This study was conducted as a randomized, double-blind, placebo-controlled, parallel-group trial with an allocation ratio of 1:1 over an 8-week intake period. No methodological changes were made after study initiation.

Paid volunteers were recruited for this study. Healthy adults who met the inclusion criteria and did not meet any of the exclusion criteria were eligible to participate. Before study initiation, prospective participants received a full explanation of the study and provided written informed consent.

The inclusion criteria were as follows: 1) Healthy females aged 40 to 59 years-old.; 2) Subjects who aware of coldness.; 3) Subjects who can make self-judgment and are voluntarily giving written informed consent.

The exclusion criteria were as follows: 1) Subjects who were diagnosed with serious disease (e.g., diabetes, liver disease, kidney disease, digestive disease, heart disease, respiratory disease and/or peripheral vascular disease); 2) Subjects who had a gastrointestinal surgery; 3) Subjects with strange finger conditions at measurement points (like inflammation or injury); 4) Subjects with a disease currently under treatment; 5) Subjects with food allergies; 6) Subjects with anemic; 7) Women who are/might be pregnant or lactating during the current study periods; 8) Subjects who play high intensity sports and/or are on a diet; 9) Subjects who have an extremely irregular eating habit, are shiftworker and/or midnight-shift worker; 10) Subjects who can’t stop using functional foods (including Food for Specified Health Uses or Foods with Function Claims) and/or Specified quasi-drugs during the current study periods; 11) Subjects who are under treatment with medications (including OTC and/or pre-scribed medications); 12) Subjects who have excessive alcohol intake more than approximately 60 g/day of pure alcohol equivalent, habit of drinking not less than 5 days a week, or can’t stop drinking from the days before each measurement; 13) Subjects who smoke 21 or more cigarettes a day, or cannot quit smoking during from waking to inspection completed; 14) Subjects who can’t stop their manicure or false nail during the current study periods; 15) Subjects who are participating in other studies or planning to participate at the start of the current study; 16) Subjects who are judged as unsuitable for the current study by the investigator for other reasons.

This study was approved by “The Ethics Committee of Miura Clinic, Medical Corporation Kanonkai” (Chair: Masaaki Nishi) (approval date: January 30, 2025) and was conducted in accordance with the “Declaration of Helsinki” (amended October 2013) and “Ethical Guidelines for Medical and Biological Research Involving Human Subjects” (partially revised March 27, 2023). The study was performed under physician supervision at the Miura Clinic. The study protocol was registered with the clinical trial registry operated by the University Hospital Medical Information Network (UMIN000057143; trial name: “A Study on the Effect of Food Containing Plant Extract on Capillary Blood Flow-A Randomized, Double-blind, Placebo controlled, Parallel-group Study-”).

During the study, the intervention consisted of continuous intake of the test food for 8 weeks. The active food was prepared by tableting a mixture of pine bark extract (Toyo Shinyaku Co., Ltd.), powdered reduced maltitol, microcrystalline cellulose, sucrose fatty acid ester, and silicon dioxide. For the placebo food, the pine bark extract in the active formulation was replaced with caramel coloring to ensure indistinguishable appearance from the active food.

The daily intake of both the active food and placebo food was two tablets (250 mg × 2 tablets). During the intake period, participants consumed two tablets of the assigned test food once daily with water or lukewarm water. The caloric and nutrient compositions per recommended daily intake are shown in Table 1. The amounts of pine bark-derived procyanidins B1 and B3 in the active food were 2.4 mg per day.

Table 1.Analysis of nutrient composition values of test food.

a. Calorie conversion factors: protein, 4; fat, 9; carbohydrates, 4. b. Nitrogen protein conversion factor: 6.25.

The primary outcome was capillary blood flow velocity, and the secondary outcome was capillary congestion. These parameters were evaluated at three time points: before intake (baseline), after 4 weeks, and after 8 weeks. No changes to the outcome measures were made after study initiation. Capillary blood flow velocity was measured at the nailfold of the ring finger on the non-dominant hand in the seated position after a rest period, using a capillary imaging device (GOKO Bscan-ZD; GOKO Imaging Devices Co., Ltd.). Image analyses were performed using analysis software (GOKO-VIP and GOKO Measure Plus; GOKO Imaging Devices Co., Ltd.). Capillary congestion was evaluated by a physician using a 100-mm visual analog scale (VAS) based on capillary images, with the left end labeled “not congested at all” and the right end labeled “very congested.” The VAS score was quantified as the distance from the left end.

Participants were provided with a diary and instructed to record daily throughout the intake period the following information: (1) test food intake status; (2) physical condition; (3) intake of medicines and health foods; and (4) alcohol consumption, exercise, and smoking status.

The target sample size was determined based on a previous report in which continuous intake of pine bark extract for 8 weeks (2.4 mg/day as pine bark-derived procyanidins B1 and B3) improved skin elasticity through improved blood flow as the mechanism of action because no randomized controlled trials (RCTs) existed evaluating capillary blood flow in healthy adults after administration of pine bark extract at the typical intake levels found in Japanese functional foods. The calculation used the change in skin elasticity from baseline (placebo food group: −0.073 ± 0.015; active food group: −0.003 ± 0.011) [16], with a significance level of 0.05 and statistical power of 80%. To account for potential dropouts and discontinuations, the sample size was set at 25 participants per group (50 participants in total).

Paid volunteers were recruited, and participants were enrolled by the principal investigator according to the inclusion and exclusion criteria. A statistical analyst used computer-generated random numbers to allocate participants by block randomization (block size of four), with age, body mass index (BMI), and capillary blood flow velocity used as adjustment factors. An allocation manager who was not directly involved in the study assigned the randomized participants to the active food group or placebo food group. The allocation manager prepared and sealed a key code table listing the allocation results and kept it sealed until the analysis population was determined, thereby ensuring blinding of all study personnel except the allocation manager. Test food were individually packaged as two-tablet sachets in plain aluminum bags and distributed to ensure blinding of both participants and intervention providers.

During the study, participants were instructed to avoid substantial changes in lifestyle habits, including diet, alcohol consumption, exercise, bedtime, and smoking; refrain from excessive exercise and extreme dieting or overeating; and, in principle, avoid the use of medicines (including OTC and prescription medicines), designated quasi-drugs, and health foods (including Foods for Specified Health Uses and Foods with Function Claims). Participants were also instructed, in principle, to avoid topical medicines and to consult the testing institution in advance if unavoidable intake was necessary due to poor physical condition. Participants were required to abstain from alcohol consumption and excessive exercise from the day before until completion of each visit; to finish eating by 22:00 on the day before each visit and fast until the end of the visit (water or lukewarm water permitted); to avoid pungent and stimulatory foods (e.g., curry, chili pepper, ginger, and Tabasco) at dinner on the day before each visit; to refrain from smoking from waking until the end of testing on visit days; and not to take the test food on visit days before coming to the clinic. In addition, participants were instructed to record the amount of water consumed from waking on visit days and consume a similar amount at subsequent visits, as well as to avoid bathing (including showers), devices that could excessively warm or cool the body, and massages before clinic visits. Except in emergency situations, participants were permitted to use medicines only with approval from the principal investigator or sub-investigator.

The population analyzed was a Per-Protocol Set (PPS). Repeated-measures analysis of variance was performed to assess group-by-time interactions. Between-group comparisons at each visit were conducted using two-tailed unpaired t-tests for both measured values and changes from baseline, and end-of-intake results were evaluated. The significance level was set at 5% for all tests. Statistical analyses were performed using IBM SPSS Statistics version 28. Participant background characteristics are presented as mean ± standard deviation, whereas other data are presented as mean ± standard error. No additional analyses were conducted.

A total of fifty participants were enrolled. No dropouts occurred after randomization, and the allocated interventions were implemented in 25 participants per group. During the study period, one participant in the active food group discontinued participation for personal reasons unrelated to the test food, resulting in 49 completers. After completion of the study, five participants met the rejection criteria, leaving 44 participants for analysis. The reasons for rejection were violations of study precautions during the study period (active food group, n = 1; placebo food group, n = 4). Analyses were performed according to the original group allocation.

Recruitment through completion of follow-up was conducted from March 2025 to July 2025, and the study was terminated after all participants completed follow-up. The baseline characteristics of the analysis population are presented in Table 2, and the flow of participants from enrollment to analysis is shown in Figure 1.

Figure 1. Flow diagram of progress through phases of randomized, double-blind, placebo-controlled, parallel-group study.

Table 2. Participant characteristics.

Values are expressed as means ± SDs. No significant difference was observed.

Capillary blood flow velocities are presented in Table 3. A significant group-by-time interaction was observed. Between-group comparisons of both the measured values and changes from baseline showed that, at 8 weeks, the measured value and change from baseline were significantly higher in the active food group than in the placebo food group (measured value, P = 0.040; change from baseline, P = 0.011).

Table 3. Test results for capillary blood flow velocity.

Values are expressed as means ± SEs. *Significantly different from the placebo food group (P< 0.05).

Capillary congestion results are presented in Table 4. A significant group-by-time interaction was also observed. Between-group comparisons indicated that, at 8 weeks, the change from baseline was significantly lower in the active food group than in the placebo food group (P = 0.025).

Table 4.Test results for capillary congestion.

Values are expressed as means ± SEs. *Significantly different from the placebo food group (P < 0.05).

During the study period, adverse events such as sore throat, rhinorrhea, and cold-like symptoms were reported. All events were judged by the principal investigator to be unrelated to the test food. No serious adverse events were observed.