Original Article

診療と新薬 2020; 57: 129‑134

Eﬀects of Glucosyl Hesperidin on
Skin Blood Flow and Temperature:

診療と新薬 WebA Randomized, Double-blind, Placebo-controlled, Crossover Study

Naoki MORISHITA ／ Saori OGIHARA ／ Shin ENDO ／Hitoshi MITSUZUMI ／ Shimpei USHIO
R&D Division, Hayashibara Co., Ltd.

● SummaryObjective: The objective of this study was to evaluate whether the intake of 100 mg of glucosyl hesperidin（GHES）promotes the recovery of blood ﬂow and the temperature of the skin after cold water loading.
Method: GHES or placebo was administered in a double-blind crossover manner to 24 healthy adult men andwomen. Cold water（15℃）was then loaded onto the subject s hand for 5 minutes, and the blood flow andtemperature of the skin were measured over time to evaluate the recovery status of each parameter.
Results: The intake of GHES significantly promoted the recovery of skin blood flow and temperature thatwere reduced（temporarily）by cold water loading compared with the placebo. The eﬀects of GHES may be
mediated by improving peripheral blood ﬂow, which has been previously reported.
Conclusion: GHES at a dose of 100 mg facilitates the recovery of skin blood ﬂow and temperature following
cold stress, such as cold water loading.
Key word: Glucosyl hesperidin, Cold water loading, Blood ﬂow, Skin temperature

INTRODUCTION
The feeling of cold is a consistent complaint of
menopausal women, and it is often complicated with stiﬀ
shoulders, lower back pain, malaise, and others 1）. It isreported that the feeling of cold is caused by local
circulatory insufficiency and decreased autonomic
activity 2,3）.
Recently, the prevalence of refrigerated air conditioning
has increased the occurrence of the feeling of cold, evenin the summer 4）, and the number of people who complainof subjective symptoms of the feeling of cold is increasing
yearly, regardless of gender and age 5）. Therefore, thefeeling of cold is considered to be one of the causes
contributing to the decreased quality of life in people
throughout the year.
Hesperidin is a type of ﬂavonoid that is often found in
citrus peels. It has been known to inhibit increased
vascular permeability 6） and to have various physiological
functions, including blood flow-improving effects 7） andantihyperlipidaemic eﬀects 8）. However, hesperidin cannot
be used in beverages or other liquids because of its low
water solubility. α-Glucosyl hesperidin（GHES）, which
is produced by adding glucose to hesperidin, has resolved
this problem. It has been shown that hesperidin and

GHES have similar pharmacokinetics in the body since
GHES is readily hydrolyzed to hesperidin and glucose by
α -glucosidase in the small intestine 9）. Also, GHES ishighly soluble in water and has a better absorption
eﬃciency 9）, and therefore, GHES exerts its physiologicalactions more eﬀectively than hesperidin.
We have previously demonstrated that GHES has
physiological actions similar to hesperidin 10,11,12）.
Speciﬁcally, we found that the long-term intake of GHES
reduces the feeling of cold and improves blood ﬂow 11,12）.
In addition, the anti-coldness eﬀect of a single dose of
GHES has been reported, demonstrating that the single
dose of GHES promotes the recovery of skin blood ﬂow
and temperature that were reduced in a cold-waterloading test, in which a transient cold stress was given 13,14）.
The dose of GHES that was required to be effective inthese reports was 250 or 500 mg, but the minimum
eﬀective dose has not been investigated.
Therefore, this study investigated the eﬀects of a single
100 mg dose of GHES, which is lower than the previously
reported effective GHES doses, on skin blood flow andtemperature after cold water loading.

診療と新薬・第 57 巻

Analysis

Main study

Acclimatization Allocation Enrolment

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第 2 号（2020 年 2 月）

Consented subjects（n = 24）

Randomly allocated subjects（n = 24）

Acclimatization trial

Intake of GHES
（n = 12）Intake of placebo（n = 12）Intake of placebo（n = 12）Intake of GHES
（n = 12）
Excluded subjects（n = 6）
Meeting the exclusion criteria（n = 3）
Excluded by the Smirnov-Grubbs test（n = 3）

Subject for analysis（n = 18）

GHES: Near-water beverage containing GHES, Placebo: Near-water beverage not containing GHES

Fig. 1

Subject ﬂow

Ⅰ SUBJECTS AND METHODS

registered in the University Hospital Medical Information
Network Clinical Trials Registry System（UMIN-CTR）.

1. Subjects
Employees of Hayashibara Co., Ltd. who were 20 to lessthan 60 years old and did not meet the exclusion criteria
described below were recruited. Among them, 24 healthymale（10）and female（14）subjects were considered
eligible for the study by the investigator and wereselected. Prior to the start of the study, the subjects
were fully informed of the nature of the study and thatparticipation in the study was on a voluntary basis. Their
informed consent was obtained in writing.
Exclusion criteria:（1）Pregnant or possibly pregnant andlactating women,（2）Men/women with a smoking habit,（3）Men/women who have difficulty in following the
instructions of the investigator, and（4）Men/women who
are judged by the investigator to be inappropriate for the
study.
This study was performed in accordance with the ethicalprinciples based on the Declaration of Helsinki and the
Ethical Guideline for Clinical Research（Ministry of
Health, Labour and Welfare Announcement）. This study
was conducted after receiving approval by the ethics
committee of Hayashibara Co., Ltd.（date of approval:
February 9, 2018; approval No. 212）and being pre-2. Study method（1）Test beverage
The test beverage（GHES beverage）was prepared bydissolving 100 mg of GHES（Hayashibara Co., Ltd.,
Okayama, Japan）in 100 mL of a commercially available
orange-flavored near-water beverage（COCA-COLA（JAPAN）COMPANY, LIMITED, Tokyo, Japan）. The
near-water beverage without GHES was used as theplacebo.
Subjects were provided with 2 types of test beverages
in a manner that they were not distinguishable byappearance and taste.
（2）Study design
The study was conducted as a randomized, double-blind,placebo-controlled, crossover study from February to
September 2018（Fig. 1）. Twenty-four subjects wererandomized using random numbers and split into 2
groups, each containing 12 subjects with an equal numberof males and females. In addition, the order of taking thetest beverage was decided for each subject group（GHES
beverage → placebo beverage or placebo beverage →GHES beverage）. The beverages were allocated by aperson responsible for allocation who was not directly

診療と新薬・第 57 巻

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Attachment of blood ﬂow meter and skin temperature sensor-55 minutes

Acclimatization

30 minutes-5 minutes 0minute-35 minutes

Skin blood ﬂow and skin temperature
were measured for 5 minutes eachat -30, -5, 0, 5, 10, 20, and 30 minutes.

Warming at 33℃

Cold water loading
15℃ for 5 minutes

Intake of beverageFig. 2

Schedule of cold-water loading test

Table 1 Subject characteristics
Item

Overall

Men

Women

Age（years）

18
41.0±9.7837.0±10.3

1048.4± 7.7

Height（cm）

163.1±8.1

170.3±3.9157.3± 5.9

Weight（kg）
BMI（kg/m2）

53.5±7.2

57.7±5.0

50.16± 7.1

20.1±2.1

19.9±1.5

20.9± 2.7

Number of subjects（mean ± standard deviation）

involved in the study, and the sealed allocation table wasstored in a secure place until the end of the study.
After 2 acclimatization trials, 4 main studies（twice eachfor GHES and placebo beverages）were conducted.
There was a washout period of at least 1 day between
each study. On the day of the study, the same breakfast
was served to control the condition of the subjects, andtheir clothing during measurements was also
standardized.
（3）Endpoint
In this study, peripheral blood ﬂow and skin temperature
were measured as the primary endpoints with reference
to the method of Takumi H. et al 15）. As part of the subject
background survey, a questionnaire survey on thesubjects awareness of the feeling of cold（Terasawa s
diagnostics criteria 16））was also conducted. The study
was performed in a constant temperature and humidity
room set at 24 ± 1℃ and 50 ± 5％ .
To stabilize the skin blood ﬂow and temperature to bemeasured, subjects were instructed to comply with thefollowing conditions:（1）no alcohol drinking on the day
before the study,（2）no eating and drinking 1 hour
before starting the study, and（3）no stand-up work 1
hour before starting the study.
In the study, the subjects were acclimated for 20
minutes after entering the constant temperature andhumidity room, and then consumed 100 mL of the test
beverage at room temperature. The subject s left hand
was then immersed in a warm bath set at 33℃ for 30
minutes to warm their hands. Subsequently, the warmed
hand was immersed in a water bath set at 15℃ for 5

minutes to temporarily decrease the peripheral blood ﬂow
and temperature（cold water loading）. The subjects werethen instructed to gently place the hand that was
subjected to cold water loading on a desk and wait for 30
minutes in a sitting posture. Skin blood flow andtemperature were measured as described below during
the study（Fig. 2）.
Skin blood ﬂow; Measurements were performed with aresearch laser blood flow meter（RBF-101, PIONEERCORPORATION, Tokyo, Japan）. Finger-tip skin blood
ﬂow was measured during acclimatization and cold waterloading at 0, 5, 10, 20, and 30 minutes, setting the timeof withdrawal from cold water as t=0.
Skin temperature; A temperature logger（LT-8A, Gram
Corporation, Saitama, Japan）was used to measuretemperature. Finger-tip skin temperature was measured
during acclimatization and cold water loading at 0, 5, 10,20, and 30 minutes, setting the time of withdrawal fromcold water as t=0.
3. Statistical analysis
Subject characteristics were presented as the mean ±standard deviation, and measured values were presented
as the mean ± standard error. The mean values of eachof the 2 measurements were used as individual data for
analysis. Outliers were tested by the Smirnov-Grubbs
test at a level of significance of 0.05, and the data for
analysis were extracted. Then, the pattern of changeover time in each test beverage group was comparedusing a two-way ANOVA. A paired t-test was used forthe comparison of values at each measurement point. The

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第 2 号（2020 年 2 月）

Skin blood ﬂow［ml/min］

70
60

†
●
●

●

50

*

*

●

●

●

●

*

●

*

40

●

Black circles（●）and white circles（●）indicate●

the intake of GHES and placebo, respectively.

●

30

●
●

●

Data are presented as the mean ± standard

20

deviation. n=18, p <0.05（Two-way ANOVA）

10*p <0.05, †p <0.10 between GHES group vs.
Placebo group（paired t-test）

0

-30

-20

-10
0
10
Time［minutes］

20

30

Fig. 3Time-dependent changes in skin blood ﬂow after cold water loading

Skin temperature［℃］

35
●

●
●

*

30

●

†

*
●

●
●

Black circles（●）and white circles（●）indicate

●

†

25

●

the intake of GHES and placebo, respectively.

●

Data are presented as the mean ± standard●

deviation. n=18, p <0.05（Two-way ANOVA）

20●●*p <0.05, †p <0.10 between GHES group vs.
Placebo group（paired t-test）

15
-30

-20

-10
0
10
Time［minutes］

20

Fig. 4Time-dependent changes in skin temperature after cold water loading

signiﬁcance level was set at less than 0.05 in a two-sided
test, and p-values less than 0.1 were considered toindicate a trend. SPSS（Ver. 25.0, IBM Japan）was usedas the test software.

Ⅱ RESULTS
1. Subjects
Twenty-four subjects were enrolled at the start of thestudy, and all of them completed the scheduled
measurements. Of these, 3 subjects who met theexclusion criteria were excluded from the analysis. In
addition, 3 subjects with low skin blood ﬂow just before
the cold-water loading, which were judged to be outliers
by the Smirnov-Grubbs test, were excluded from theanalysis.
Finally, the analysis was performed with the dataobtained from 18 subjects（8 men and 10 women）（Table1）. The investigation of the presence or absence of the
feeling of cold based on the results of the questionnaire
survey administered before the start of the study showedthat the number of subjects with and without the feelingof cold was 8（6 men and 2 women）and 10（2 men and 8
women）, respectively.
No carry-over eﬀect of the test beverages was observed30

in any of these tests. Therefore, it was judged that thecrossover method worked appropriately in this study.
2. Skin blood ﬂow
The measurements of skin blood ﬂow are shown in Fig. 3.
An interaction was observed between the placebo and. The significance test at each
GHES groups（p <0.05）
measurement point showed that the ﬁnger-tip skin blood
ﬂow at 5, 10, 20, and 30 minutes after cold water loading
was significantly higher in the GHES group than in theplacebo group（p <0.05）. Immediately before cold waterloading（-5 minutes）, the blood flow decreased in theplacebo group, while it remained constant in the GHES.
group（p <0.10）
3. Skin temperature
The measurements of skin temperature are shown in Fig.
4. An interaction was observed between the placebo and. The significance test at each
GHES groups（p <0.05）
measurement point showed that the finger-tip skintemperature was signiﬁcantly higher（p <0.05）at 20 and
30 minutes and tended to be higher（p <0.10）at 0 and 5
minutes after cold water loading in the GHES group thanin the placebo group.

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4. Adverse events
No adverse events were observed throughout the study,
including in subjects that were excluded from theanalysis.

Ⅲ Discussion
This study examined if the ability of GHES to promote
the recovery of skin blood flow and temperature after
cold water loading, which has been conﬁrmed at 250 or
500 mg, was observed at a lower dose of 100 mg. The
study evaluated the effects of 100 mg of GHES on therecovery of skin blood flow and temperature that weretransiently reduced by cold water loading in healthy adultmen and women. We found that the reduced skin bloodflow and temperature after cold water loading wasmarkedly recovered in the GHES group compared withthe placebo group. Previously, we demonstrated that asingle dose of 50, 100, and 200 mg of GHES inhibited thedecrease of skin blood ﬂow in a dose-dependent mannerin a slow-cooling study conducted at 24℃ where bloodﬂow decreased in a resting state 17）. In addition, a dosedependent eﬀect on the feeling of cold has been reported
for hesperetin（from 17 mg）, which is the aglycone of
GHES 15）. This study, which was conducted based onthese results, showed a similar eﬀect at a dose of 100 mg,
which is lower than the previously reported effective
GHES doses that promoted the recovery of skin bloodflow and temperature after cold water loading. In 3
subjects who were judged as outliers by the SmirnovGrubbs test, the blood ﬂow in the skin was much lower
compared with other subjects before cold water loading
and did not decrease further following cold water loadingthat was performed to temporarily decrease blood ﬂow.
For these subjects, the pre-set temperature of the warm
bath or duration of bathing（33℃ for 30 minutes）
performed before cold water loading to reduce variationsin skin blood flow and temperature may have been
insuﬃcient.
Two potential mechanisms are proposed that may havecontributed to the ability of GHES to improve blood ﬂow
in this study. One is through autonomic nerves, and theother is via nitric oxide（NO）production, which havebeen previously reported. For the former, it is generally
known that increased sympathetic activity causes
vasoconstriction and decreased blood flow, whereas
increased parasympathetic activity causes vasorelaxationand increased blood flow. In the human clinical study
conducted by Takumi H et al., the analysis of heart ratevariability revealed that the intake of GHES inhibits
sympathetic activity and enhances parasympathetic
activity compared with the intake of a placebo 14）. On theother hand, Takumi H et al. investigated the effects ofhesperetin on the production of NO, a vasodilator, using
human umbilical vein endothelial cells and reported that
increased NO production was observed following

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administration of hesperetin. They concluded that GHES
promotes NO production in vascular endothelial cells andimproves blood flow through capillary dilation 15）.
Therefore, in this study, we suggest that changes in skinblood ﬂow aﬀected skin temperature, and it is likely thatthe intake of GHES promoted the increase in skin blood
ﬂow, which resulted in the recovery of skin temperature.
To date, the effect of GHES has been reported only ingroups of subjects who have the feeling of cold, but thisstudy was conducted in subjects regardless of thepresence or absence of the feeling of cold. These results
conﬁrm that GHES promotes the recovery of skin blood
ﬂow and temperature in all subjects. If the magnitude andcharacteristics of the effect of GHES depending on
diﬀerences in gender, age, and the degree of the feelingof cold are clarified in the future, GHES can be used
more eﬀectively as an anti-coldness agent. We would liketo further investigate these issues.

CONCLUSION
The results of this study showed that the intake of 100
mg of GHES signiﬁcantly accelerates the recovery of skinblood flow and temperature that was transiently
decreased by cold water loading compared with theplacebo. Since similar eﬀects were observed at 100 mg,
which is lower than the previously reported effective
doses of GHES（250 or 500 mg）, a wider range of foodapplications is expected in the future. Furthermore, since
it was shown that GHES exerts its eﬀect in people otherthan women who have the feeling of cold, it may relieve
the discomfort associated with reduced body temperatureand blood ﬂow under cooled conditions and help improve
the quality of life for more people.
ACKNOWLEDGMENTS
We are grateful to the study participants for their cooperation. We would
like to thank Junichi Hiramatsu M.D for his valuable medical advice as adoctor throughout the study.

COI
Hayashibara Co., Ltd. performed all operations related to the conduct of
the study.

REFERENCES
1）Melby MK. et al. Chilliness: a vasomotor symptom in Japan.
Menopause; 2007; 14: 752-759
2）Takatori A. et al. Thermological study on the coldness women Second report. The relationship between the change of atmospherictemperature and body surface temperature in women with complaintsof coldness in limbs. Papers of the Institute for Environmental
Medicine, Okayama University Medical Scool. 1991; 62: 16-223）Kondo M. et al. Cold Constitution : Analysis of the Questionnaire.
Acta obstet. gynaec. Jpn.（in Japanese）1987; 39: 2000-2004
4）Miyamoto N. et al. Relationship between Chilliness of the Limbs andDaily-life Conditions in Young Females. Japanese Journal of Hygiene.
1995; 49: 1004-1012
5）Ministry of Health, Labour and Welfare. Summary Report ofComprehensive Survey of Living Conditions 2010. 2010: 42-45

70 （134）

診療と新薬・第 57 巻

6）Garg A. et al. Chemistry and pharmacology of citrus bioflavonoid
hesperidin. Phytother Res. 2001; 15: 655-659
7）Morand C. et al. Hesperidin contributes to the vascular protective
effects of orange juice: a randomized crossover study in healthy
volunteers. Am. J. Clin. Nutr. 2011; 93: 73-80
8）Rathi AB. et al. Action of vitamin P like compounds on lysosomal
status in hypercholesterolemic rats. Acta Vitaminol. Enzymol. 1983;
5: 255-261
9）Yamada M. Bioavailability of Glucosyl Hesperidin in Rats. Biosci
Biotechnol Biochem. 2006; 70: 1386-1394
10）Yamada M. et al. Antioxidant Activity of Glycosylated Vitamin P and
Its Suppressive Eﬀect on Oxidative Stress in Hyperlipidemic Mice.
Jpn. Soc. Nutr. Food Sci. 2003; 56: 355-363
11）Endo S. et al. Dietary Glucosyl Hesperidin Improves Skin Color andSkin Conditions in Women̶Placebo-controlled Double-blind
Comparative Study̶. Jpn. Pharmacol. Ther. 2015; 43: 1687-1699
12）Yasuda A. et al. Improvement of physiological function by

第 2 号（2020 年 2 月）
administration of citrus polyphenol "Glucosyl hesperidin".
Kajukyoukaiho. 2010; 5: 1-12
13）Yoshitani K. et al. Eﬀect of α-Glucosylhesperidin on Poor Circulationin Women. J.Jpn. Soc. Nutr. Food Sci. 2008; 61: 233-239
14）Takumi H. et al. Effects of α-Glucosyl Hesperidin on the Blood
Circulation. Bulletion of Applied Glycoscience. 2011; 1: 186-193
15）Takumi H. et al. Bioavailability of orally administered waterdispersible hesperetin and its effect on peripheral vasodilatation inhuman subjects: implication of endothelial functions of plasma
conjugated metabolites. Food Funct. 2012; 3: 389-393
16）Terasawa K. On the Recognition and Treatment of "Hie-sho"（Chillphobia）in the Traditional Kampoh Medicine. Shoyakugaku
Zashi. 1987; 41: 85-96
17）Mizote A. et al. Improvement of blood flow circulation by a single
intake of Glucosyl hesperidin. Lecture Summaries of the 70th Annual
Meeting of the Japanese Society of Nutrition and Food Science 2016:
227