2.3. Study Protocol, Procedure, Instrumentation, and Measurements
COLD, HEAT EFFECT
The study was conducted between January and March 2022 to eliminate the seasonal heat effect on the study measurements. As per protocol, the participants arrived at the designated clinical study facility at noon (i.e., 12:00 h) after having abstained from lunch or any food and beverage for at least 2 h before the start of the experimental measurements. Participants were also asked to abstain from exercise, caffeine, and alcohol for at least one day. Each measurement was performed without lunch between 12:00 and 14:30 h to avoid any influence of diurnal changes in the body temperature.Participants wore light, comfortable clothing for each measurement to eliminate the possibility of the clothing’s influence on body surface temperature and other measured parameters. The participants were quietly seated in the measurement chamber for 15 min to acclimate them to the constant ambient conditions within the measurement room. The mean temperature and relative humidity in the measurement chamber were 25 ± 1 °C and 30.0 ± 5%, respectively. After becoming accustomed to the experimental chamber conditions, the participants were given the two study capsules, each containing either a placebo or varied doses (i.e., 300 mg and 150 mg) of HEPT7G/βCD inclusion complex with 100 mL of distilled water for oral ingestion. The doses and timing of their intake were selected because they can produce a significant increase in plasma hesperetin metabolite levels within the physiological range. After 45 min from time of intake, the measurements were carried out with the study participants calmly resting in a spine position throughout each testing period. The order of the experiments was randomized at intervals of three days during the washout period. Whereas, for the initial screening, the same protocol was performed except that the participants were given only 100 mL of distilled water as pre-ingestion.The peripheral skin temperature of the middle finger on the left hand was measured using a Type-K thermometer equipped with a sensor probe sealed with non-permeable tape. After that, the left hand was immersed up to the ulnar and radial styloid in a tank containing cold water precisely maintained at 15 ± 2 °C by means of a cooling system controller, and a pump continuously stirred the water. The cooling stress was maintained for one minute, and then the hand was removed from the cold water tank, and a spontaneous rewarming phase ensued with continued measurement of the peripheral skin temperature of the finger at regular intervals for the following 30 min upon completion of the cooling stress to assess the temperature recovery, i.e., rewarming (During the visit to the clinical trial facility, the height was measured using a calibrated portable stadiometer with an accuracy of 0.1 cm (Seca, Japan), and body mass (weight) was measured using a digital weighing scale with a sensitivity of 0.1 kg (DP-7200, Daiwa Seihei Co., Ltd., Tokyo, Japan) for each participant. The body mass index was estimated as body weight in kilograms divided by height in meters squared. The blood pressure and pulse rate were recorded using a sphygmomanometer (a digital blood pressure monitor) equipped with a double air bladder and an irregular pulse wave detection function (Omron HEM-1000; Omron Corporation, Kyoto, Japan). All measured variables were recorded by a data logger.