Biochemical and Biophysical Research Communications 484 (2017) 740e745

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Biochemical and Biophysical Research Communications
journal homepage: www.elsevier.com/locate/ybbrc

Acute improvement of endothelial functions after oral ingestion of
isohumulones, bitter components of beer
Junko Tomita a, f, *, Seiichi Mochizuki b, Sohachi Fujimoto c, Naoki Kashihara d,
Takashi Akasaka e, Mitsune Tanimoto f, g, Kiyoshi Yoshida haDepartment of Cardiology, Kasaoka City Hospital, Japan
Department of Medical Engineering, Kawasaki University of Medical Welfare, Japan
Department of Health Care Medicine, Kawasaki Medical School, Japand
Department of Nephrology, Kawasaki Medical School, Japane
Department of Cardiology, Wakayama Medical University, Japanf
Department of Hematology, Oncology, and Respiratory Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical
Sciences, Japang
Department of Medicine, Iwakuni Medical Center, Japanh
Department of Cardiology, Sakakibara Heart Institute of Okayama, Japanbca r t i c l e i n f o

a b s t r a c t

Article history:
Received 19 January 2017
Accepted 24 January 2017
Available online 26 January 2017

Isohumulones, principal components of the bitter taste of beers, have antioxidant capacity. We studied i)the effects of oral ingestion of isomerized hop extract (IHE) on the endothelial functions in smokers aswell as non-smokers and ii) the effects of IHE on cultured endothelial cells in high oxidative stress state.
Twelve cigarette smokers and eleven non-smokers ingested IHE and placebo in a randomized crossoverdesign. Flow-mediated vasodilatation (FMD) was measured using ultrasonography. We also studied theeffects of isohumulones on i) the cell viability under hypoxia and ii) the levels of angiotensin II (AT-II)induced reactive oxygen species (ROS) in the cultured human aortic endothelial cells (HAECs). At
baseline, the FMDs of the smokers were signiﬁcantly lower than those of the non-smokers. The FMDs
increased signiﬁcantly after 30 min and 120 min of IHE ingestion in both the smokers and the nonsmokers. IHE protected the HAECs from hypoxia-induced cell death as assessed by cell viability. IHE also
reduced the AT-II-induced intracellular ROS level. Oral ingestion of IHE appears to exert acute beneﬁcial
effects on the endothelial functions in both the smokers and non-smokers, and the in vitro experiments
using HAECs suggested that the effect be through reducing intracellular oxidative stress.
© 2017 Elsevier Inc. All rights reserved.

Keywords:
Beer hop
Cigarette smoker
Endothelial cell
Endothelial dysfunction
Isohumulones
Oxidative stress1. Introduction
The vascular endothelium plays important roles in the regulation of vasomotor tone, platelet activation and inﬂammation [1].
Endothelial dysfunction (EDF) including the suppressed synthesis
and/or the reduced bioavailability of nitric oxide (NO) occurs whenvarious diseases and smoking injure the endothelium [2,3].
Beer hops, the female inﬂorescences of the hop plant (Humulus
lupulus L.), are known as good preservatives and ﬂavoring in beers.
Isohumulones, the main compounds of the bitter ﬂavor of beer, areconverted from humulons by isomerization during a brewing* Corresponding author. Department of Cardiology, Kasaoka City Hospital, 5628-1
Kasaoka, Kasaoka-city, Okayama, 714-0081, Japan.
E-mail address: tomita-j@kasaokacityhsp.jp (J. Tomita).
http://dx.doi.org/10.1016/j.bbrc.2017.01.1330006-291X/© 2017 Elsevier Inc. All rights reserved.

process. Various biological effects of isohumulones have been reported [4,5]. Namikoshi et al. reported that isohumulones reduced
the reactive oxygen species (ROS) production in the rat kidney andled to the restoration of bioavailable NO [6]. Accordingly, we
speculated that isohumulones may ameliorate EDF through the
reduction ROS under high oxidative stress conditions.
The purpose of this study was thus to assess the effects of isohumulones on the endothelial functions in smokers and nonsmokers as well as in cultured endothelial cells under high oxidative stress.

J. Tomita et al. / Biochemical and Biophysical Research Communications 484 (2017) 740e745

2. Materials and methods

741

NTGMD was calculated in the same way as FMD, i.e., the percent
change before and after NTG.

2.1. Ethical issues and approval
2.6. Biochemical analyses
This study was approved by the Kasaoka City Hospital Ethics
Committee and was conformed to the Declaration of Helsinki. All
the participants received the oral and written explanations about
the details of this study before signing the agreement forms.
2.2. Subjects
Twelve smoking men (smokers; smoking period: 8.2 ± 4.2
years) and eleven non-smoking men (non-smokers) were enrolled.
All the subjects were asymptomatic, normotensive and nondiabetic. They were instructed not to change their dietary habitsduring this study. Food intake and smoking were allowed before
19:00 on the day before each study. When needed, they drank only
ion-exchanged water containing neither nitrite nor nitrate (NOx),
both of which are oxidative products of NO.
2.3. Hop extract
Commercially available isomerized hop extract (IHE) was used
(ISOHOPCON2; English Hop Ltd., Tonbridge, Kent, UK). IHE contains
three kinds of water-soluble isohumulones: isohumulone, isocohumulone and isoadhumulone at a ratio of 37:48:15. IHE contained isohumulones of a purity of 79%, and the chemical structuresof these isohumulones were already reported [7]. One IHE capsulecontains 10.8 mg of IHE. Participants were instructed to take 1
capsule (10.8 mg IHE)/10 kg-body weight.

Serum levels of total cholesterol, HDL-cholesterol, triglycerides,
insulin, glucose, free fatty acid, and uric acid were measured bystandard blood test methods. Malondialdehyde-modiﬁed lowdensity lipoprotein (MDA-LDL), an oxidative stress marker, wasmeasured by an ELISA kit (Sekisui Medical Co., Ltd., Tokyo, Japan).
To measure the plasma levels of NOx, blood samples of 2 mLduring hyperemia were collected from the radial vein at the
maximal vasodilatation time point. After centrifugation of blood
samples, plasma was mixed with an equivalent volume of ethanol
for deproteinization. After another centrifugation, the NOx concentrations in each sample (supernatant plasma) were measured
by a NOx analyzer (ENO-20; Eicom, Kyoto, Japan) as reported previously [9]. Since NO is stable mostly as nitrate in blood, we used
nitrate as an index of NO level.
We calculated the NO production rate based on the following
equations:
Flow volume (cm3/beat) ¼ CSA (cm2)  TVI (cm/beat) ¼ (D/2)2  p  TVI(1)

where CSA (cross section area) was calculated from the diameter ofthe brachial artery (D), and TVI (time velocity integral) was
measured automatically by pulse-Doppler echosonography.
NO production rate (mol/sec) ¼ C (mol/mL)  Flow volume (mL/beat)  HR/60 (beat/sec)(2)

2.4. Protocols
We performed a randomized double-blind, cross-over study. All
the subjects proceeded to the second study within one week after
the ﬁrst study. Each study was scheduled to start before 8 a.m. After
15-min resting, hemodynamics parameters were measured, andvenous blood samples of 10 mL were obtained for the analyses ofbiochemical makers. Flow-mediated dilatation (FMD) andnitroglycerin-mediated dilatation (NTGMD) were assessed by ultrasonography. After 60-min resting, all the volunteers took IHE
capsules or placebos with NOx-free water. Blood sampling andmeasuring FMD were performed after 30 min and 120 min. NTGMD
was measured after NTG application.
2.5. Measurement of brachial artery reactivity
FMD measurement was performed in accordance with the JACC
guidelines [8]. After a 15-min rest in a temperature-controlled
room, the diameter of the right brachial artery and brachial blood
ﬂow were measured with a 15 MHz linear-array vascular ultrasound transducer and a HDI 5500 ultrasound system (Hitachi
Medico, Chiba, Japan). Hyperemic stress was applied by inﬂating
the cuff of a tonometer that was set on the proximal portion of theforearm to occlude arterial ﬂow (>200 mmHg) for 5 min, and thenthe cuff was rapidly deﬂated. Pulse-Doppler blood ﬂow velocity
was measured at a 60 angle to the vessel, and the range gateadjusted to each vessel diameter and the position in the center of
the artery. The maximum dilatation appeared about 1 min after cuff
deﬂation. The FMD was calculated as the ratio of the change inbrachial artery diameter before and after hyperemic stress over thebaseline diameter and expressed as a percent change. After 20-min
resting, to determine the maximal dilatation of the brachial artery,scanning was started 3 min after the sublingual administration of
nitroglycerin (NTG; 300 mg) and was kept for another 3 min.

where C is the plasma nitrate concentration and heart rates (HR)was obtained by echography.
The ratio of the NO production rates between baseline and hyperemia (NO ratio; shown as eq. (3)), was used as an index ofchanges in NO production and bioavailability.
NO ratio (%) ¼ NOHE (mol/sec) /NOBASE (mol/sec)  100(3)

where NOHE was the NO production rate of hyperemia, and NOBASE
was the baseline NO production rate.
2.7. Cell culture
Human aortic endothelial cells (HAEC; passage 3) were purchased from Clonetics Corp. (San Diego, CA, USA) and cultured in
EBM-2 SingleQuots medium (Cambrex Bio Science WalkersvilleInc., Walkersville, MD, USA) supplemented with 5% fetal calf serum,hydrocortisone, human ﬁbroblast growth factor-B, human vascularendothelial growth factor (VEGF), recombinant long R insulin-likegrowth factor-1 (R3-IGF-1), ascorbic acid, human epidermal
growth factor (rhEGF), gentamaicin sulfate, amphotericin-B, andheparin. The cells were incubated at 37  C in the humidiﬁed atmosphere of 95% air/5% CO2 in the medium that was replaced every
2 days. Morphological observation demonstrated that these cells
exhibited the speciﬁc characteristics of the endothelial cells. All thecells used in the experiments were within passage 6e8.
2.8. Hypoxic treatment
After reaching conﬂuence, the cell culture medium was replacedwith the fresh serum free medium. HAECs of about 2$104 wereseeded in each well of 24-wells (Corning Inc., Corning, NY, USA) andfurther cultured for 24 h. HAECs were kept under the hypoxic

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J. Tomita et al. / Biochemical and Biophysical Research Communications 484 (2017) 740e745

condition for 24 h with or without IHE. A hypoxic condition wasachieved using a CO2 incubator (99% CO2/1% air; Panasonic
Healthcare Co., Ltd., Tokyo, Japan).
2.9. Cell counting assay
An index of cell proliferation was obtained by the method using
a CellTiter-Blue™ Cell Viability Assay kit (Promega Corporation,
Madison, WI, USA). An increase in cell number is correlated with anincrease in the absorbance at 570 nm. In our experimental condition, an increase of 0.2 in absorbance reﬂects an increase of
20,000 cells/well in cell culture.
2.10. Oxidative stress treatment
HAECs cultured for 24 h in serum free medium were exposed toeither vehicle or 1 mg/mL IHE for 12 h, and then 1 mmol/L angiotensin II (AT-II; WAKO Pure Chemical Industries, Tokyo, Japan) was
added to the cell culture medium and incubated for 3 h. After
40 min, intracellular level of ROS was measured by using the 20 , 70 dichlorodihydroﬂuorescein-diacetate (DCFH-DA) method. HAECs
from each group were incubated with 10 mmol/L DCFH-DA (Invitrogen, Tokyo, Japan) for 1 h. After permeating the cells, DCFH-DA ishydrolyzed to DCFH by esterase. Intracellular ROS if any oxidize
DCFH to ﬂuorescent DCFH, which is detected by confocal laser
microscopy (Leica-Microsystems, Tokyo, Japan; excitation at
495 nm, emission at 530 nm). The mean ROS ﬂuorescence intensity
was analyzed by Leica TCS-NT system software (LeicaMicrosystems).
2.11. Statistical analysis
Comparisons of the baseline characteristics between thesmokers and the non-smokers were performed by one-way
ANOVA. The data for pre- and post-ingestions of IHE wereanalyzed by paired t tests. A repeated measure ANOVA was performed to evaluate for signiﬁcant time-course changes of thecomposite effect of the IHE versus placebo in each group. All in vitro
data were from at least three independent experiments. Continuous variables were described by means ± SD. P values < 0.05 wereconsidered statistically signiﬁcant. Data analysis was performed
using SPSS 23 (IBM SPSS Advanced Statics; IBM Japan, Tokyo,
Japan).
3. Results
3.1. Subjects characteristics
Twenty six volunteers were enrolled. Three volunteers were
excluded because their ultrasound images were inadequate foranalysis. The results of the baseline characteristics of the subjects
for the placebo studies are summarized in Table 1. The averages of
total cholesterol, LDL-cholesterol, triglyceride, and MDA-LDL of thesmokers were signiﬁcantly higher than those of the non-smokers.
The HDL level of the smokers was lower than that of the nonsmokers.

3.3. Biochemical parameters
As shown in Table 1, triglyceride levels decreased signiﬁcantly
from the baseline after ingesting IHE in both the groups. In thesmokers, triglyceride decreased remarkably even after taking placebo. Glucose levels decreased signiﬁcantly compared with thebaseline at each time point in both the groups after IHE intake.
Insulin levels decreased signiﬁcantly after ingesting IHE and placebo at 120 min compared with the baseline. In the non-smokers,
glucose levels did not change signiﬁcantly after taking placebo,
although insulin levels decreased signiﬁcantly at 120 min
compared with other time points. However, there was no statisticaldifference in the triglyceride, glucose and insulin levels between
IHE and placebo at each time point in both the smokers and nonsmokers. The other parameters did not show any signiﬁcantchanges between IHE and placebo in both the groups.
3.4. Brachial artery responses
As shown in Fig. 1, the FMDs of the smokers at the baseline
before taking IHE were signiﬁcantly lower than those of the nonsmokers (3.1 ± 2.1% and 6.3 ± 2.2%, p < 0.001). At 30 min and
120 min after ingestion of IHE, the FMDs of the smokers increased
signiﬁcantly up to 6.2 ± 1.2% (p < 0.0001vs baseline) and 7.7 ± 1.0%
(p < 0.001 vs baseline; p < 0.01 vs 30 min). The FMDs of the nonsmokers increased further to 8.4 ± 2.5% (p < 0.01 vs baseline) and10.7 ± 2.1% (p < 0.001 vs baseline; p < 0.05 vs 30 min). The FMDs of
the non-smokers were signiﬁcantly higher than those of thesmokers at every time point (p < 0.05). No signiﬁcant changes in
the FMDs were observed after ingestion of placebos in both thegroups. The effect of IHE almost disappeared after 4 h in both thegroups (data not shown).
NTGMDs were equivalent at baseline in the smokers and nonsmokers. (20.2 ± 6.7% and 18.9 ± 4.9%; p ¼ NS). NTGMDs did notchange signiﬁcantly 120 min after treatment with IHE or placebo inthe smokers (20.0 ± 6.7% and 18.7 ± 7.6%; p ¼ NS vs baseline) andnon-smokers (19.3 ± 5.9% and 15.5 ± 6.3%; p ¼ NS vs baseline).
There were no signiﬁcant differences of NTGMDs between the twogroups.
3.5. Effect of IHE on NO production
Blood samples for NO production analysis were obtained fromten volunteers (7 smokers and 3 non-smokers). As in Fig. 2A, after
IHE intake, the NO ratios (NOHE/NOBASE  100) of the smokers
increased from baseline of 179 ± 94% to 235 ± 106% at 30 min(p < 0.05), while after 120 min to 289 ± 170% (p ¼ NS vs baseline or
30 min). In contrast, the NO ratios after placebo did not change
signiﬁcantly. The NO ratios at 30 min after IHE intake signiﬁcantly
increased compared with placebo (235 ± 106% vs 169 ± 69%;
p < 0.05), while no signiﬁcant difference between the two groups
was observed after 120 min.
Fig. 2B shows the results for 7 smokers and 3 non-smokers.
Including non-smokers, the NO ratios of IHE differed signiﬁcantly
from those of placebo. After taking IHE, the NO ratios increased
from the baseline values of 183 ± 81% to 281 ± 197% (30 min;p < 0.05) and 351 ± 273% (120 min; p < 0.05 vs baseline, NS vs
30 min). In addition, the NO ratios of IHE were signiﬁcantly higher
than those of placebo at 30 min and 120 min (p < 0.05).

3.2. Hemodynamic parameters3.6. Effect of IHE on hypoxia-induced endothelial cell death
As shown in Table 1, at baseline, blood pressure, heart rate andbrachial artery diameter under the resting condition were nearly
equivalent in both the groups. These parameters were not signiﬁcantly changed even after ingesting either IHE or placebo.

Since IHE is dissolved in ethanol for avoiding crystallization at
the pH for cell culture, we checked if there was any effect of ethanol
in this experimental model. The exposure to IHE or ethanol did not

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743

Table 1
Hemodynamic and biochemical parameters of the smokers and non -smokers. IHE: isomerized hop extract, BMI: body mass index, BP: blood pressure, MDA-LDL:
Malondialdehyde-modiﬁed low-density lipoprotein, LDL: low density lipoprotein, HDL: high density lipoprotein. yp < 0.05 and zp < 0.01 for smokers vs non-smokers.
*
p < 0.05 and **p < 0.01 vs baseline. ap < 0.05 for 120 min vs 30 min #p < 0.05 for IHE vs placebo.

Age (year)
BMI (kg/m2)

Smokers (n¼12)Non-smokers (n¼11)29 ± 321.7 ± 1.7

28 ± 220.9 ± 1.3

Before intake

After intake

Before intake

30 min

120 min

108.5 ± 9.4
59.4 ± 6.2
60.7 ± 10.9

104.9 ± 7.4
58.8 ± 5.3
58.2 ± 8.7

107.3 ± 8.0
60.3 ± 6.0
60.1 ± 7.8

Total cholesterol (mg/dL)
LDL-Cholesterol (mg/dL)
HDL-Cholesterol(mg/dL)
Triglycerides (mg/dL)
Glucose (mg/dL)
Insulin (mU/mL)
Free fatty acid (mEq/L)
MDA-LDL (U/L)
Placebo
Systolic BP (mmHg)
Diastolic BP (mmHg)
Heart rate (beats/min)

177 ± 23z
113 ± 21z
46.± 9y95 ± 41y
97 ± 96.8 ± 2.8
0.4 ± 0.3
78 ± 35

172 ± 20110 ± 18
46 ± 975 ± 18*
97 ± 86.4 ± 3.4
0.3 ± 0.1
80 ± 29109.5 ± 8.4
59.3 ± 5.5
62.3 ± 10.0

Total cholesterol (mg/dL)
LDL-Cholestrol(mg/dL)
HDL -Cholestrol(mg/dL)
Triglycerides (mg/dL)
Glucose (mg/dL)
Insulin (mU/mL)
Free fatty acid (mEq/L)
MDA-LDL (U/L)

171 ± 26z
111 ± 20z
44 ± 10y108 ± 57y
102 ± 78.8 ± 4.0
0.4 ± 0.2
82 ± 20y

Hop extract capsule
Systolic BP (mmHg)
Diastolic BP(mmHg)
Heart rate (beats/min)

After intake
30 min

120 min

103.9 ± 9.9
59.8 ± 10.1
58.1 ± 5.4

102.3 ± 11.6
62.9 ± 10.1
56.4 ± 5.2

104.9 ± 12.5
62.9 ± 7.8
67.8 ± 5.2

174 ± 18111± 1547 ± 1069 ± 16*,a93 ± 7**,a4.9 ± 2.2**,a,#0.3 ± 0.1
89 ± 35

156 ± 2188 ± 1054 ± 1355 ± 1799 ± 56.4 ± 1.8
0.4 ± 0.1
66 ± 16154 ± 18
88 ± 1053 ± 1245 ± 10*94 ± 4*5.6 ± 2.2
0.4 ± 0.2
71 ± 16153 ± 1988 ± 1053 ± 1144 ± 10*94 ± 3**5.2 ± 1.4
0.4 ± 0.1
71 ± 16102.5 ± 8.3
55.2 ± 5.4
58.5 ± 8.1

105.3 ± 8.4
57.1 ± 6.0
56.8 ± 8.5

104.5 ± 8.5
58.5 ± 5.1
56.9 ± 6.2

103.3 ± 6.7
61.5 ± 5.7
60.3 ± 5.1

104.6 ± 6.3
63.6 ± 5.5
59.3 ± 4.1

168 ± 23112 ± 2044 ± 1087 ± 51*
100 ± 67.0 ± 4.3*
0.4 ± 0.2
90 ± 31168 ± 23112 ± 2044 ± 976 ± 41*,a98 ± 5**,a,#6.6 ± 4.1*
0.3 ± 0.2
87 ± 25

151 ± 1785 ± 1154 ± 1263 ± 35
95 ± 46.7 ± 2.6
0.4 ± 0.2
61 ± 15151 ± 1685 ± 1054 ± 1153 ± 2193 ± 76.8 ± 1.7
0.3 ± 0.2
61 ± 11152 ± 1586 ± 954 ± 1151 ± 1692 ± 66.1 ± 2.2*,#
0.3 ± 0.1
58 ± 7

Fig. 1. Effects of IHE on FMDs. FMDs were shown for (A) smokers and (B) non-smokers treated with IHE (black bar) and placebos (white bar). **p < 0.01 vs baseline. #p < 0.05 and##p < 0.01 for IHE vs placebo. a p < 0.05 for 120 min vs 30 min.

affect the HAEC viability signiﬁcantly in our study. As in Fig. 3A,
exposure to IHE resulted in an attenuation of the hypoxia-induced
HAEC death in a doseedependent manner, although a statisticallysigniﬁcant difference in cell number was observed only for 2 mg/mL
IHE.

3.7. Effect of IHE on AT- II-induced intracellular generation of ROS
Fig. 3B shows the effect of IHE on the level of ROS in HAECs.
HAECs exposed to either vehicle or IHE for 12 h were further
treated with AT-II for 3 h. AT-II enhanced the level of ROS (2.4 ± 0.6
fold, p < 0.01 vs control), and it was signiﬁcantly reduced by IHE(1.7 ± 0.4 fold, p < 0.01 vs AT-II).

4. Discussion
In this study, we demonstrated that ingestion of IHE, the bitter
compounds extracted from hops, acutely increased the FMDs inboth the smokers and non-smokers. In addition, the in vitro experiments using HAECs demonstrated that IHE attenuated intracellular ROS levels.
EDF is one of the major risk factors for various cardiovasculardiseases [10]. Especially, smoking causes EDF of healthy young
people [11]. The average value of the FMDs of healthy males in their
30s was reported to be about 6.6% [12]. In our study, three of thesmokers (25%) and seven of the non-smokers (70%) were over 6.6%.
After IHE intake, the FMDs of eleven of the smokers (92%) wereameliorated and exceeded the average value of normal subjects of
6.6%. Surprisingly, the FMDs of the non-smokers was also improved

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J. Tomita et al. / Biochemical and Biophysical Research Communications 484 (2017) 740e745

Fig. 2. Changes in total NO ratio. NO ratio (the ratio of NO production rate between reactive hyperemia and baseline) for (A) smokers only and (B) seven smokers and three nonsmokers at 0, 30 and 120 min after IHE (black bar) or placebo (white bar) intake. *p < 0.05 vs baseline. #p < 0.05 IHE vs placebo.

from 6.3% to 10.7%. However, no signiﬁcant changes were observedin blood pressures and heart rates.
Some previous studies reported the acute positive effects of beer
on EDF [13]. Karatzi et al. reported that beer improved the FMDs ofhealthy non-smokers from 2.5% (95% CI 1.4, 3.5) to 4.2% (95% CI 2.8,5.5) after drinking 400 mL of beer. In our study, the subjects with anaverage body weight (59.8 ± 6.6 kg) intook IHE of about 64.8 mg,
equivalent to almost 2 L of beer.
Smoking increases oxidative stress that causes EDF and arteriosclerosis and interferes with the production and bioactivity of
NO by various kinds of mechanisms [14,15]. Endothelium-derived
NO plays a vital role in the regulation of vascular tone by
opposing the effect of AT-II [16,17]. Though NO is quickly oxidized
or inactivated into nitrate by oxyhemoglobin and/or ROS, the direct
in vivo measurement of NO levels during reactive hyperemia is
difﬁcult [18]. Here, we measured the nitrate (an oxidative productof NO) concentration in each sample collected from the radial veinat the maximal vasodilatation time point reﬂecting the immediatechange of NO generation induced by increasing shear stress. The
baseline samples were obtained after 14-hr fasting, based on ourprevious study to exclude possible effects of nitrates derived fromfood and drinks [9]. As shown in Fig. 2, we observed a signiﬁcant
increase in the NO ratio during hyperemic stress from the baseline,
suggesting the ﬂow-induced NO generation from the endothelium.
IHE further increased the NO ratio compared to placebo, suggestingthat IHE may improve the production and/or bioavailability ofendothelium-derived NO. Here, blood pressures did not change
signiﬁcantly, and thus isohumulones may not exert any further
antihypertensive effects within the range of normal blood pressurein the young subjects.
We also studied the effects of isohumulones on the cultured
HAECs in vitro. Previously, it was reported that hypoxia induced theapoptosis of endothelial cells through increasing oxidative stress[19]. The activation of angiotensin I receptor by AT-II also promoted

Fig. 3. Effects of IHE on HAECs. (A) Hypoxia: endothelial cells cultured under hypoxic condition for 24 h; IHE: IHE-treated endothelial cells cultured under hypoxic condition for
24 h. **p < 0.001 vs control (hypoxia-untreated cells). #p < 0.01 vs hypoxia without IHE. (B) AT-II-induced intracellular production of ROS visualized by DCF: HEACs werepreincubated for 12 h (control) and then followed by a 3-hr incubation with 1 mmol/L angiotensin II (AT-II) or by a 3-hr incubation with 1 mmol/L AT-II and 1 mg/mL IHE (AT-II þ IHE).
(C) Summary of DCF ﬂuorescence intensities. *p < 0.01 vs control. #p < 0.01 vs AT- II.

J. Tomita et al. / Biochemical and Biophysical Research Communications 484 (2017) 740e745

the increase of ROS in vascular smooth muscle cells and induced theapoptosis of endothelial cells [20]. In our study, isohumulones
decreased the hypoxia-induced ROS as well as AT-II-induced ROS,
resulting in the protection of HAECs from the cell injury. Theseﬁndings suggest that isohumulones protect endothelial cells fromthe cytotoxic effects of ROS, maintaining the cell viability under theconditions of high oxidative stress.
Leikart et al. showed that alcohol-free red wine polyphenol (awell-known antioxidant) extract of 400 mg/mL increased theendothelial NO synthase expression and the endothelium-derived
NO release in cultured human umbilical vein endothelial cells[21]. Here, the polyphenol concentration was almost at the samelevel as the plasma concentration as reported by Nigdikar et al. [22].
Rodda et al. reported that the plasma levels of trans-isohumulones
reached ~0.1 mg/mL after consumption of 600~800 mL of beercontaining isohumulones of ~40 mg/L (50 mg IHE) [23]. In ourin vitro study, the effective concentration of isohumulones was
2.0 mg/mL, and this is much higher than 0.2 mg/mL which wasestimated from the results by Rodda et al. Although the cis-isomers
are more abundant in beer, Rodda et al. measured only transisomers of isohumulones, and thus the overall plasma concentration of isohumulones in vivo may be higher than their estimations.
Rodda et al. also reported the trans-isohumulones concentrations
peaked at 30 min and decreased to baseline by 120 min after
consumption of about 600~800 mL of beer. In our study, capsulized
isohumulones may need longer time to reach the peak plasma
concentration along with concomitant peak of the FMDs. Further
study is necessary to determine the metabolism and the bioavailability of isohumulones.
It was reported that isohumulones have agonistic effects on theactivities of peroxisome proliferator-activated receptors (PPARs)[24]. The activators of PPARg such as pioglitazone protected thevascular functions in both the diabetic and non-diabetic patientswith cardiovascular risk factors [25,26]. In our study, there were no
statistical time-course differences in glucose, insulin, and triglyceride levels between IHE and placebo in both the groups (Table 1).
The prolonged fasting periods may affect those values. Since we did
not measure the activities of PPARs simultaneously, it is still unclear
whether the PPARs activation by isohumulones ameliorates the
FMDs in an acute phase.
In conclusion, the FMD measurements and HAECs studies suggested that even a single ingestion of isohumulones acutely
ameliorate the endothelial functions of smokers and non-smokers
by reducing ROS.

Acknowledgements
We thank Dr. Aruto Yoshida of the Central Laboratories for Key
Technology, Kirin Co., Ltd. for his kind support including donationof IHE. This study was supported in part by Kawasaki University of
Medical Welfare Scientiﬁc Research Fund. We would also like tothank Dr. Yoji Neishi of Department of Cardiology, Kawasaki Medical School for recruiting the volunteers.

Transparency document
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