663 0.000
76.63 § 8.85 73.85 § 9.12 0.000 26.82 § 2.61 25.83 § 2.66 0.000 96.56 § 8.33 94.02 § 7.93 0.000 95.08 § 7.38 93.59 § 7.04 0.003 1.01 § 0.03 1.00 § 0.05 0.024 136.62 § 18.99 132.72 § 15.74 0.431 86.05 § 9.14 84.54 § 8.38 0.513 2432 § 678 2091 § 753 0.000
0.814 0.710 — 0.158 0.163 — 0.614 0.516 — 0.634 0.489 — 0.067 0.102 — 0.793 0.819 — 0.395 0.510 — 0.803 0.744 —
0.522 0.796 — 0.050 0.123 — 0.621 0.470 — 0.531 0.472 — 0.034 0.078 — 0.885 0.927 — 0.556 0.512 — 0.590 0.855 —
BMI D body mass index, WC D waist circumference, HC D hip circumference, WHR D waist-to-hip ratio, SBP D systolic blood pressure, DBP D diastolic blood pressure, IPAQ D International Physical Activity Questionnaire. a Data are expressed as mean § SD. P1 are results from independent sample t tests; P2 are results from analysis of covariance in the adjusted models (adjusted for age, sex, energy, and physical activity); P3 are results from paired sample t tests.
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Hesperidin Supplementation Table 2. Dietary Intakes of the Study Population during the Interventiona
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Variables Energy (kcal) Time 1 Time 2 Time 3 P3 Protein (g) Time 1 Time 2 Time 3 P3 Carbohydrate (g) Time 1 Time 2 Time 3 P3 Fat (g) Time 1 Time 2 Time 3 P3 SFA (g) Time 1 Time 2 Time 3 P3 MUFA (g) Time 1 Time 2 Time 3 P3 PUFA (g) Time 1 Time 2 Time 3 P3 Cholesterol (mg) Time 1 Time 2 Time 3 P3 Fiber (g) Time 1 Time 2 Time 3 P3
Hesperidin N D 38 Placebo N D 37
P1
P2
1221 § 372 1559 § 525 1906 § 485 0.000
1164 § 204 1472 § 375 1892 § 392 0.000
52.23 § 24.33 60.02 § 26.80 73.40 § 40.50 0.000
42.27 § 19.18 0.053 0.669 50.44 § 18.97 0.079 0.069 76.94 § 39.95 0.704 0.715 0.000
153.97 § 54.72 210.60 § 52.70 263.92 § 52.95 0.000
148.00 § 64.06 0.665 0.538 198.17 § 73.77 0.403 0.369 258.33 § 62.68 0.678 0.720 0.000
36.60 § 19.98 49.78 § 24.31 64.12 § 23.93 0.000
42.24 § 28.59 0.325 0.336 55.82 § 31.61 0.356 0.400 66.89 § 28.92 0.652 0.700 0.000
10.18 § 6.63 13.88 § 6.90 20.11 § 12.00 0.000
9.91 § 7.38 14.71 § 8.02 21.46 § 12.93 0.000
0.870 0.630 0.642 —
7.84 § 4.54 11.75 § 6.01 19.22 § 6.55 0.000
7.05 § 5.28 12.39 § 7.14 19.13 § 6.96 0.000
0.490 0.326 0.677 0.844 0.954 0.812
8.23 § 9.87 12.90 § 12.25 8.23 § 9.87 0.000
11.02 § 10.84 0.248 0.356 16.10 § 13.79 0.293 0.408 29.37 § 12.31 0.306 0.358 0.000
131.68 § 66.59 207.84 § 96.64 230.16 § 69.57 0.000
114.54 § 52.39 0.220 0.245 172.97 § 66.43 0.073 0.080 208.98 § 55.99 0.151 0.141 0.000
2.02 § 1.46 3.23 § 2.08 7.05 § 5.01 0.000
2.37 § 2.19 3.77 § 2.89 7.56 § 5.42 0.000
0.416 0.253 0.414 0.277 0.892 0.764
0.804 0.692 0.691 —
0.414 0.435 0.350 0.382 0.673 0.621
Time 1 D at the baseline, Time 2 D middle of the intervention, Time 3 D end of the intervention, SFA D saturated fatty acid, MUFA D mono-unsaturated fatty acid, PUFA D polyunsaturated fatty acid. a Data are expressed as mean § SD. P1 are results from independent sample t tests; P2 are results from analysis of covariance in the adjusted models (adjusted for age, sex, energy, and physical activity); P3 are results from repeated measures analysis of variance.
(TG), total cholesterol (TC), and LDL-C significantly decreased and the mean concentration of adiponectin and HDL-C significantly increased postintervention in both groups (p < 0.05). Percentage changes in inflammatory markers and lipid profile are shown in Table 4. Results showed significant differences in percentage changes of adiponectin, HDL-C, and low-
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density lipoprotein (LDL)-C between the 2 groups (p D 0.040, p D 0.001, p D 0.014, respectively). However, after adjustment for age, sex, energy, and physical activity, this significant effect did not remain for adiponectin (p D 0.081). There were no significant differences in percentage changes of leptin, E-selectin, IL-6, hs-CRP, TG, and TC between the 2 groups (p > 0.05).
DISCUSSION In this clinical trial, we have shown for the first time the effect of daily consumption of pure hesperidin (600 mg) on inflammatory markers, lipid profile, and blood pressure in patients with MI. The main findings of this study was that consumption of a hesperidin supplement, the major orange flavonoid constituent, for 4 weeks significantly decreased E-selectin and elevated adiponectin and HDL-C levels in patients with MI. An improvement in other inflammatory markers, such as IL-6, hs-CRP, leptin, and other lipids, was also observed at the end of the intervention, compared to baseline values, but the difference between the hesperidin and placebo groups was not statistically significant (p > 0.05). Although the beneficial effects of hesperidin on lipid profile have been shown in several animal models [15–18], studies regarding the anti-inflammatory effects of hesperidin are very limited and its effect in humans, especially in patients with MI, remains unclear. The results from a crossover study that was conducted in healthy subjects have shown that consumption of orange juice or hesperidin for 4 weeks did not affect plasma concentrations of CRP and IL-6 in healthy subjects [9]. Contrary to this finding, one study reported that oral consumption of hesperidin (500 mg once daily for 3 weeks) reduced E-selectin and hs-CRP in subjects with metabolic syndrome [19]. Adhesion of leukocytes to endothelial cells is the initial event in the acute inflammatory response, which is thought to play an important role in myocardial reperfusion injury. E-selectin is a cell-surface glycoprotein that belongs to the selectin family, and it appears to be the initial adhesion molecule for leukocytes during the inflammatory process [20]. Results of a previous study suggested that an increase in plasma E-selectin level may reflect enhanced endothelial cell activation in patients with MI [21]. It is transiently expressed in vascular endothelial cells in response to pro-inflammatory cytokines (such as TNF-a and IL-1) and its serum levels decreased slowly during the chronic phase of MI [3]. Therefore, it seems that the decreased levels of E-selectin in the intervention group in this study might be due to the antioxidant properties and inhibitory effects of hesperidin on generation of TNF-a [10,13]. Several studies demonstrated the decreased levels of adiponectin in patients with MI [4,5], but the effects of flavonoids
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Hesperidin Supplementation Table 3. Effects of Hesperidin Supplementation on Serum Levels of Inflammatory Markers and Lipid Profile in Patients with Myocardial Infarctiona Variables Adiponectin (mg/L)
E-selectin (ng/ml)
IL-6 (pg/ml)
hs-CRP (mg/L)
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Leptin (ng/ml)
TC (mg/dl)
TG (mg/dl)
HDL-C (mg/dl)
LDL-C (mg/dl)
Baseline End P3 Baseline End P3 Baseline End P3 Baseline End P3 Baseline End P3 Baseline End P3 Baseline End P3 Baseline End P3 Baseline End P3
Hesperidin N D 38
Placebo N D 37
P1
P2
6.71 § 1.05 8.10 § 1.24 0.000 41.32 § 5.27 33.21 § 3.85 0.000 54.76 § 7.38 16.85 § 4.31 0.000 5.99 § 1.55 1.77 § 0.84 0.000 13.95 § 4.32 6.52 § 2.91 0.000 207.6 § 58.9 186.3 § 51.3 0.003 148.8 § 38.6 139.9 § 31.7 0.096 34.7 § 7.2 42.9 § 9.8 0.000 143.0 § 61.1 115.4 § 56.0 0.000
6.82 § 1.07 7.57 § 0.93 0.000 40.21 § 7.59 36.26 § 7.34 0.041 52.45 § 8.02 17.27 § 6.00 0.000 6.37 § 1.55 1.82 § 0.85 0.000 13.53 § 3.90 7.23 § 3.49 0.000 203.5 § 49.1 193.4 § 48.5 0.041 151.7 § 41.3 140.6 § 35.2 0.014 35.3 § 8.7 37.7 § 10.8 0.037 137.8 § 52.5 127.5 § 51.6 0.000
0.646 0.043 — 0.463 0.027 — 0.200 0.728 — 0.290 0.837 — 0.662 0.342 — 0.749 0.541 — 0.759 0.930 — 0.776 0.034 — 0.698 0.333 —
0.896 0.049 — 0.632 0.036 — 0.126 0.658 — 0.328 0.925 — 0.665 0.383 — 0.733 0.517 — 0.953 0.992 — 0.931 0.031 — 0.732 0.309 —
IL-6 D interleukin-6, TC D total cholesterol, TG D triglycerides, HDL-C D high-density lipoprotein cholesterol, LDL-C D low-density lipoprotein cholesterol. a Data are expressed as mean § SD. P1 are results from independent sample t tests; P2 are results from analysis of covariance in the adjusted models (adjusted for age, sex, energy, and physical activity); P3 are results from paired sample t test.
on adiponectin levels in these patients are limited and rare. The present study also indicated a significant increase in adiponectin level in the intervention group compared with the control group after 4 weeks. Adiponectin is regarded as a protective adipokine associated with a lower risk of MI [22]. Therefore, the increased levels of adiponectin in the hesperidin group compared to the placebo group in this study could be explained in part by the anti-inflammatory, anti-oxidative, and enzyme inhibitory effects of hesperidin [12]. In the present study, the serum level of HDL-C in the hesperidin group was significantly increased compared to the control group; however, there was no significant difference in serum levels of other lipid profile between the hesperidin and placebo groups. Previous studies have shown conflicting results regarding the effect of hesperidin on lipid profile [23–26]. Our results concur with those of Yasim and colleagues, who showed that diosmin– hesperidin administration to rats fed a high-cholesterol diet significantly increased HDL-C levels but did not significantly affect other lipid parameters [16]. In another study, Kurowska et al. showed that consumption of orange juice (750 ml once a day for 4 weeks) increased HDL-C levels in hypercholesterolemic individuals [23]. Kim et al. also reported that 12 weeks’ administration of hesperetin and its metabolite, ferulic acid, in
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hypercholesterolemic hamsters increased the HDL-C/total cholesterol ratio and paraoxonase levels [27]. The correlation between paraxonase activity and protective role of HDL-C in oxidative stress and atherosclerosis pathogenesis has been suggested in
Table 4. Percentage Changes in Inflammatory Markers and Lipid Profile in Patients with MIa
Variables Adiponectin (mg/L) E-selectin (ng/ml) IL-6 (pg/ml) hs-CRP (mg/L) Leptin (ng/ml) TC (mg/dl) TG (mg/dl) HDL-C (mg/dl) LDL-C (mg/dl)
Hesperidin N D 38 23.32 ¡18.26 ¡68.66 ¡70.93 ¡50.33 ¡8.31 ¡3.54 24.92 ¡16.97
Placebo N D 37
P1
P2
§ 27.55 12.39 § 15.52 0.040 0.081 § 14.72 ¡4.73 § 34.88 0.055 0.077 § 9.17 ¡66.84 § 10.67 0.151 0.061 § 9.85 ¡72.09 § 9.96 0.182 0.250 § 27.90 ¡45.04 § 28.46 0.270 0.298 § 18.87 ¡5.19 § 1.02 0.091 0.110 § 21.25 ¡5.90 § 15.89 0.750 0.788 § 21.80 8.44 § 21.24 0.001 0.000 § 29.87 ¡8.23 § 7.81 0.014 0.017
IL-6 D interleukin-6, hs-CRP D high-sensitivity C-reactive protein, TC D total cholesterol, TG D triglycerides, HDL-C D high-density lipoprotein cholesterol, LDL-C D low-density lipoprotein cholesterol. a Data are expressed as mean § SD. P1 are results from independent sample t tests; P2 are results from analysis of covariance in the adjusted models (adjusted for age, sex, energy, weight change, and physical activity).
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Hesperidin Supplementation several studies [12]. Therefore, the significantly higher levels of HDL-C in the hesperidin group compared to the placebo control group in our study might be partly explained by the antioxidant properties of hesperidin. Contrary to these findings, some animal and clinical trials demonstrated that hesperidin or orange juice administration could decrease total cholesterol [15,24] and triglyceride levels [25]. The mechanisms by which hesperidin can affect the lipid profile are largely unknown, but previous studies have shown that in a human liver cell line (HepG2), principal flavanones from oranges and grapefruit, hesperetin and naringenin, both reduced the medium content of Apo lipoprotein B, the main protein component of LDL [15,24]. However, in the present study, comparison of groups (before and after intervention) showed that the serum levels of all inflammatory biomarkers and lipid profile significantly improved at the end of study in both groups (p < 0.05). This result might be masked by the effect of hesperidin on some inflammatory markers, adipocytocines, and lipid profile. In other words, one possible reason for the lack of a significant effect of hesperidin on leptin, IL-6, hs-CRP, TG, TC, and LDL-C in our study may be that the concurrent drug therapy (such as statins and aspirin) [28] and lifestyle modifications (such as weight loss) have powerful effects on modulating these factors after MI, similar to the way in which the same effect of hesperidin is masked. In our study, no significant changes in systolic or diastolic blood pressure were observed during the intervention. Our results are consistent with data reported by Wang and colleagues, who showed that consumption of hesperidin in rats fed a high-cholesterol diet did not cause a significant change in blood pressure [15]. On the other hand, these finding contrast with a study that demonstrated that consumption of orange juice as well as hesperidin for 4 weeks in healthy subjects resulted in a significantly lower diastolic blood pressure compared to that measured after consumption of placebo [9]. The possible mechanisms by which flavonoid-rich foods lower blood pressure may involve a chronic increase in the production of NO by vascular endothelium [29]. Other mechanisms, such as an inhibitory effect on angiotensin-converting enzyme, could also be responsible for the blood pressure–lowering effects of flavanones [30]. In conclusion, the present study showed that the intake of 600 mg/day hesperidin could compensate decreased levels of adiponectin and HDL-C and increased levels of E-selectin in patients with MI. The effect of hesperidin on blood pressure and other inflammatory factors and lipid profile in individuals with MI, however, was not significant in this study. The natural process of the disease and acute changes in these parameters following MI might be responsible for masking the effect of hesperidin. Further investigation to explore the effect of a higher dosage of hesperidin and to define its clinical efficacy would be highly desirable. Furthermore, the search for safe anti-inflammatory and anti-oxidative foods must continue.
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