Valls RM, Pedret A, Calderón-Pérez L, et al. Hesperidin in orange juice improves human endothelial function in subjects with elevated blood pressure and stage 1 hypertension: a randomized, controlled study (Citrus study). J Funct Foods. October 2021;85:104646. doi: 10.1016/j.jff.2021.104646.
Endothelial dysfunction is a contributing factor and response to atherosclerosis and cardiovascular disease (CVD). It involves the impairment of vasodilation, restricting proper relaxation and contraction of endothelium tissue, inflammation, and disrupted endothelial integrity and homeostasis. Hesperidin, a flavonoid in Citrus spp. (Rutaceae) fruits and peels and other fruits, has demonstrated vasodilatory, hypotensive, antithrombotic, antilipemic, anti-inflammatory, and antioxidant activities in preclinical models. Hesperidin's bioactivity is associated in vivo with hesperetin-7-O-β-d-glucuronide conjugate (H7Oβd-Glu). Data on human intake of hesperidin differ. Trials in different populations report benefits or null effects.
The authors tested acute and sustained effects of hesperidin intake on endothelium-dependent vasodilation, and of sustained use on acute effects, in a randomized, parallel groups, double-blind, placebo (PLA)-controlled, clinical trial (RCT) in January 2016 – June 2017 at Hospital Universitari St. Joan (HUSJ-Eurecat; Reus, Tarragona, Spain). Participants were recruited via newspaper advertising. Inclusion criteria were systolic blood pressure (SBP) ≥ 120 mmHg, no family history of CVD or chronic disease, and age 18-65 years. Exclusion criteria were SBP < 120 mmHg or ≥ 160 mmHg; diastolic blood pressure > 100 mmHg; use of hypertensive drugs; fasting blood glucose > 125 mg/dL; body mass index ≥ 35 kg/m2; hyperlipidemia/use of antilipemic drugs; smoking; pregnancy/plans to become pregnant; use of other drugs, antioxidants, or vitamins; chronic alcoholism; vegetarian diet; physical activity > five hours (h)/week (w); intestinal disorders or anemia. Of 311 potential participants, 159 met criteria and were randomized to three groups. This sample gave > 80% power to detect statistically significant differences (P < 0.05) of eight units ischemic reactive hyperemia (IRH), standard deviation 11, assuming 20% dropouts and based on intention-to-treat. After enrollment, participants completed a one-w run-in, eating a control diet based on their eating habits and nutritionist suggestions. During the RCT, they maintained their dietary habits but refrained from Citrus spp.-containing foods and limited intake of flavonoid-rich foods.
Study products provided by the Department of Citrus, State of Florida, included orange (C. × sinensis) juice (OJ) with 690 mg/L hesperidin, enriched orange juice (EOJ) with 1200 mg/L, and a control drink (CD) with none. Hesperidin, narirutin, and vitamin C content were analyzed with liquid chromatography-tandem mass spectrometry (LC-MS/MS). Participants and researchers were blinded to allocation. Participants drank one 500 mL can daily of their assigned product for 12 weeks. At baseline (week 1) and week 12, dose-response (D-R) RCTs were nested into the overall RCT.
Participants visited HUSJ-Eurecat at baseline (week 1) and every two weeks thereafter for 12 weeks. Food diaries verified dietary adherence. Fasting blood and urine samples were taken at weeks 1, 4, 8, and 12. Levels of plasma H7Oβd-Glu, as per LV-MS/MS, verified study drink adherence. At all visits, participants had physical examinations, including anthropometric measures, and completed the Physical Activity Questionnaire Class AF (PAQ). Resting blood pressure (BP) was measured three times at one-minute intervals and averaged. IRH, a measure of endothelial-dependent vasomotor function, was assessed using a laser-doppler linear flowmeter. Acute D-R studies at weeks 1 and 12 were conducted from 8 am – 2 pm. The authors do not state here how many participants were selected from each group for these, or on what basis; however, of 52 allocated, 50 completed the first D-R; 42, the second. The number of completers is given for each group. In the D-R studies, a 500 mL drink was administered, and fasting blood samples taken 0, 2, 4, and 6 hours after. IRH was measured at weeks 1, 4, 8, and 12 in the sustained intake study; in D-R studies, 2, 4, and 6 hours after intake.
Of 53 participants randomized to each group, 43 in CD; 46, OJ; and 40, EOJ completed the sustained intake RCT (total = 129). There were no significant between-group differences in demographic or anthropometric factors, blood glucose or lipid levels, or PAQ scores at week 1. Plasma H7Oβd-Glu rose in both sustained and acute studies in OJ and EOJ groups. At week 12, H7Oβd-Glu had increased in a dose-dependent manner (P < 0.0001 for linear trend), significantly so in OJ and EOJ groups vs. CD (P < 0.001 for both) and significantly higher in EOJ vs. OJ (P < 0.05). There were no significant changes in IRH at weeks 4 and 8. At week 12, IRH was significantly higher for EOJ vs. CD (P = 0.043). Increases in IRH by week 12 were directly correlated with increases in H7Oβd-Glu (P = 0.009). In acute D-Rs, at week 1 IRH rose significantly (P < 0.05) after all treatments, but most after EOJ, and at six hours post-intake was significantly higher with EOJ vs. CD and OJ (P < 0.05 for both). In the week 12 D-R study, increases in IRH were seen at six hours post-intake in all groups (P < 005) with no significant among-group differences.
Serum glucose, insulin, nitrites and nitrates, interleukin (IL)-6, IL-18, high-sensitivity C-reactive protein, myeloperoxidase, and endothelin were measured, with demonstratable links between nitric oxide and IRH values. In a subset of participants (n = 37; 11, 15, and 11 in CD, OJ, and EO groups, respectively), gene expression in peripheral mononuclear cells was assessed in fasting conditions at weeks 1 and 12. Functional and biochemical analyses of roles of differently expressed genes were performed. Four genes related to endothelial function, validated by polymerase chain reaction (PCR), were differently expressed at week 12 for EOJ, with interrelated pro-endothelial effects. This is the first RCT reporting dose-dependent effects of Citrus flavanones on endothelium-dependent vasodilation in individuals with elevated BP and stage 1 hypertension. Besides hesperidin, vitamin C and/or narirutin in study drinks could have affected results. The authors declare that they had no conflicts of interest.