The Effect of Food Containing Pueraria Flower Extract on Energy Metabolism
―A Randomized, Double-blind, Placebo-controlled, Parallel-group Study―

Introduction

Obesity can induce hyperglycemia, dyslipidemia, and high blood pressure, and has been associated with the onset of lifestyle-related diseases.¹⁾ Therefore, prevention of obesity is important for health promotion. Obesity is defined as an excessive accumulation of body fat,^{1, 2)} which is caused by an imbalance between energy intake and energy expenditure (EE), and it is important to maintain this balance to prevent obesity.³⁾
Increasing EE through physical activities (exercise or nonexercise activities) in daily life has been shown to be an effective method for preventing obesity.^{1, 4)} Recent clinical trials have shown that capsinoids and Kaempferia parviflora extract from food can efficiently increase EE, ^5-7） and have anti-obesity effect of reducing abdominal fat.^{8, 9）}
Kudzu are perennial climbing plants belonging to the Fabaceae family. The kudzu flower is known to be a rich source of isoflavones.¹⁰⁾
Pueraria flower extract (PFE) is a hot-water extract of the kudzu (Pueraria thomsonii) flower. Several clinical trials have shown that PFE has anti-obesity effects such as reducing abdominal fat.^11)-15)　The active ingredient of PFE causing the anti-obesity effect is thought to be isoflavones, namely tectorigenin, tectoridin, and tectorigenin 7-O-xylosylglucoside.^{14, 16)} Furthermore, PFE appears to exert its effects by suppressing lipogenesis in the liver and promoting lipolysis in white adipose tissue (WAT) and thermogenesis in brown adipose tissue (BAT).17) Moreover, it has been reported that isoflavones from PFE increase EE, and exert anti-obesity effects in animal model.¹⁶⁾ Based on these previous research results, intake of Pueraria thomsonii flower isoflavones tectorigenin, tectoridin, and tectorigenin 7-O-xylosylglucoside (PFIF) is thought to exert anti-obesity effects such as reduction of abdominal fat by increasing EE. However, we have not verified the effects of PFIF oral intake on energy metabolism in humans.
Therefore, in the present study, we performed a randomized, double-blind, placebo-controlled, parallel-group study in healthy males ingesting foods containing PFE to investigate the effects of PFIF oral intake on energy metabolism in healthy adults.

Materials and methods

Test food

Tablets containing PFE (TOYO SHINYAKU Co., Ltd.), maltitol, sucrose esters of fatty acids,silicon dioxide, and cellulose were used as the test food (active food). The placebo food consisted of tablets containing maltitol instead of PFE, and caramel color to render the tablet types indistinguishable. To maintain blinding of the study subjects and the care providers, the active and placebo foods were distributed with plain aluminum packets sealed individually containing daily intake, 2 tablets (0.25 g per tablet) per packet. The nutritional compositions of the test foods per daily intake are shown in Table 1. PFIF contained in the active food was calculated to be 35 mg per daily intake.

Subjects

The target sample size in the present study was set to 80 subjects (40 subjects per group) based on the sample size (significance level 0.05, power 0.80) calculated using visceral fat area as an index referred to previous reports that confirmed the anti-obesity effects of PFE,13), 14) and taking into consideration potential future dropouts or discontinuation. Subjects were recruited as paid volunteers, and the investigator enrolled 80 men who met the following inclusion criteria and did not violate the exclusion criteria.

Inclusion criteria：(1) Healthy males aged 30 to 39 years-old; (2) Subjects whose body mass index (BMI) ≥ 20 and < 25; (3) Subjects who could make self-judgments and voluntarily provide written informed consent.

Exclusion criteria：(1) Subjects who had chronic disease and were under treatment; (2) Subjects who had a history of and/or contracted serious diseases (e.g., liver and kidney disease, digestive disease, heart disease, respiratory disease, endocrine disease, thyroid disease, adrenal disease, and/or metabolic disease); (3) Subjects who were suspected to be diabetic, judged to have hypertension and/or have symptoms of anemia by screening tests; (4) Subjects who declared allergic reaction to ingredients of test foods; (5) Subjects who could not stop using medicines, quasi-drugs, supplements and/or functional foods (including Food for Specified Health Uses or Foods with Function Claims) affecting energy metabolism during test periods; (6) Subjects who displayed excessive alcohol intake (more than approximately 60 g/day of pure alcohol equivalent) or a habit of drinking not less than 5 days a week; (7) Subjects who had a history of and/or showed drug addiction and/or alcoholism; (8) Subjects who could not stop drinking from 2 days before each measurement; (9) Subjects who had a habit of smoking; (10) Subjects who had a history and/or a surgical history of digestive disease affecting digestion and absorption; (11) Subjects who were judged as unsuitable for the current study by the investigator through the screening tests; (12) Subjects who were shift workers and/or a midnight-shift worker; (13) Subjects who did regular exercise for 2 or more days a week; (14) 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 last three months prior to the current study; (15) Subjects who were planning to participate and/or had participated in other clinical studies within the last one month prior to the current study; (16) Subjects who were judged as unsuitable for the current study by the investigator for other reasons.

Experimental design

A randomized, double-blind, placebo-controlled, parallel-group study (allocation ratio of 1: 1) was conducted, and consisted of a 1-week pre-observation period, an examination at the start of ingestion (0-week examination), an 8-week test period during consumption of test foods, and a post-ingestion examination (8-week examination).

The investigator enrolled the subjects in this study according to the inclusion criteria and the exclusion criteria, and the statistical analysis manager randomized the subjects into two groups by a block-randomized method using age, height, BMI, body fat, and estimated muscle mass as adjusting factors. The controller not directly involved in the study assigned the two groups to the active group and the placebo group. In addition, to ensure blindness, the controller prepared and sealed a table (key code) describing the assignment result, and kept the container closed until the key code was disclosed after the analysis for determining the subjects for further evaluation.

The test subjects took each test food 1 packet (2 tablets) once daily with water or lukewarm water during the test period. During the test period,, all the test subjects were instructed to have similar life style as before the study, except for instructions of this study, to avoid a large amount of alcohol consumption, supplements and/or functional foods affecting energy metabolism, and massive caffeine-containing beverage and food consumption for one week before the start of ingestion, not to participate in other studies, and to prohibit from engaging in alcohol consumption 2 days before each examination, from spontaneous exercise (except daily living) from 1 day before each examination, before each examination.

All the test subjects were given a food diary and a subject diary. The contents of the daily diet for 7 days before each examination were entered in a food diary. The following survey items were entered in the subject diary from 1 week before the start of ingestion to the day before the 8-week examination.

Survey item：(1) test food ingestion; (2) changes in physical condition; (3) changes in living conditions; (4) functional food, and supplement intake; (5) pharmaceutical use; (6) alcohol or caffeine consumption.

Evaluation of energy metabolism

In each of the 0-week and 8-week examinations, an exercise test was performed, and an evaluation of energy metabolism was performed by measuring respiratory metabolism. On the day before each examination, the subjects were given the same dinner by 22:00 and they were asked to stay at a designated accommodation. After dinner on the day before each examination, the subjects were prohibited from eating and drinking, except water.

On the day of the examination, the subjects were provided a breakfast (one rice ball) 1.5 h prior to the commencement of the exercise, and then provided active food or placebo food. Prior to exercise, the subjects wore a respiratory metabolism measurement mask, and put a heart rate measurement electrode on their chest, and then were asked to be in a sedentary position for 15 min (rest). Following that, the subjects exercised for 40 min, and then were again in a sedentary position for 15 min (recovery).

Respiratory metabolism was assessed according to the breath-by-breath method using a respiratory gas analyzer (AE-300S; Minato Medical Science Co., Ltd.). The exercise in the examination was a pedaling exercise using a bicycle ergometer (Aerobike 75XLII; Combi Wellness), and consisted of 2 min at 15 watts and 2 min at 25 watts, which served as a warm-up, and then 36 min at a constant load of 35 watts, making the total exercise time 40 min. The rotation speed of the pedal was 60 rpm under any load. The measurement was performed in a constant temperature (set to 20 ± 2 ° C) and humidity (set to 48 ± 5%) room. The subjects were prohibited from eating and drinking during measurements. Before and after the measurement, the physical condition was confirmed by interview, blood pressure and pulse rate measurement, and body temperature measurement.

Outcome measures of energy metabolism were EE18）and respiratory quotient (RQ), which were calculated from the volume of oxygen uptake (VO₂) and volume of carbon dioxide output (VCO₂), determined from the respiratory metabolism measurement using the following formula:

EE（kJ/day）＝（3.9×VO₂＋1.1×VCO₂）×4.184×60×24/1000

RQ＝VCO₂/VO₂

Study design

This study was registered in the UMIN clinical trial registration system (ID UMIN000036693). The study was reviewed and approved by the Ethical Committee of Nihonbashi Cardiology Clinic (Chairman: Mamoru Nishimuta, Date of approval: April 25, 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 from all subjects. The study was conducted in Meiji Gakuin University Yokohama Campus, and Shinagawa Season Terrace Health Care Clinic (from May, 2019 to August, 2019). There were no changes to study protocol after the study commenced.

Statistical analysis

Primary outcome measure was energy metabolism. Statistical analyses were performed in per-protocol set (PPS). To compare differences among the groups, mixed model and unpaired t-test for each examination was used. In addition, paired t-test was used to compare differences among periods (in each group). A p-value of < 0.05 in a two-sided test was used as the criterion for statistically significant differences. Statistical analysis was carried out using PASW Statistics 18 (IBM). Missing data were treated as missing values, and statistical data were expressed as mean ± standard error.

Results

Subjects

Eighty men were enrolled in the study, with no drop outs after randomization until commencement of study. During the test period, two subjects in the active group dropped out of the study due to personal problems unrelated to the study; thus, the number of subjects who completed the study was 78. In addition, among the 78 subjects, 17 were excluded from analysis for evaluation; therefore, 61 subjects analyzed for evaluation. The exclusion reasons and the number of subjects were as follows. (1) big changes in lifestyle (6 subjects; 4 and 2 subjects in the active and placebo groups, respectively); (2) violation of spontaneous exercise (4 subjects; 3 and 1 in the active and placebo groups, respectively); (3) violation of prohibited food (2 subjects; 1 each in the active and placebo groups); (4) exclusion criteria after the test (2 subjects; 1 each in the active and placebo groups); (5) large differences in physical condition between each examination (lack of sleep or toothache; 2 subjects; 1 each in the active and placebo groups); and (6)insufficient measurement data (1 subject in the active group).

The flow diagram of the study design showing the flow of subjects in each group until analysis for evaluation in this study is shown in Fig.1. The subjects’ characteristics are shown in Table 2. There were no significant differences in the value of age, height, body weight, BMI, body fat, and estimated muscle mass between the groups.

Energy metabolism

An interaction effect was observed in case of EE (P<0.05), with EE at 8-week examination being higher in the active group compared with that in the placebo group (active group, 20200.1±229.0 kJ/day; placebo group, 19642.6±217.9 kJ/day, p < 0.1). In the active group, the EE at the 8-week examination was significantly increased compared with that at 0-week examination (8-week examination, 20200.1±229.0 kJ/day; 0-week examination, 19861.6±212.7 kJ/day, p < 0.05). In addition, changes in EE at 8 weeks after ingestion [(8-week examination)–(0-week examination)], i.e. ΔEE was significantly increased in the active group compared with that in the placebo group (active group, 338.5±157.2 kJ/day; placebo group, -253.4±208.8 kJ/day, p < 0.05, Fig. 2). As for RQ, there was no interaction effect, and there were no significant differences in the comparisons between groups and within groups.

Safety

During the study, there were no adverse events related to the test food, including abnormal laboratory values.

Discussion

In the present study, we performed a randomized, double-blind, placebo-controlled, parallel-group study in healthy adults ingesting foods containing PFE (active group) or foods not containing PFE (placebo group) over an 8-week period to investigate the effects of PFIF oral intake on energy metabolism in healthy adults. The results indicated that PFIF oral intake significantly increased EE during exercise. According to the National Institute of Health and Nutrition documents about revised edition of METs table of physical activities (2012), intensity of exercise in the present study was approximately 3.5 METs. According to the documents, the intensity of 3.5 METs is known to be equivalent to the intensity of physical activities in daily life such as exercise or nonexercise activities such as housework. Thus, it is considered that PFIF oral intake can increase EE in healthy adults during exercise or nonexercise activities such as housework in daily life.

In our previous animal study, isoflavones from PFE showed anti-obesity effects such as abdominal fat reduction, and increased EE.^{16, 17）} In addition, isoflavones from PFE intake significantly increased expression of thermogenic gene (UCP-1) and significantly increased UCP-1-positive area in BAT.^{16, 17）} UCP-1 in BAT is known to produce heat using carbohydrates and lipids as an energy source. In this study, there was no change in RQ, and only EE was increased; this was thought to be caused by the involvement of UCP-1, which produces heat using carbohydrates and lipids as an energy source.

Meanwhile, it has been reported that PFE increases lipolysis gene (HSL) expression in WAT in animal model, as well as shows an anti-obesity effect such as reduction of abdominal fat.^17）Since it is known that the promotion of lipolysis by HSL activity in WAT enhances energy utilization of lipids, the PFIF in this study were expected to have the effect of lowering RQ. However, no significant difference in RQ was observed compared with the placebo food. It is known that carbohydrates and lipids as energy sources are used to approximately the same extent during low-intensity exercise.¹⁹⁾As the intensity of the exercise in the study was low (~ 3.5 METs), the effect may be less visible. Therefore, the effect of ingestion of PFIF on RQ should be examined in a future study using more optimal exercise load conditions.

In this study, the increase in EE due to the intake of PFIF was approximately 140 kcal/day when converted to kcal. According to previous reports, improving EE and intake of 100 kcal per day is useful for preventing weight gain.^20） It is also thought that a 1-cm reduction in waist circumference is equivalent to a reduction in body weight of about 1 kg, and it is necessary to improve EE and intake of 7000 kcal to reduce 1 kg of visceral fat. ^21）In our previous study, the reduction of waist circumference in healthy adults after 12 weeks of ingestion of PFIF was approximately 0.8 cm to 1.2 cm; ^13-15）this can be achieved by improving EE and intake by approximately 67 to 100 kcal/day per day. In this study, the increase in EE due to PFIF intake showed an effect exceeding this value. Based on these findings, it is suggested that an intake of PFIF in healthy adults increases EE and might thereby prevent obesity. In addition, no adverse events attributable to the test food were observed, suggesting that there are no safety issues associated with the long-term ingestion of the food containing PFE.

Conclusion

In summary, our results indicate that intake of PFIF can increase EE on energy metabolism in healthy adults. In addition, no safety issues were observed with the intake of PFE-containing tablets.

Conflicts of interest

The cost of conducting this study was borne by TOYO SHINYAKU Co., Ltd. The test food for this study was supplied by TOYO SHINYAKU Co., Ltd.

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