This pilot prospective, single-arm intervention study took place at the Nephrology Clinic “C. Katsinas” of the General Hospital of Ptolemaida “Mpodosakeio” from November 2021 to April 2022. Patients meeting inclusion criteria [aged ≥ 18 years, cognitively competent, diagnosed with arterial hypertension (per European Society of Cardiology/European Society of Hypertension 2018 guidelines or on antihypertensive medication), type 2 diabetes mellitus, and CKD stage I-IV) participated. Exclusion criteria encompassed chronic atrial fibrillation, congestive heart failure New York Heart Association class III-IV, recent acute myocardial infarction or anginal symptoms, mental illness or dementia, active malignancy, alcohol abuse, inability/unwillingness to provide consent, or known flavonoid allergies.

Participants received a daily oral dietary supplement rich in phenols (200 mg) for 3 months. The supplement composition included fats (36.5 g/100 g), proteins (10 g/100 g), and carbohydrates (40.8 g/100 g), providing 532 kcal/100 g. Patients underwent clinical examinations, laboratory tests, and arterial stiffness assessments via carotid-femoral pulse wave velocity (cfPWV) at enrollment and after 3 months. Oxidative stress indicators (total phenols, total antioxidant capacity, protein carbonyls) were also evaluated.

All patients provided written informed consent after receiving comprehensive study information. The Ethics Committee of the General Hospital of Ptolemaida approved the study (80/18-11-2021), which adhered to the Declaration of Helsinki (1989).

Arterial stiffness was evaluated by measurement of cfPWV at enrollment and 3 months later by an experienced operator blinded to the aims of the study. PWV measures the speed at which a pressure wave travels along the aorta and great arteries, reflecting the elasticity and compliance of these vessels. cfPWV is generally accepted as the “gold standard” measurement in evaluating aortic stiffness and is an important predictor of cardiovascular events. It can be calculated by dividing the distance traveled between two recording sites by the pulse transit time (PWV = distance in meters/by transit time in seconds). In this study, cfPWV was measured using the validated SphygmoCor device (AtCor Medical, IL, United States)[5-8]. All participants were positioned in a quiet environment with stable temperatures and rested in a supine position for a minimum of 10 min.

The measurements were conducted on the right common carotid and the right femoral artery. Participants were instructed to maintain silence during the measurements. Distances were measured directly with a tape measure: From the suprasternal notch to the femoral artery, and from the carotid artery to the suprasternal notch. The difference between these two measurements was calculated[6]. PWV was calculated by determining the pulse transit time and the distance between the two measurement sites. An electrocardiogram was recorded concurrently to align the pressure waves. All measurements were performed by the same experienced operator.

The radial pressure waveforms were calibrated using sphygmomanometric systolic and diastolic blood pressures taken from the brachial artery. The augmentation index of the central (aortic) pressure waveform was then estimated to assess wave reflection. This index reflects the interplay between wave reflection magnitude and arterial stiffness, with arterial stiffness influencing the timing of wave reflections.

The concentration of total phenols was estimated according to the Folin-Ciocalteu method[9]. For the definition of the plasma total antioxidant capacity (TAC), the 2,2-diphenyl-1-picrylhydrazyl method was used. It is based on the use of the free radicals used for assessing the potential of substances to serve as hydrogen providers or free-radical scavengers. The results were shown as a percentage of antioxidant ability according to the following equation: AC% = (AT–AD) × 100/AT, where AT (means) represents the blind absorption to photometry to 520nm and AD represents sample absorption to photometry to 520 nm.

Finally, the concentration of carbonyls was detected based on their reaction with 2,4-dinitrophenylhydrazine to form a 2,4-dinitrophenylhydrazone, and it was expressed as nmol/mL[10].

The normality of the distribution of continuous variables was assessed by the Shapiro-Wilk test. Continuous variables that follow a normal distribution were presented as mean ± SD. In the case of non-normally distributed continuous variables, data were presented as median with the interquartile range. Categorical variables were displayed as percentages. For the over-time comparison of normally continuous variables, we used the parametric paired sample t-test and the non-parametric Wilcoxon signed ranked test, accordingly. For the assessment of differences in cfPWV across the different CKD stages, a repeated measures analysis of variance was conducted, using the Benferroni correction.

All statistical calculations were performed using SPSS software (version 25.0; SPSS Inc., Chicago, IL, United States). All reported P values were based on two-sided hypotheses, with a P value of 0.05 being considered statistically significant.

The alterations in arterial stiffness measurements following the administration of flavonoids are presented in Table 3. After the intervention, there was a statistically significant reduction in the peripheral systolic blood pressure by 14 mmHg (P < 0.001). The mean cfPWV at baseline was estimated at 8.9 m/s (6.7, 11.8) and was markedly reduced to 8.2 m/s (5.1, 9.2); a difference that was statistically significant (P < 0.001) (Figure 1). The central pulse pressure was reduced after flavonoid administration from 59 mmHg (44, 69) at baseline to 48 mmHg (37, 60) at the end of the study (P = 0.003).

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Figure 1 Scatter plot demonstrating the significant differences in carotid-femoral pulse wave velocity at baseline (first visit) and at the end of the study (3 months later). cfPWV: Carotid-femoral pulse wave velocity.

We also examined whether the alteration of cfPWV differed proportionally according to the stage of CKD. The analysis showed that the changes in cfPWV differed significantly among the CKD stages (P = 0.037) (Figure 2). After pairwise analysis among the CKD stages and the alteration of cfPWV, a statistically significant difference was noted between stages I and IIIb-IV (P = 0.042).

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Figure 2 Boxplots of the alteration in carotid-femoral pulse wave velocity according to chronic kidney disease stage. cfPWV: Carotid-femoral pulse wave velocity; CKD: Chronic kidney disease.

The changes in parameters of oxidative stress before and after flavonoid administration are displayed in Table 4. After the flavonoid administration, a reduction of plasma proteinic carbonyl was observed (73.50 ± 18.65 nmol/mL at baseline vs 52.54 ± 25.04 nmol/mL at the 3-month follow-up, P < 0.001). Moreover, the total phenolic compound concentration was significantly enhanced [25.11 mg/mL (16.95, 30.29) at baseline vs 31.91 mg/mL (30.49, 47.51) at the 3-month follow-up, P = 0.001]. The TAC also appeared augmented after the intervention [3.55% (1.15, 6.38) at baseline vs 12.51% (6.26, 17.66) at the 3-month follow-up, P = 0.013].