Objectives: Smoking causes endothelial dysfunction, but it still remains unclear whether oral administration of anthocyanin brings benefits to endothelial function and redox states in young healthy smokers. We tested the hypothesis that supplement anthocyanin may rescue endothelial dysfunction and redox states in young healthy smokers. Methods: Young healthy male non-smoker (n = 8) and smokers (n = 14) were enrolled to measure the derivatives of reactive oxygen metabolites (d-ROMs) and biological antioxidant potential (BAP) levels and brachial artery flow-mediated dilation (FMD) before and 2 hours after one cigarette smoking or resting under administration of blackcurrant supplements (anthocyanins 210 mg) or not. Results: FMD, d-ROMs, and BAP at baseline were similar between non-smokers and smokers. One cigarette smoking induced a decrease in FMD at 2 hours (9.0% ± 2.6% vs 7.5% ± 2.3%, p < 0.05) without affecting plasma levels of d-ROMs and BAP in young healthy smokers. Single oral administration of anthocyanin at 210 mg abolished smoking-induced decrease in FMD (8.3% ± 3.4% vs 9.5% ± 3.6%, p = ns) without changes in plasma levels of d-ROMs and BAP in young smokers. In non-smokers, however, administration of anthocyanin at 210 mg slightly but significantly elevated plasma level of d-ROMs at 2 hours (249.6 ± 30.3 vs 265.5 ± 36.3 U CARR, p < 0.05) without affecting plasma level of BAP and FMD. Conclusions: Single oral administration of anthocyanin rescued smoking-induced endothelial dysfunction in young healthy smokers, but facilitated oxidative stress in non-smokers. When anthocyanin is taken as supplements, it seems important to pay attention to the dose and timing of administration.
Lifestyle-related disease has become an object of public concern, and arteriosclerosis is attributable to the pathogenesis of lifestyle-related diseases such as cardiovascular disease. Smoking is predisposed to arteriosclerosis and regarded as one of the major risk factors for the development of cardiovascular diseases [
Blackcurrant is rich in many health-beneficial substances, such as polysaccharides, flavonoids and anthocyanins [
The blackcurrant supplement, which was made with solvent free, freeze-dried extraction methods to preserve quality, was purchased from Just the Berries Limited, New Zealand. It consists of not lower than 35.0 g total anthocyanin, 13.6 mg carbohydrate, 0.5 g lipid, and 0.1 g protein in total 100 g, and energy is 1650 Kj (394 Kcal)/100 g.
Young healthy male non-smoker (n = 8, mean age 23 ± 2 years) and smokers (n = 14, mean age 22 ± 1 years) were recruited from students of Hirosaki University. Exclusion criteria are having any disease such as hypertension, hyperlipidemia or diabetes, and a weight reducing dietary regimen or taking any dietary supplements. All subjects were asked to complete a health and lifestyle questionnaire, and suitable subjects were asked to sign an informed consent form. All study protocols were approved by The Committee of Medical Ethics of Hirosaki University Graduate School of Medicine, Hirosaki, Japan (2013-357).
The subjects were asked to follow a low-flavonoid diet for 3 days before each determination and to refrain from taking hard exercise (
Brachial artery flow-mediated dilation (FMD) was measured, and blood samples were taken from cubital vein to measure free radical and antioxidant capacity. Smokers were undergoing two series of experiments: one cigarette smoke alone, and take blackcurrant supplements after one cigarette smoke. Non-smokers were undergoing two series of experiments: no supplements or blackcurrant supplements alone.
Brachial artery FMD were measured two hours after one cigarette smoking and/or blackcurrant supplements alone. Blood samples were taken two hours after one cigarette smoking and/or blackcurrant supplements alone.
The derivatives of reactive oxygen metabolites (d-ROMs) and biological antioxidant
potential (BAP) levels were measured using a Free Radical Elective Evaluator (Wismerll Co. Ltd, Tokyo, Japan) [
Endothelium-dependent vasodilation and endothelium-independent vasodilation were measured according to methods that have been described previously. Brachial artery FMD was measured by a trained technician according to the guidelines for the ultrasound assessment. A linear-array transducer operating at 10 MHz was used to acquire longitudinal images of the right brachial artery. A standard blood pressure cuff was positioned around the right arm, 5 cm below the antecubital fossa, and the artery was imaged 5 to 9 cm above the antecubital fossa. After baseline images were obtained, the cuff was inflated to 30 mmHg above systolic blood pressure for 5 minutes. The diastolic pre-beat diameter of the brachial artery was determined semi-automatically using an instrument equipped with software for monitoring the brachial artery diameter (Unex Co. Ltd., Nagoya, Japan), and the validity and reproducibility of the equipment were confirmed. The participants rested on beds in supine position for at least 20 min before the study.
Results were expressed as mean ± SEM. Data were analyzed by using IBM SPSS Statistics 22.0. Statistical significance was defined as p < 0.05. For the analysis of between groups with or without taking blackcurrant supplements, a two-way repeated-measures ANOVA was applied. The intra-group comparison between pre-intervention and post-intervention was done by the Bonferroni correction for multiple comparisons.
As shown in Figures 2(a)-(d), d-ROMs, BAP, BAP/d-ROMs ratio, and FMD at baseline were all similar between young healthy non-smokers and smokers.
BAP/d-ROMs was changed 2 hours after one cigarette smoking in young healthy smokers (d-ROMs: 260.8 ± 45.0 vs 262.3 ± 52.3 U CARR in
taking blackcurrant supplements (gray dotted lines in
The major findings of this study are as follows. Oxidative stress d-ROMs, antioxidant capacity BAP, and endothelial function at baseline were similar between young healthy smokers and non-smokers. One cigarette smoking induced transient endothelial dysfunction without affecting oxidative stress and antioxidant capacity in young healthy smokers. Single oral administration of anthocyanin rescued smoking-induced endothelial dysfunction without affecting oxidative stress and antioxidant capacity in young healthy smokers, but facilitated oxidative stress in young healthy non-smokers.
Oxidative stress d-ROMs, antioxidant capacity BAP, and endothelial function assessed by FMD measurement were all similar between young healthy smokers and non-smokers. In clinical studies, long-term exposure to cigarette smoke was reported to induce systemic lipid peroxidation with depletion of antioxidants such as vitamins A and C and elevation of inflammatory responses including C-reactive protein, fibrinogen, and interleukin (IL)-6 [
Cigarette smoke oxidants can directly impair endothelial function, primarily through a lack of nitric oxide bioavailability which results both from decreased nitric oxide formation by the dysfunctional endothelium and increased nitric oxide scavenging by oxygen free radicals [
In general, the beneficial effects of antioxidant-rich food with respect to cancer are widely reported [
The potential efficacy of dietary antioxidants at protecting against smoking-induced oxidative stress was indicated as follows. Decrease in plasma vitamin C was ameliorated by supplements of a vitamin cocktail containing vitamin C, α-tocopherol, and folate in smokers [
To date, in young healthy smokers who are at lower risk than old continuous smokers but maintain a higher risk than non-smokers, the clinical efficacy of dietary or supplementary antioxidants remains to be established. It is noted that single oral administration of anthocyanin unexpectedly elevated the serum level of d-ROMs slightly in young healthy non-smokers. In contrast, there was no effect of anthocyanin supplements on its level in young healthy smokers who were already protected by an adaptive response in antioxidant system. Furthermore, endothelial function was unchanged during two hours with or without blackcurrant supplements in non-smokers. Taken together, these suggest that an excessive increase in antioxidant system by single oral administration of anthocyanin may facilitate oxidative stress as a reactive response, thereby the state of redox balance is unchanged compared with that before administration of anthocyanin. To our knowledge, this is the first report that single oral administration of anti-oxidant supplements could cause the unexpected increase in the serum radical level in young healthy non-smokers.
The authors thank Mr. Syuhei Yamada (Hirosaki University) and Mr. Hiroshi Tomisawa (Central Chemical Co., Ltd.) for their support. This work was supported in part by Grant-in-Aid for Scientific Research (C) (16K00844) and a Hirosaki University Grant for Exploratory Research by Young Scientists and Newly-appointed Scientists.
Yoshizaki, A., Tomisawa, T., Osanai, T., Nanashima, N., Kitajima, M., Mikami, K., Fujita, T., Maeda, H. and Kato, Y. (2018) Single Oral Administration of Anthocyanin Rescues Smoking-Induced Endothelial Dysfunction in Young Smokers but Facilitates Oxidative Stress in Non-Smokers. Food and Nutrition Sciences, 9, 179-190. https://doi.org/10.4236/fns.2018.93014