A study on the effect of food containing pine bark extract on vascular endothelial function
―A randomized, double-blind, placebo-controlled, parallel-group study―

Introduction

The vascular endothelium is the innermost cell layer of the blood vessel and functions to maintain efficient blood transport to peripheral organs by controlling contraction and expansion of blood vessels, blood coagulation, inflammation, oxidation, etc. ¹⁾. The functional degradation of vascular endothelium (endothelial dysfunction) is linked to cardiovascular events and is considered to be present at the preclinical stage of arteriosclerosis ²⁾. A community-based cohort study revealed that increasing age, BMI, smoking, and blood pressure were especially associated with endothelial dysfunction ³⁾. On the other hand, it has been reported that consumption of antioxidants such as polyphenol ⁴⁾ or vitamin E ⁵⁾ improves vascular endothelial function. Therefore, improving vascular endothelial function by reconsidering lifestyle and using functional food materials is thought to be important for maintaining healthy blood vessels.

Pine bark extract (PBE) is an extract from the bark of the pine tree that is rich in polyphenols, represented by oligomeric proanthocyanidins ⁶⁾. It is known that the major components of the procyanidin dimer contained in pine bark are procyanidin B1 and procyanidin B3 ⁷⁾. PBE has been reported to have various effects such as antioxidant ⁸⁾ and antihypertensive effects ⁹⁾, blood flow improvement ¹⁰⁾, and vascular enhancement ¹¹⁾. Concerning the effect on vascular endothelial function in subjects with risk factors of endothelial dysfunction (high fasting blood glucose, high blood pressure, or climacteric symptoms), it has been reported that consumption of tea beverages containing PBE significantly improves vascular endothelium function ¹²⁾.

In the present study, to investigate the effect of food containing PBE in healthy subjects on vascular endothelial function, we conducted a randomized, double-blind, placebo-controlled, parallel-group study using tablet-form food containing PBE.

Materials and methods

Test food

A tablet-form food containing PBE (TOYO SHINYAKU Co., Ltd.), maltitol, microcrystalline cellulose, sucrose esters of fatty acids, and silicon dioxide was used as the active food (PBE). As the control food, in order to make it indistinguishable from PBE, a food containing an equivalent amount of caramel pigment instead of pine bark extract in PBE was used (placebo). To maintain blinding of the subjects and the care providers, PBE and the placebo were distributed with plain aluminum packets sealed individually containing daily intake: two tablets (0.25 g per tablet) per packet.

Table 1 shows the analysis values of nutrient composition. The amount of PBE-derived procyanidin, which is the sum of procyanidin B1 and procyanidin B3 contained in PBE, was calculated to be 2.4 mg per daily intake.

Subjects

The target sample size in the test was set to 40 subjects (20 subjects per group) based on the sample size (significance level 0.05, power 0.80) calculated from a previous report ¹²⁾. Subjects recruited were paid volunteers, and the investigator enrolled 40 subjects (16 men and 24 women) who met the inclusion criteria and did not violate the exclusion criteria.

Inclusion: (1) Healthy men and women aged 40 to 64 years old. (2) Subjects with a BMI < 30. (3) Subjects who can make self-judgment and voluntarily give written informed consent.

Exclusion: (1) Subjects who contract chronic disease and are under treatment.(2) Subjects who cannot stop using medicines, quasi-drugs, supplements, and/or functional foods (including Food for Specified Health Uses or Foods with Function Claims) affecting blood pressure, lipid metabolism, blood sugar level, body fat, and so on during the test periods.(3) Subjects with a history and/or presence of serious diseases (e. g., liver disease,

kidney disease, digestive disease, heart disease, respiratory disease, endocrine disease, thyroid disease, adrenal disease, and/or metabolic disease).(4) Subjects who had a history of digestive diseases and/or underwent a surgical procedure for digestive disease.(5) Subjects who were judged as unsuitable for the current study by screening tests.(6) Subjects who were diagnosed with arrhythmia by screening tests.(7) Subjects who are shift workers and/or whose shift encompasses midnight.(8) Subjects who declared allergic reactions to ingredients of test foods.(9) Subjects who had excessive alcohol intake of > 60 g/day of pure alcohol equivalent or habit of drinking not less than five days a week.(10) Subjects with a history of and/or current drug addiction and/or alcoholism.(11) Subjects unable to cease drinking for two days before each measurement.(12) Subjects who were pregnant or planned to become pregnant or breastfeed during the study period.(13) Subjects who had donated over 200 mL of blood and/or blood components within the last one month prior to the current study or over 400 mL of blood and/or blood components within the three months prior to the current study.(14) Subjects who were planning to participate and/or had participated in other clinical studies within the month prior to the commencement of the current study.(15) Subjects who were judged as unsuitable for the current study by the investigator for other reasons.

Study design

It was a randomized, double-blind, placebo-controlled, parallel-group study. The study consisted of a pre-observation period (two weeks) and a test period (eight weeks) for a total of ten weeks. The investigator enrolled the subjects in this test according to the inclusion and exclusion criteria, and the statistical analysis manager randomized the subjects into two groups by a block-randomized method with a 1:1 allocation using random block sizes of four with sex, age, and FMD%. The controller not directly involved in this study assigned the two groups to the PBE group and the placebo group. In addition, to ensure blindness, the controller prepared and sealed a table (key code) describing the assignment result. The container was kept closed until the key code was disclosed after determining the subjects included in the statistical analysis.

During the test period, the subjects in each group were required to consume the allotted food: one packet (two tablets) once daily with cold water or lukewarm water. The subjects were allowed to chew and consume the test food.

The subjects were instructed in precautionary notes during this test not to use drugs or health foods that may affect blood pressure, lipid metabolism, blood sugar level, body fat, etc., not to change their lifestyle from before the start of the test, not to consume a large amount of alcohol, and not to participate in other studies. In addition, the subjects were prohibited from consuming alcohol for two days before each examination, exercising hard from the day before each examination until the end of the examination, and from eating, drinking (other than drinking water), or smoking from 21:00 the day before each examination until the end of the examination.

The protocol of this study was registered with the UMIN Clinical Trial Registry (ID: UMIN000036602). The study was reviewed and approved by the Ethical Committee of Kobuna Orthopedics Clinic (Chairman: Toshio Kawata, Date of approval: April 19, 2019) and conducted under the supervision of physicians in accordance with the World Medical Association Declaration of Helsinki 64th WMA General Assembly, Fortaleza, Brazil, October 2013, and the Ethical Guidelines for Medical and Health Research Involving Human Subjects. The subjects were fully informed of the study, and informed consent to participate in the study was obtained in writing. The study was conducted in Maebashi North Hospital (April 2019 to August 2019). There were no changes to the trial protocol after the trial commenced.

Examination

①  Diet, living conditions, physical condition survey
Subjects were asked to keep their daily record from one week before intake until the end of the test period. Subjects were distributed with subject diaries and were asked to fill out the following survey items every day from one week before the start of intake until the end of the test period: (1) test food intake, (2) changes in physical condition, (3) changes in living conditions, (4) bedtime, (5) alcohol/supplements/health food intake, (6) smoking habit, (7) use of drugs and quasi-drugs

②  Physical Examination
During the physical examination, height, and body weight (BMI) and vascular endothelial function were evaluated. The vascular endothelial function in this study was evaluated by flow-mediated dilation (FMD) with ultrasonic inspection equipment before intake (baseline), four weeks after intake (4w), and eight weeks after intake (8w) as the primary outcome. Briefly, after measuring the resting arterial vascular diameter of the upper arm, the forearm was squeezed with a cuff for 5 min, and the maximum arterial vascular diameter after releasing the cuff was measured. The examination values (FMD%) were calculated as the percentage change in the maximum arterial vessel diameter from the resting arterial vessel diameter.

③  Interview
Interviews by a physician were conducted before intake, four weeks after intake, and eight weeks after intake.

Statistical Analysis

The primary outcome of this study was FMD%. The effect of the test foods on FMD was evaluated using the per-protocol set (PPS). The interaction between the PBE group and the placebo group was compared by two-way repeated measures analysis of variance. If the interaction was observed, the between-group comparison at each examination of both measured values and change from baseline was performed with a two-tailed unpaired t-test. All statistical analyses were performed using the Statistical Package for Social Science (PASW Statistics18, IBM, NY, USA) and statistical significance was defined as p < 0.05. The subject characteristics were expressed as mean ± standard deviation (SD), and the other data were expressed as mean ± standard error (SE).

Result

Subjects

40 subjects (male=16, female=24) were included in this study. After randomly allocated to either the PBE or placebo group (20 subjects in each), all 40 subjects received allocated intervention. All 40 subjects completed an 8-week consumption. Five subjects (male=1, female=4) were excluded because of ingestion of foods that affected FMD (any form) during the test period. Thus, 35 subjects (male=15, female=20) were included in the analyses. The CONSORT flow diagram of the study subjects is shown in Fig 1. The subject characteristics are shown in Table 2.

Efficacy for FMD

The FMD results are shown in Table 3. Between the PBE group and placebo group, the interaction of FMD% was observed (p<0.05), and there were significant increases in the PBE group compared to the placebo group in both measured values in 8w and change from baseline to 8w (p<0.05 in each).

Discussion

We conducted a randomized, double-blind, placebo-controlled, parallel-group study in healthy males and females aged 40 to 64 years old, using food containing PBE or placebo, measuring FMD% as the outcome for vascular endothelial function.

The results showed that FMD% in the PBE group was significantly higher than that in the placebo group after 8 weeks of consumption. In a previous study, the effect of PBE on vascular endothelial function in subjects with risk factors of endothelial dysfunction was shown by plethysmography ¹²⁾. In the FMD in this study, changes in vessel diameter caused by ischemic reactive hyperemia (increased blood flow after opening compressed blood vessels, in which blood flow stopped temporarily) were measured ¹³⁾. In a previous plethysmography study, changes in the volume of blood flow caused by ischemic reactive hyperemia were measured ¹²⁾. Both FMD and plethysmography were used to evaluate vascular endothelial function¹⁴⁾. Superior vascular endothelial function, which expands blood vessels caused by blood flow, is associated with higher FMD% ¹³⁾. Hence, we conclude from the results of this study that, in healthy adult males and females, PBE improved vascular endothelial function.

Maintaining a healthy vascular endothelium is thought to be important because endothelial dysfunction can degrade normal vascular tone and structure and lead to the progression of atherosclerosis ¹⁴⁾. ApoE-deficient mice used as a model for atherosclerosis are known to have very high blood cholesterol levels, and in ApoE-deficient mice, it has been reported that administration of PBE suppressed the enlargement of arteriosclerotic lesion area in blood vessels by lowering blood cholesterol ¹⁵⁾. LDL, one of the lipoproteins that carry cholesterol in the body, was reported to damage vascular endothelial function by being oxidized¹⁶⁾. In a cohort study, the level of LDL oxidization in blood was reported to be related to a decrease in FMD% ¹⁷⁾. Regarding the effect of PBE on LDL oxidation in humans, following a single dose of PBE, an enhancement of the antioxidant defense capacity of LDL was observed ¹⁸⁾. These results suggest that blood cholesterol and lipoprotein may be involved in the improvement of vascular endothelial function by PBE.

Conclusion

These results indicate that food containing PBE leads to an improvement in vascular endothelial function in healthy subjects.

Conflicts of interest

This study was funded by TOYO SHINYAKU Co., Ltd. The test food was supplied by TOYO SHINYAKU Co., Ltd. The co-author doctor did not receive any research expenses or rewards from TOYO SHINYAKU Co., Ltd.

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