Effects of iso‐α‐acids, the hop‐derived bitter components in beer, on the MRI‐based Brain Healthcare Quotient in healthy middle‐aged to older adults

Abstract Aim Neurological disorders are a major public health issue worldwide and are often associated with structural changes in the brain. We have previously demonstrated that iso‐α‐acids (IAAs), the hop‐derived bitter components in beer, improve memory impairment in aged and Alzheimer's disease mouse models. In this study, we evaluated the effects of IAA intake on the brain structure in healthy middle‐aged to older adults. This study was conducted under the Impulsing Paradigm Change through Disruptive Technologies Program (ImPACT) study launched by the Cabinet office of Japan. Method This study employed an open‐labeled, single‐arm, before and after design. Healthy middle‐aged to older adults consumed a beverage containing IAAs (3 mg/190 mL) for 4 weeks.Recently developed magnetic resonance imaging‐based brain health indicators were used to evaluate the following brain conditions: the Brain Healthcare Quotient (BHQ) based on gray matter volume (GM‐BHQ) and white matter fractional anisotropy (FA‐BHQ). Results In total, 25 subjects were recruited, and GM‐BHQ and FA‐BHQ were measured before and after intervention. In all subjects, no significant differences in GM‐BHQ and FA‐BHQ were observed. In subjects aged ≥ 60 years (mean 54.5; standard deviation 3.9) (n = 8), GM‐BHQ was significantly increased 4 weeks after intervention compared with that before intervention. Conclusion Intake of beverages containing IAAs might affect brain aging, particularly in healthy older adults, which may prevent the development of neurological disorders. Future studies employing more robust designs can elucidate the effects of IAAs on GM‐BHQ and cognitive functions.


| INTRODUC TI ON
With an aging society, the number of patients exhibiting neurological disorders has rapidly increased worldwide, which is burden on the patients and society. 1 Maintenance of brain health and prevention of neurological disorders are important aspects required to decrease social costs. Disease pathologies of neurological disorders, including Alzheimer's and Parkinson's diseases, are associated with structural changes in the brain, primarily atrophy. 2 Brain structure atrophy is associated with negative neurological symptoms, such as cognitive decline. 3 Thus, maintaining a healthy brain structure is important for the prevention of various neurological disorders, and structural brain changes are a useful indicator for the health status of the brain. Daily food-derived nutrients have acquired increasing attention as a preventive approach for neurological diseases.
The herb hop (Humulus lupulus L.) is a well-known brewing material and is a primary ingredient in beer. Hops have long been used in brewing to add bitterness and flavor. Iso-α-acids (IAAs) are the main bitter components in beer produced from hops. Commercially available beer typically contains IAAs at 10-30 ppm; furthermore, nonalcoholic beverages simulating beer contain IAAs. Our group has previously demonstrated that IAAs suppress brain inflammation and prevent cognitive impairment in Alzheimer's transgenic and aged mice. 4 In aged mice, IAA intake improved hippocampal atrophy and memory impairment. In addition, we recently revealed that IAAs activate the vagal nerve and improve memory function in mice through the dopamine system. 5 Overall, IAA intake is expected to improve or maintain brain function. However, no studies have evaluated the effects of IAA intake on brain health in humans.
Recently, the Impulsing Paradigm Change through Disruptive Technologies Program (ImPACT) initiated by the Japanese cabinet office has developed the Brain Healthcare Quotient (BHQ) based on gray matter (GM) volume (GM-BHQ) and the white matter fractional anisotropy (FA; FA-BHQ) as the standardized health index of brain structure in healthy individuals using magnetic resonance imaging (MRI). 6 (GM-BHQ and FA-BHQ of each subjects were shown in Table S1). The GM volume reflects the number of dendrite expanse and neural cell synapses, 7 and the white matter FA that is measured using diffusion tensor imaging (DTI) reflects the plasticity of white matter and the efficacy of neural transmissions in the brain. 8 In an ImPACT study, GM-BHQ and FA-BHQ have been developed and standardized based on the MRI images of approximately 150 healthy subjects. These indexes are simple and are highly correlated with aging and various physical and social factors (eg, body mass index or subjective well-being, respectively) that are associated with the development of neurological disorders. Thus, these can be useful indicators of brain health in healthy subjects. Preventative approaches improving GM-BHQ and FA-BHQ are required to maintain brain health and prevent neurological disorders.
This study is the first clinical trial evaluating the effects of IAAs on human brain health as indicated by GM-BHQ and FA-BHQ. We recruited 25 healthy middle-aged to older adults, and they consumed beverages containing 3.4 mg of IAAs each day for 4 weeks. This study employed an open-labeled, single-arm, before and after design and was conducted under the ImPACT study.

| Subjects
We recruited 45 right-handed healthy Japanese adults, aged 50-70 years, who were not habitual alcohol drinkers (ie, consuming

| Experimental supplements
In hops, α-acids predominantly consist of three congeners: cohumulone, humulone, and adhumulone. During the brewing process, these are isomerized into two epimeric isomers, namely cis-and trans-IAAs. In the present study, we used isomerized hop extract (Hopsteiner) with 30.5% (w/v) IAAs, comprised of trans-isocohumu- The test formula consisted of a 190-mL beverage containing isomerized hop extract and containing 3.4-mg IAAs; this beverage was ingested every day for 4 weeks, and it contained acidulant, sweetener, and flavor to mask the bitterness of IAAs. The IAA dose in this study is approximately equivalent to the IAA content in 500 mL of commercially available beer.

| Statistics
The Kolmogorov-Smirnov test was performed in order to check the normality of the data set. Then, changes in GM-BHQ and FA-BHQ

| Subject characteristics
The flow of subjects through this study is shown in Figure 1.  Table 2.

| Changes in GM-BHQ and FA-BHQ
Changes in GM-BHQ and FA-BHQ before and after intervention are shown in Table 3; no significant differences were observed.
GM-BHQ and FA-BHQ were previously reported to be sensitive to aging. 4 Thus, we classified subjects into two groups, <60 (54.5 ± 3.9, n = 17) or ≥60 (64.9 ± 3.3, n = 8) years of age, and evaluated the effects of IAAs in older subjects. Changes in GM-BHQ and FA-BHQ among subjects classified according to age are shown in Table 3.
GM-BHQ, but not FA-BHQ, was significantly increased after intervention compared with that before intervention in older subjects (Table 4, Figure 2). In younger subjects, GM-BHQ and FA-BHQ remained unchanged before and after intervention.

| D ISCUSS I ON
In this study, we first evaluated the effects of IAAs on brain health in humans. GM-BHQ, but not FA-BHQ, significantly increased following IAA intake, particularly in older subjects. GM-BHQ reflects the volume of GM associated with an expanse of dendrites, whereas FA-BHQ is associated with white matter plasticity. 7,8 Thus, IAA intake may improve brain health by affecting GM volume.
GM-BHQ is reported to decrease with age. 6 Therefore, older subjects were predicted to be more sensitive to IAA intake. Amyloid-β (Aβ) accumulation and inflammation in the brain increases with age, even in subjects without symptoms of neurological disorders. 10,11 These events damage neural cells, resulting in a decrease in GM volume. 10,12 IAA intake has been reported to suppress inflammation and Aβ accumulation in the hippocampus and frontal cortex in Alzheimer's disease transgenic and aged mouse models. 4 A decrease in hippocampal weight in aged mice was suppressed by IAA intake (unpublished data). IAA intake at 1 mg/kg for 7 days was shown to reduce microglial inflammation and improve neural hyperactivation, which was detected using manganese MRI in an Alzheimer's disease mouse model. 4 In addition, IAAs have been reported to activate the peroxisome proliferator-activated receptor (PPAR)-γ. 13 Low-dose administration of the PPAR-γ agonist pioglitazone improved resting-state functional connectivity in rat brains. 14 These reports suggest that the suppression of inflammation by IAAs through PPAR-γ may be associated with GM-BHQ improvement.
Furthermore, GM-BHQ exhibits a negative correlation with body mass index. 6 Obesity and obesity-associated metabolic disorders such as insulin resistance are strong exacerbating factors of dementia. 15 In addition, an association between obesity and brain atrophy has been reported. 16 Obesity exacerbates brain inflammation, resulting in neural cell damage. 15 IAA intake has been reported to improve metabolic syndromes, including obesity [17][18][19] ; obesity-induced cerebral inflammation; brain atrophy; and cognitive decline in a high-fat diet-induced obesity mouse model. 20 In the clinical trial, IAA intake at 16 mg/d for 4 weeks resulted in decreased HbA1c levels, and IAA intake at 48 mg/d for 12 weeks resulted in decreased body mass indexes in subjects with prediabetes. 19 Thus, IAAs may be associated with improved body metabolism and increased GM-BHQ. However, we could not obtain metabolic factors in this study, so we could not assert that the improvement of GM-BHQ by the intake of IAAs was due to the TA B L E 4 Changes in GM-BHQ and FA-BHQ before and after intervention within subjects classified by age Note: Data are the mean ± SD. Abbreviations: FA-BHQ, the Brain Healthcare Quotient based on white matter fractional anisotropy; GM-BHQ, the Brain Healthcare Quotient based on gray matter volume. *Shows P < .05 performed by paired t-test.
F I G U R E 2 Changes in GM-BHQ in subjects ≥60 y of age. The each line shows the changes in GM-BHQ in subjects ≥60 y of age. Data represent the changes in each subject. *shows the P < .05 performed by paired t test. GM-BHQ, the Brain Healthcare Quotient based on gray matter volume improvement of metabolic conditions. Further study evaluating the relationship between the effects of IAAs on GM-BHQ and metabolic factors are needed to elucidate the underlying mechanisms in detail.
In addition to cognitive decline or dementia, GM-BHQ is affected by psychological conditions. It was reported that GM-BHQ was declined in the group with the subjective fatigue, which is associated with fatigue and stress. 21 Recently, it has been reported that psychological stress causes microglial activation, leading to the productions of inflammatory cytokines such as tumor necrosis factor-α and interleukin-6 in the brain 22  In conclusion, IAA intake improves the decrease in GM-BHQ associated with aging and contributes to brain health maintenance, which may prevent the development of neurological disorders. The IAA dose utilized in this study is easily consumed daily in alcoholic or nonalcoholic beverages. Thus, IAA intake could be a practical method to maintain brain health.

ACK N OWLED G M ENT
We appreciate Mr. Hideyuki Wakabayashi for preparing the test beverage.

CO N FLI C T O F I NTE R E S T
M.K., S.Y., K.K., and Y.A. are employed by the funder, and Y.Y. is the member of ImPACT. The test beverage was prepared by the funder.
The MRI data collection and analysis were performed by ImPACT.

AUTH O R CO NTR I B UTI O N S
M.K. and Y.A. designed and conducted the study. S.Y. and K.K. supervised the study. Y.Y. conducted this study as ImPACT manager.
M.K. wrote the manuscript.

E TH I C A L A PPROVA L
The study was conducted in accordance with the Declaration of Helsinki and the recommendations of Ethical Guidelines for Medical and Health Research Involving Human Subjects, Ministry of Health, Labor and Welfare. This study was approved by ethics committee of Huma R&D CO. Ltd (Tokyo, Japan) and the University of Tokyo (Tokyo, Japan).

DATA R E P O S I TO RY
I agree to publish part of it as supplementary information.

A PPROVA L O F TH E R E S E A RCH PROTO CO L BY A N I N S TITUTI O N A L R E V I E WER B OA R D
This study was approved by the ethics committee of Huma R&D Co Ltd. (Tokyo, Japan) and the University of Tokyo (Tokyo, Japan).

I N FO R M E D CO N S E NT
Written informed consent was obtained from all participants.

R EG I S TRY A N D TH E R EG I S TR ATI O N N O. O F TH E S T U DY
The study was registered on November 30, 2015, in the database of the University Hospital Medical Information Network (UMIN) (Registration No. UMIN000020006; Registration title: A study for the effect of intake of bitter component derived from hops on cognitive functions).

A N I M A L S TU D I E S
n/a.