Research Article

Received: 24 November 2020 Revised: 26 January 2021 Accepted article published: 31 January 2021 Published online in Wiley Online Library:

(wileyonlinelibrary.com) DOI 10.1002/jsfa.11132

Daily Brazilian green propolis intake elevates

blood artepillin C levels in humans
Jiangli Ding,a Tomoh Matsumiya,a* Ryo Hayakari,a Yuko Shiba,a
Shogo Kawaguchi,a Kazuhiko Seya,a Kayo Uenob and Tadaatsu Imaizumia

Abstract
BACKGROUND: Propolis is a natural product collected by worker bees from a variety of plant species. As a type of propolis, Bra-
zilian green propolis contains a large amount of artepillin C. Artepillin C is a cinnamic acid derivative and has been shown to
have a wide variety of biological functions, including anti-inflammatory, antiviral and antitumor activities, in both cell culture
and animal models. However, how propolis is digested and absorbed remains to be elucidated. Moreover, blood artepillin C
levels after propolis intake have not been shown in human studies.
RESULTS: A randomized, single-blind placebo-controlled study on the effect of Brazilian green propolis on serum artepillin C
levels was conducted with healthy volunteers. The participants (n = 133) were randomly allocated in an approximately 2:1 ratio
to two groups: propolis (n = 91) and placebo (n = 42). The participants took daily propolis or placebo, and blood tests were
performed on day 0 (before propolis intake) and days 1, 3 and 7. Artepillin C was detected in serum in almost all individuals
in the propolis groups. No serum artepillin C was detected in the placebo group. Serum artepillin C levels in the female group
tended to be higher than those in the male group. In the female group, menstrual status was unrelated to serum artepillin C
levels.
CONCLUSION: These results suggested that propolis intake might be more effective for females than for males.
© 2021 Society of Chemical Industry

Keywords: propolis; artepillin C; human study

INTRODUCTION
Propolis, referred to as bee glue, is produced by honey bees (Apis
mellifera) to seal their hives.1 This salivary and enzymatic
secretion-enriched material is used by bees to cover hive walls, fill
cracks or gaps, and embalm invader insects that have been killed.2
A constituent of propolis is derived from bud exudates collected
by worker bees from a variety of buds of different plant species,
including palm, pine, alder, poplar, beech, conifer and birch.3
The constituent also varies depending on the climatic and geo-
graphical regions it was collected. Therefore, propolis from
Europe, North and South America, Asia and Africa is altered with
respect to its chemical composition.4 For example, poplar-type
propolis is derived from the poplar tree (Populus nigra) and is
found in North America, Europe, nontropical regions of Asia and
New Zealand.5 Similarly, the main source of Brazilian propolis is
Baccharis dracunculifolia leaf resin.6
To date, more than 300 compounds have been identified in
propolis.7 The biological activities of propolis are mainly attrib-
uted to its flavonoids and cinnamic acid derivatives, such as caf-
feic acid phenethyl ester and 3,5-diprenyl-4-hydroxycinnamic
acid (artepillin C).8 Brazilian green propolis contains a large
amount of artepillin C.9 Artepillin C has a wide variety of biological
effects, including anti-inflammatory, antiviral and antitumor
properties.10–12 Such effects of artepillin C have been extensively
studied with in vitro cell culture and in vivo animal models. By con-
trast, limited information is available about the role of artepillin C
in the human body. Moreover, no study has measured blood arte-
pillin C levels after propolis intake.
In the present study, we measured the daily artepillin C level in
serum from volunteers taking Brazilian green propolis. We also
addressed the influences of sex on serum artepillin C.
MATERIALS AND METHODS
Participants
Because of future analyses that will be conducted using data from
the present study, the study protocol basically followed the CON-
SORT 2010 statement guidelines for randomized clinical trials.13 A
randomized, single-blinded control study was conducted in Hiro-
saki City, Japan. Participants with missing data, a medical history
of cancer, stroke, ischemic heart disease or Parkinson's disease
were excluded from the analysis.
* Correspondence to: T Matsumiya, Department of Vascular Biology, Institute of
Brain Science, Hirosaki University Graduate School of Medicine, 5 Zaifu-cho,
Hirosaki 036-8562, Japan. E-mail: tomo1027@hirosaki-u.ac.jp
a Department of Vascular Biology, Institute of Brain Science, Hirosaki University
Graduate School of Medicine, Hirosaki, Japan
b Department of Pharmaceutical Science, Hirosaki University Graduate School
of Medicine, Hirosaki, Japan
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Inclusion criteria for volunteers were (i) healthy male or female;
(ii) aged > 18 years; (iii) no history of allergy to flavonoids; (iv) no
history of propolis intake; (v) all related biomedical parameters in
the normal range; (vi) ability to provide informed consent and
willingness to take propolis; and (vii) agreement to follow up for
the duration of the study. Exclusion criteria included
(i) volunteers' unwillingness to have their biomedical parameters
assessed and (ii) being pregnant. In total, 200 volunteers were
recruited. The participants were free to decide whether to volun-
teer for the study and written informed consent was obtained
from all volunteers before starting the study.
Procedures
Only one investigator was involved in the randomization process,
which employed the random number generator in Excel
(Microsoft Corp., Redmond, WA, USA). The participants were ran-
domly allocated in a two-to-one ratio to receive Brazilian green
propolis tablets (4 tablets day−1) or placebo (4 tablets day−1)
(Yamada Bee Farm Company, Inc., Okayama, Japan). Four propolis
tablets contained 406.8 mg of propolis powder. The propolis
powder (lot. LY-009), standardized to contain 10.4 mg per 4 tab-
lets of artepillin C, was obtained from Yamada Bee Company,
Inc. The detailed compounds in the propolis that we were pro-
vided with have been reported previously.14 At the first visit,
blood samples were collected from the participants (day 0). After
blood collection, the participants received either propolis or pla-
cebo tablets in white and opaque small packages. For 1 week,
starting on the following day, the tablets were taken within
30 min after breakfast, and blood was collected within 2 h after
propolis intake on days 1, 3 and day 7.
To monitor adverse reactions, the levels of serum lactate dehy-
drogenase (LDH), alanine transaminase and aspartate amino-
transferase (liver); total cholesterol, triglycerides, low-density
lipoprotein, high-density lipoprotein and glucose (metabolism);
and C-reactive protein and leukocytes (immunity and inflamma-
tion) were monitored in all blood samples.
Liquid chromatography-tandem mass spectrometry
Quantification of serum artepillin C was conducted as described
by Sparidans et al.15 Briefly, artepillin C (molecular weight = 300.39)
was separated on an Acquity UPLC I-class system (Waters, Milford,
MA, USA) with an Acquity UPLC HSS C18 column (1.8 μm,
2.1 × 100 mm; Waters). The column temperature was maintained
at 40 °C. Gradient elution was employed using a mobile phase of
0.1% formic acid in water (buffer A) and 0.1% formic acid in CH3CN
(buffer B) as: buffer A = 95% at 0–1 min, then from 95 to 5% over
5 min, and held at 5% for 2 min. The entire flow rate was
0.4 mL min−1. Xevo TQD tandem quadrupole mass spectrometry
(Waters) was used for artepillin C detection in positive mode using
the optimum transitional daughter ion mass: m/z 301.2 → 189.1
(retention time = 4.86).
Serum samples (100 μL) were added to 150 μL of CH3CN and
centrifuged at 17500 × g for 5 min at room temperature. The
obtained supernatant was diluted with water. Aliquots (10 μL) of
diluted supernatant were injected into the analytical system
described above. An external standard was applied to generate
a calibration curve for artepillin C by measuring serum containing
serial dilutions of standard artepillin C (Wako, Tokyo, Japan), cov-
ering the concentration range of 10–100 ng mL−1. Analysis was
performed in triplicate.
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Statistical analysis
Data are shown as the mean ± SD or the median and the lower
and upper quartiles (defined as the 25th and 75th percentiles).
Data gathered were statistically analyzed using the Mann–
Whitney nonparametric U-test. P < 0.05 was considered statisti-
cally significant. Data were analyzed using Origin Pro 2020
(Lightstone, Tokyo, Japan).
RESULTS
Study population
Following the approval of this study by the ethics committee, the
recruitment of volunteers began on 4 September 2017 and the
study ended on 11 September 2017. In total, 134 subjects
(72 males and 62 females) were enrolled in the study (Fig. 1). After
implementing the evaluation and exclusion criteria, 133 volun-
teers were randomly assigned to either the propolis group
(n = 91) or the placebo group (n = 41). One hundred and eighteen
participants completed the study. The demographics of the par-
ticipants who completed the study are shown in Table 1. The
mean age of all the participants was 42.3 years and the median
age was 41 years (range 20–66 years). There was no significant
difference between the placebo group (median age = 44 years)
and the propolis group (median age = 40 years) (P = 0.66) or
between males (median age = 41 years) and females (median
age = 42.5 years) (P = 0.96). Additionally, no significant difference
between the placebo group and propolis group in the number of
males (P = 0.75) or females (P = 0.4) was observed.
Serum artepillin C levels are dependent on propolis intake
Artepillin C is a major active component of Brazilian green propo-
lis.16 Therefore, we measured serum artepillin C levels to monitor
the absorption and digestion of propolis. At the start of the study
(i.e. before propolis intake), serum artepillin C from all participants
was undetectable (data not shown). Additionally, no artepillin C
was detected in blood samples from the placebo group during
the study. By contrast, serum artepillin C was detected in the sam-
ples in the propolis group on at least 1 day during the study,
except in one female whose artepillin C levels were below the
detection limit (Fig. 2). The ranges of serum artepillin C concentra-
tions on days 1, 3 and 7 were 0 to 115.5 ng mL−1 (median
5.0 ng mL−1), 0 to 357.6 ng mL−1 (median 12.0 ng mL−1) and
0 to 274.1 ng mL−1 (median 12.2 ng mL−1), respectively. We
found that alterations in blood artepillin C levels were dependent
on individual participants. For example, the serum artepillin C
concentration in a participant on day 1 was 23.1 ng mL−1,
whereas the concentration on day 7 showed the highest serum
artepillin C level (274.1 ng mL−1). Similarly, the concentration in
a participant on day 1 (111.5 ng mL−1) was the highest, whereas
the concentration on day 7 was 11.7 ng mL−1.
Next, differences between males and females in the level of
serum artepillin C were examined (Fig. 3). The ranges of serum
artepillin C concentrations in males on days 1, 3 and 7 were 0 to
70.2 ng mL−1 (median 6.4 ng mL−1), 3.0 to 62.4 ng mL−1 (median
11.1 ng mL−1) and 0 to 110.6 ng mL−1 (median 11.0 ng mL−1),
respectively. Similarly, the ranges of serum artepillin C concentra-
tions in females on days 1, 3 and 7 were 0 to 115.5 ng mL−1
(median 4.8 ng mL−1), 0 to 357.6 ng mL−1 (median 15.2 ng mL−1)
and 0 to 274.1 ng mL−1 (median 12.3 ng mL−1), respectively.
We further analyzed the data and found that there were two
patterns of serum artepillin C levels after propolis intake. In the
transient pattern, serum artepillin C reached its maximal level on
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Figure 1. Flow chart showing the number of participants in the enrollment and allocation phases and the final number of cases analyzed in the present
study.

Table 1. Participant population

Placebo (n = 35) Propolis (n = 83)

Age (years)
Mean 43.9 41.7
Median 44 40
Range 26–62 20–65
Sex Male Female Male Female
(n = 18) (n = 17) (n = 43) (n = 40)
Age (years)
Mean 43.8 43.9 42.9 40.5
Median 44 44 41 37.5
Range 26–62 28–66 23–65 20–65

day 1 and gradually decreased (Fig. 4A). For this pattern, the
ranges of serum artepillin C concentrations in males on days 1, 3
and 7 were 10.1 to 57.1 ng mL−1 (median 25.5 ng mL−1), 3.0 to
30.5 ng mL−1 (median 14.2 ng mL−1) and 1.2 to 44.8 ng mL−1
(median 11.6 ng mL−1), respectively. Similarly, the ranges of
serum artepillin C concentrations in females on days 1, 3 and
7 were 6.2 ng mL−1 to 99.7 ng mL−1 (median 57.3 ng mL−1), 4.4
to 54.3 ng mL−1 (median 20.3 ng mL−1) and 5.0 to 75.5 ng mL−1
(median 14.3 ng mL−1), respectively. There was a significant
difference between males and females on day 1 (P = 0.03). In
the incremental pattern, the serum artepillin C concentration
increased gradually and reached a maximal level on day 3 or
day 7 (Fig. 4B). For this pattern, the ranges of serum artepillin C
concentrations in males on days 1, 3 and 7 were 0 to 11.8 ng mL−1
(median 3.5 ng mL−1), 3.1 to 48.2 ng mL−1 (median 10.9 ng mL−1)
and 0 to 22.0 ng mL−1 (median 7.3 ng mL−1), respectively. Simi-
larly, the ranges of serum artepillin C concentrations in females
on days 1, 3 and 7 were 0 to 115.3 ng mL−1 (median 4.4 ng mL−1),
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Figure 2. Box plots of serum artepillin C levels (ng mL−1) on days 1, 3 and
7 in the total propolis group.
Figure 3. Box plots of serum artepillin C levels (ng mL−1) on days 1, 3 and
7 in the male and female propolis groups. P values were calculated using
the Mann–Whitney nonparametric U-test.
6.1 to 357.6 ng mL−1 (median 17.8 ng mL−1) and 2.6 to
122.9 ng mL−1 (median 12.2 ng mL−1), respectively.
Effect of menstruation state on serum artepillin C levels
Because the serum artepillin C levels at almost all time points were
higher in females than in males, we further analyzed the kinetics
of serum artepillin C levels in females. We focused on the effect
of menstruation status because menstruation status has been
known to influence nutrient uptake, such as iron absorption or
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Figure 4. Box plots of transient (A) or incremental (B) patterns of serum
artepillin C levels (ng mL−1) on days 1, 3 and 7 in the male and female
propolis groups. P values were calculated using the Mann–Whitney non-
parametric U-test.
glucocorticoid metabolism, in females.17,18 The demographics of
the female participants based on menstruation status are shown
in Table 2. The mean age of premenopausal participants
(n = 44) was 37.1 years, and the median age was 35 years (range
20–55 years). The mean age of postmenopausal participants
(n = 13) was 59.3 years and the median age was 55 years (range
50–66 years). There was a significant difference in age between
the pre- and postmenopausal groups (P < 0.01).
Figure 5 shows the effect of menstruation status on the serum
artepillin C level in females. The ranges of serum artepillin C con-
centrations in menopausal women (n = 9) on days 1, 3 and 7 were
2.5 to 115.5 ng mL−1 (median 5.7 ng mL−1), 3.9 to 163.3 ng mL−1
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Relationship between serum artepillin C levels and propolis intake
Table 2. Female participants' menstruation status
Placebo (n = 17)
Premenopause
Menstruation status (n = 13)
Age (years)
Mean 39.4
Median 42
Range 26–62
Figure 5. Box plots of serum artepillin C levels (ng mL−1) on days 1, 3 and
7 in the premenopausal and postmenopausal propolis groups. P values
were calculated using the Mann–Whitney nonparametric U-test.
(median 17.1 ng mL−1) and 3.0 to 70.3 ng mL−1 (median
15.8 ng mL−1), respectively. Similarly, the ranges of serum artepil-
lin C concentrations in females on days 1, 3 and 7 were 0 to
100.5 ng mL−1 (median 4.4 ng mL−1), 0 to 357.6 ng mL−1 (median
14.7 ng mL−1) and 0 to 274.1 ng mL−1 (median 12.5 ng mL−1),
respectively. These results suggested that menstruation status
did not affect serum artepillin C levels.
Adverse events
During the study, neither propolis, nor placebo influenced the
levels of routine blood tests we conducted. Additionally, no seri-
ous adverse events were reported.
DISCUSSION
Clinical trials of propolis have been reported. More than half of
them focused on the effect of propolis on oral diseases, including
dental caries,19 stomatitis20 and periodontitis.21 Topical usages of
propolis, such as in mouthwash,19 are likely effective as a result of
its antimicrobial and anti-inflammatory properties. By contrast,
only a few studies have demonstrated the effect of propolis intake
on human metabolic disorders.22 According to previous reports,
propolis affects oxidative stress in type 2 diabetes patients.23 Oral
administration of commercially available propolis solution in Chile
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Propolis (n = 40)
Post menopause Premenopause Post menopause
(n = 4) (n = 31) (n = 9)
58.8 36.1 55.6
57 34 55
55–66 20–65 50–65
has also been shown to decrease lipid peroxidation and increase
glutathione levels.24 In both reports, artepillin C and p-coumaric
acid in propolis were speculated to exert the anti-oxidative effects
of propolis. Indeed, in vitro studies have shown that such cinnamic
acid derivatives contribute effective anti-oxidant properties.12,25
In addition, only a single clinical study has examined the effect
of pure artepillin C on colorectal adenoma polyps.26 In that study,
the patients took artepillin C (165 μmol) for 3 months. It was con-
cluded that the results of the study did not provide evidence for
artepillin C being effective in preventing changes occurring dur-
ing early stages of colon cancer. By contrast, elevated
8-hydroxy-20 -deoxyguanosine and creatine phosphokinase (CPK)
levels were observed in blood tests from the propolis group, indi-
cating the detrimental side effects of artepillin C on muscle tissue,
including myocardial cells. Although our study was able to follow
up participants for 1 week, we did not observe any side effects,
including elevated blood CPK levels, in the propolis group during
this study (data not shown). Furthermore, no such detrimental
side effects of propolis have been reported. Therefore, it is sug-
gested that, with respect to consuming artepillin C, crude propolis
might be a better option than pure components when aiming to
avoid severe side effects.
Artepillin C has been extensively studied using cell culture and
animal models rather than clinical studies. Indeed, artepillin C
has been shown to have anti-inflammatory, anti-oxidant, antimi-
crobial and immunomodulatory properties in both in vitro and
in vivo studies.27 In many in vitro studies, artepillin C concentra-
tions greater than 25 μmol L−1 have been shown to function as
anti-inflammatory and anti-oxidant agents.27 In a rat model, arte-
pillin C levels in blood withdrawn from the portal vein and
abdominal artery were measured after the administration of arte-
pillin C by gastric intubation.28 In that study, artepillin C was
shown to have a lower absorption efficiency and higher hepatic
clearance than other phenolic acids, including p-coumaric acid.
In the present study, the range of blood artepillin C concentra-
tions after daily propolis intake was found to be 0 to
357.6 ng mL−1 (0 to 1.19 μmol L−1); therefore, the effects of arte-
pillin C in such in vitro studies might be overestimated by the high
doses of artepillin C. On the other hand, 0.72 μmol L−1 artepillin C
was able to inhibit lipid peroxidation in rat liver mitochondria,29
allowing us to consider physiologically relevant levels of artepillin
C that still have biochemical functions.
In the present study, we found that serum artepillin C levels in
females were higher than those in males after propolis intake.
To date, no study has examined sex differences in the absorption
of artepillin C after propolis intake. On the other hand, a few stud-
ies have investigated sex differences in the absorption of poly-
phenols in humans.30–32 Polyphenol theaflavin consumption
resulted in maximum theaflavin concentrations of 1.0 and
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0.5 μg L−1, respectively, being detected in the plasma of the
female and male volunteers. By contrast, oral consumption of
unprocessed apple juice resulted in higher total polyphenolic
content in male plasma than in female plasma.33 Although both
studies were conducted to collect plasma samples only up to
8 h after each intake, sex differences in the efficiency of polyphe-
nol metabolism and absorbance may be dependent on individual
polyphenols. Because no study has been conducted on long-term
alterations in serum/plasma polyphenol concentrations in
humans, we are unable to conclude why there were two different
patterns of serum artepillin C concentrations after propolis intake.
Future studies will aim to clarify what factors are involved in such
differences.
A woman's risk of cardiovascular disease increases after meno-
pause, suggesting cardioprotection by estrogen.34 A recent clini-
cal study demonstrated the beneficial effects of cacao
polyphenols in the prevention of cardiovascular disease in post-
menopausal women.35 In the present study, no significant differ-
ence in serum artepillin C levels was observed in the
menopausal state. This might explain the beneficial role of propo-
lis in protecting against oxidative stress involved in cardiovascular
disease in postmenopausal women.
Propolis contains a variety of chemical ingredients, including
cinnamic acid derivatives (e.g. p-coumaric acid, drupanin, artepil-
lin C and baccharin).16 Because no study has measured the blood
level of such chemical ingredients of propolis after propolis inges-
tion, we are unable to conclude that blood artepillin C level is the
most appropriate indicator of the digestion and absorption of
propolis in humans. Although blood artepillin C could be mea-
sured in most propolis intake groups, future studies measuring
other propolis components in serum will be able to show which
chemical ingredients should be used for the evaluation of propo-
lis digestion and absorption. In addition, we only observed artepil-
lin C levels up to 7 days after propolis intake in the present study,
and long-term observation will clearly reveal the kinetics of arte-
pillin C levels.
In summary, we found that propolis intake elevates the levels of
blood artepillin C, which is able to exert biochemical functions. In
humans, artepillin C levels are higher in females than in males,
suggesting that daily propolis intake is more effective in females
than in males. Propolis, a natural product, is considered to be safe
and has been approved as a health food or medicine, at least in
the context of artepillin C in the human body.
ACKNOWLEDGEMENTS
This work was supported by Takeda Science Foundation (TM); a
Hirosaki University Institutional Research Grant for Young Scien-
tists (TM); and a Yamada Research Grant (TM). We thank Yamada
Bee Farm Company, Inc. (Okayama, Japan) for providing Brazilian
propolis and placebo tablets. We also thank Dr Makoto Hayakari
for his advice regarding the analysis of serum artepillin C.
AUTHOR CONTRIBUTIONS
JD and TM designed the study. RH, YS and KU analyzed serum
artepillin C. SK and TI conducted the clinical examination during
the study. JD, KS and TM analyzed the data and wrote the paper.
CONFLICT OF INTERESTS
The authors declare that they have no conflicts of interest.
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ETHICAL APPROVAL
This study was approved by the Hirosaki University School of
Medicine Ethics Committee (approval no. 2016–260 and
no. 2018-178) and all residents voluntarily provided written,
informed consent to participate. This study was conducted in
accordance with the Declaration of Helsinki.
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