Evaluation of Urolithin A and Fisetin on Improving Sleep and Aging Biomarkers

We will conduct blood routine and urine routine tests, and report the number of people with abnormal tests results. The tests indicators include: Red Blood Cell Count (RBC), Hemoglobin (Hb), Hematocrit (Hct), White Blood Cell Count (WBC), Neutrophils (Neut# and Neut%), Lymphocytes (Lym# and Lym%), Monocytes (Mono# and Mono%), Platelet Count (PLT), Mean Corpuscular Volume (MCV), Mean Corpuscular Hemoglobin (MCH), Mean Corpuscular Hemoglobin Concentration (MCHC), Urine Physical Characteristics (Appearance, specific gravity, pH), Urine Biochemical Analysis (Protein, Glucose, Occult blood, Leukocytes, Erythrocytes, and Bacteria). Any abnormal results will be marked on the laboratory report.

Plasma samples collected at different time points will be classified and labeled with two sets of each sample. An automated hematology analyzer in the hospital will be used.The degree of Insulin Resistance (IR) and sensitivity will be calculated by the Homeostatic Model Assessment (HOMA-IR) using the formula:

HOMA-IR=(Fasting Insulin (μIU/mL) × Fasting Glucose (mmol/L) )/22.5

Frailty status will be evaluated using the Fried Frailty Phenotype. The Fried Frailty Phenotype includes exhaustion, weight loss, low physical activity, slow gait speed, and grip strength measurement.For consistency with previous literature, participants who meet 3 or more criteria will be diagnosed as frailty, those who meet 1 or 2 criteria will be classified as prefrailty, and those who meet none of the criteria will be considered as nonfrailty.

The Fatigue Severity Scale will be used to measure the fatigue score of participants.Evaluates fatigue severity over the past week through 9 items, with each item scored from 1 (strongly disagree) to 7 (strongly agree). The total score ranges from 9 to 63. Higher scores indicate more severe fatigue.

The Patient Health Questionnaire-9 (PHQ-9) is a brief self-report scale commonly used in clinical practice to assess depressive symptoms. It consists of 9 questions, primarily asking about the experience of depressive symptoms over the past two weeks and their frequency. Each response is set from 0 (not at all) to 3 (nearly every day), with a total score range of 0 to 27. Higher scores indicate more severe depressive symptoms.

The Generalized Anxiety Disorder Scale (GAD-7) is a brief self-report scale used to assess anxiety symptoms over the past two weeks. It includes 7 questions, with responses rated from 0 to 3. The total score ranges from 0 to 21. Higher scores indicate more severe anxiety symptoms.

The levels of immunoglobulin A (IgA), immunoglobulin G (IgG), and immunoglobulin M (IgM) in plasma samples will be measured using a fully automated biochemical analyzer, with all units expressed as g/L.

The DNA methylation clock is an algorithm that integrates DNA methylation measurements across the genome to quantify biological age changes. It is considered a highly accurate molecular marker of human biological age. This clock can detect DNA methylation age reversal up to three months in advance, thus showing potential for quantifying biological aging, assessing expected lifespan, or evaluating the efficacy of interventions.

The Profile of Mood States-Short Form (Chinese Version) will be used to measure the energy score of participants. It measures energy levels using a 5-point scale ranging from 0 (never) to 4 (extremely), total score ranges from 0 to 160. Higher scores indicate greater vitality.

Plasma interleukin levels are primarily used to reflect the inflammatory and immune status. In this study, the concentrations of interleukins (IL-1β, IL-6, IL-8, IL-10, IL-11, IL-33, IL-23R, and IL-17A) will be measured in fasting plasma samples of participants using ELISA kits, with units expressed as pg/mL. Notably, IL-11, IL-33, IL-23R, and IL-17A have recently gained attention as key molecules associated with aging.

Stool samples (10g) from participants will be collected. These samples will be rapidly frozen in dry ice to stop metabolic reactions. DNA will be extracted from the frozen stool samples using the GNOME DNA isolation kit, and 16S rRNA sequencing will be performed using the IlluminaHiSeq2500 platform. 16S rRNA gene sequencing is a common method for analyzing the composition of the gut microbiota by high-throughput sequencing technology, widely used to assess the diversity and structural changes of the gut microbiota. The process includes DNA extraction from stool samples, amplification of specific regions of the 16S rRNA gene (such as the V3-V4 regions), and sequencing of the amplicons using a high-throughput sequencing platform (e.g., Illumina). Data quality control and microbiome structure analysis will be performed using bioinformatics tools (such as QIIME, Mothur and USEARCH) to assessing diversity.

D-lactic acid level (μmol/L) in peripheral blood will be measured as an indicator of intestinal mucosal barrier integrity. Under physiological conditions, circulating D-lactic acid remains at trace concentrations in micromolar range. Elevated serum level reflects increased intestinal permeability due to mucosal damage from inflammation or ischemia, allowing bacterial-derived D-lactic acid to enter circulation.

Nicotinamide adenine dinucleotide (NAD+) plays a crucial role in cellular energy metabolism, redox reactions, and aging, which will be evaluated to monitor metabolic status and intervention efficacy. Methods include high-performance liquid chromatography (HPLC), mass spectrometry (MS), HPLC-MS/MS, and ELISA. Although ELISA has relatively lower accuracy, it is convenient to use. For this study commercial ELISA kits will be used.

The World Health Organization Quality of Life Brief Version (WHOQOL-BREF) will be administered at baseline (week 0) and the end of intervention (week 12) to assess changes across four domains: physical health, psychological status, social relationships, and environmental factors. This instrument assesses the overall impact of the intervention on participants' quality of life.The standardized scores range from 0 to 100, with higher scores indicating a better quality of life.

Arterial stiffness will be assessed as a secondary outcome using an automated, noninvasive oscillometric vascular screening system. Bilateral brachial and ankle blood pressures and pulse waveforms will be recorded to derive brachial-ankle pulse wave velocity (baPWV) and the ankle-brachial index (ABI), along with peripheral blood pressure and heart rate, following standardized resting conditions and the manufacturer's recommended procedures.

Cardiopulmonary exercise testing (CPET) will be conducted using an incremental, symptom-limited protocol on an electronically braked cycle ergometer. Participants will complete standardized pre-test screening and resting measurements, followed by a brief warm-up and stepwise increases in workload until volitional exhaustion or predefined termination criteria are met. Breath-by-breath gas exchange will be measured continuously to derive oxygen uptake (VO₂), carbon dioxide production (VCO₂), and minute ventilation (VE), alongside concurrent monitoring of heart rate, blood pressure, and electrocardiography. Key outcomes will include peak oxygen uptake (VO₂peak) and ventilatory threshold-based indices, with test procedures and interpretation aligned with established CPET guidelines.