Effects of lifelong intake of lemon polyphenols on aging and intestinal microbiome in the senescence-accelerated mouse prone 1 (SAMP1)

Abstract

Polyphenols have been examined for their beneficial effects on health, particularly in rodents, but their lifelong effects are unclear. Lemons (Citrus limon), containing lemon polyphenols (LPP), are widely consumed but the effects of LPP on aging are unknown. Therefore, we examined the effects of LPP on aging such as aging-related scores, locomotor activity, cognitive functions, and intestinal microbiome using senescence-accelerated mouse prone 1 (SAMP1) and senescence-accelerated resistant mouse 1 (SAMR1). All mice had ad libitum access to water (P1_water group, SAMR1) or 0.1% LPP (P1_LPP group). In the P1_LPP group, LPP intake prolonged the lifespan by approximately 3 weeks and delayed increases in aging-related scores (e.g., periophthalmic lesions) and locomotor atrophy. The P1_water group showed large changes in the intestinal microbiome structure, while the R1 and P1_LPP groups did not. The phylum Bacteroidetes/Firmicutes, which is associated with obesity, in the P1_water group was significantly lower and higher than that in the P1_LPP and R1 groups, respectively. Although the relative abundance of Lactobacillus significantly increased in both P1 groups with aging, the P1_LPP group showed a significantly lower increase than the P1_water group. Thus, lifelong intake of LPP may have anti-aging effects on both phenotypes and the intestinal environment.

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Eects of lifelong intake of lemon
polyphenols on aging and intestinal
microbiome in the senescence-
accelerated mouse prone 1
(SAMP1)
Chikako shimizu1, Yoshihisa Wakita
1, Takashi Inoue2, Masanori Hiramitsu1, Miki okada3,
Yutaka Mitani1, Shuichi segawa1, Youichi Tsuchiya1 & Toshitaka Nabeshima4,5
Polyphenols have been examined for their benecial eects on health, particularly in rodents, but their
lifelong eects are unclear. Lemons (Citrus limon), containing lemon polyphenols (LPP), are widely
consumed but the eects of LPP on aging are unknown. Therefore, we examined the eects of LPP on
aging such as aging-related scores, locomotor activity, cognitive functions, and intestinal microbiome
using senescence-accelerated mouse prone 1 (SAMP1) and senescence-accelerated resistant mouse
1 (SAMR1). All mice had ad libitum access to water (P1_water group, SAMR1) or 0.1% LPP (P1_LPP
group). In the P1_LPP group, LPP intake prolonged the lifespan by approximately 3 weeks and delayed
increases in aging-related scores (e.g., periophthalmic lesions) and locomotor atrophy. The P1_water
group showed large changes in the intestinal microbiome structure, while the R1 and P1_LPP groups
did not. The phylum Bacteroidetes/Firmicutes, which is associated with obesity, in the P1_water group
was signicantly lower and higher than that in the P1_LPP and R1 groups, respectively. Although the
relative abundance of Lactobacillus signicantly increased in both P1 groups with aging, the P1_LPP
group showed a signicantly lower increase than the P1_water group. Thus, lifelong intake of LPP may
have anti-aging eects on both phenotypes and the intestinal environment.
Lemon fruit (Citrus limon) is one of the most widely consumed fruits, either directly or used in so drinks,
alcoholic drinks, and cooking. Lemons are rich in citric acid, vitamin C, and polyphenols, which confer vari-
ous health benets, such as the alleviation of fatigue1 and lipid-lowering eects2,3. Eriocitrin, the main lemon
polyphenol (LPP), is a yellow and water-soluble antioxidant2,4 that is abundant in lemon juice and peel. Although
the anti-aging eects of polyphenols have been suggested, few studies in rodents have been conducted until
animal death, such as studies of tea and wine polyphenols5,6. However, humans are likely to consume the same
habitual diets throughout their lifespan. Data obtained for a limited period may not reect the benecial eects
or safety of a food. us, studies of the lifelong eects of foods are needed.
Senescence-accelerated mouse prone (SAMP) strains were established by Takeda et al.7. e SAMP substrain
SAMP1 shows early decits in age-associated pathological features such as senile amyloidosis, impaired immune
response, and impaired motor function8,9 and has been widely used to analyse various antioxidants, such as
reduced coenzyme Q1010. As an age-matched control, the senescence-accelerated resistant mouse 1 (SAMR1) is
frequently used for comparison with SAMP1 to detect age-related changes.
1Frontier Laboratories for Value Creation, SAPPORO HOLDINGS LTD., 10 Okatome, Yaizu, Shizuoka, 425-
0013, Japan. 2Foodtechnology Laboratories for Value Creation, SAPPORO HOLDINGS LTD, 1189-4 Nippa-cho,
Kouhoku-ku, Yokohama, 223-0057, Japan. 3POKKA SAPPORO Food & Beverage Ltd., 45-2 Juniso, Kumanosho,
Kitanagoya, Aichi, 481-8515, Japan. 4Fujita Health University, 1-98 Dengakugakubo, Kutsukake-cho, Toyoake,
Aichi, 470-1192, Japan. 5NPO Japanese Drug Organization of Appropriate Use and Research, 3-1509 Omoteyama,
Tanpaku-ku, Nagoya, 468-0069, Japan. Yutaka Mitani is retired. Correspondence and requests for materials should
be addressed to C.S. (email: Chikako.Shimizu@sapporoholdings.co.jp)
Received: 12 October 2018
Accepted: 12 February 2019
Published: xx xx xxxx
opeN
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Studies of the intestinal microbiome in the last decade have demonstrated its major eects on the host11–13.
Turnbaugh et al.14 reported the relationship between the phylum Firmicutes/Bacteroidetes ratio and capacity
to harvest energy from the diet. Additionally, changes in the intestinal microbiome throughout life have been
reported15,16. In these contexts, changes in the microbiome involving aging may aect host health and function
as markers of aging.
In this study, we investigated the lifelong eects of LPP in SAMP1 to evaluate healthy aging. From the per-
spective of welfare and management of animals, we did not sacrice the mice, but evaluated their lifespan using
non-invasive pharmacological methods and faeces to investigate the intestinal microbiota in P1 mice drinking
LPP throughout life. For example, we examined aging-related phenotypes every month and cognitive functions
and locomotor activity every 3 months. Additionally, the intestinal microbiome was evaluated.
is is the rst report of the eects of LPP during the lifespan of mice.
Results and Discussion
Nutrition facts, polyphenols, and the anti-oxidative activities of LPP. LPP was obtained as a yel-
low solid. e nutritional components of LPP were as follows (w/w%): moisture content, 7.8; protein, 4.3; fat, 1.7;
carbohydrates, 85.4; and ash, 0.8. e contents of structurally identiable polyphenols contained in carbohydrates
within LPP were as follows (w/w%): eriocitrin, 21.7; hesperidin, 3.5; eriodictyol-7-glycoside, 1.2; and eriodictyol,
0.4. Miyake et al.17 isolated and identied six avanone glycosides and three avone glycosides from lemon fruits
(juice and peel). Eriocitrin showed the highest content of polyphenols in lemon fruits. us, we conformed that
the main polyphenol component in LPP was eriocitrin.
e total phenol content in LPP according to the Folin-Ciocalteu method (reference compound: gallic acid)18
was 15.9% (w/w%). LPP showed anti-oxidative potency. e α-diphenyl-β-picrylhydrazyl (DPPH) free radical
scavenging activity19 and oxygen radical absorbance capacity (ORAC)20 were 560 μmol Trolox equivalent (TE)/g
and 5400 μmol TE/g, respectively. e anti-oxidative results of LPP appeared to contribute to the anti-oxidative
activities and mechanisms.
We chose a dose of 0.1% LPP based on a previous study by Miyake et al.21. e previous study reported that
aer a 28-day feeding period in rats, the daily eriocitrin intake via pellets containing 0.2% eriocitrin was approx-
imately 267 mg/kg/rat (e.g. body weight; 240 g, food intake; 32 g/day) and anti-oxidative eects were signicantly
increased. In our study, the mice were throughout life; therefore, we decreased the dose of LPP (containing 21.7%
eriocitrin). An SAMP1 mouse (e.g. 30 g body weight) consumed 6 mL of 0.1% LPP solution each day, which cor-
responded to LPP 200 mg/kg/day (eriocitrin dose of 43 mg/kg/day).
Food consumption, liquid consumption, body weight, and number of surviving mice in P1
mice drinking water or 0.1% LPP during the lifespan. e results for food consumption, liquid con-
sumption, body weight, and number of surviving mice are shown in Fig.1a–d. Food and liquid consumption is
expressed as the consumption of each mouse per day, which was calculated by dividing the total consumption by
the number of mice per cage.
Food consumption in all groups gradually decreased with age (Fig.1a). ere were no signicant dierences in
food consumption, liquid consumption, and body weight between the P1_water and P1_LPP groups (Fig.1a–c).
Although spikes in liquid intake were occasionally observed (Fig.1b), we predicted that the spikes were caused by
contact between the top of the bottle nozzle and the cage paper. e contents of protein and fat in LPP were low
(less than 5%). e caloric intake of a mouse in the P1_LPP group was 14.3 kcal from CRF-1 (approximately 4 g/
day/mouse) and 0.02 kcal from LPP (approximately 6 mL/day/mouse). us, LPP accounted for only 0.16% of the
total daily calories. We therefore assumed that the contributions of nutrient components and calories of LPP were
negligible. In the R1 group, the liquid consumption was lower, but the average body weight was higher than in
both P1 groups; however, food intake was nearly equal to that in the P1 groups (Fig.1a–c). Body weight increased
until 30 weeks old, but then gradually decreased in both P1 groups; body weight decreased from approximately
65 weeks old in the R1 group. We conrmed the dierences in body weight and feed eciency (body weight/food
consumption) between the SAMR1 and SAMP1 groups in a previous report22. e results suggest that there was
a major dierence in metabolism between the two groups.
e mean lifespans of the groups were as follows: R1, 90.9 ± 4.7 weeks; P1_water, 81.8 ± 4.7 weeks; and P1_
LPP, 85.0 ± 4.6 weeks (Fig.1d). e average lifespan in the P1-LPP group was approximately 3 weeks longer
than that in the P1-water group. At 74 weeks old, there was no signicant dierence in the numbers of surviving
mice between the R1_group (16/18, number of surviving mice/number of mice used) and P1_LPP group (14/18)
(P = 0.658), but mice in the R1 group tended to be larger than those in the P1_water group (10/18) (P = 0.060), as
determined by Fisher’s exact test. Our ndings showed that the lifelong intake of LPP extracts has positive eects
over the lifespan.
Changes in aging-related scores in P1 mice drinking water or 0.1% LPP during the lifespan. We
examined the physical activity, skin conditions, eye inammation, and spinal curvature in all groups nearly every
month from 6 to 88 weeks old. ere were signicant dierences in aging-related scores between the P1_water
and P1_LPP groups in periophthalmic lesions, hair coarseness, and hair loss (Fig.2a–c). Other non-signicant
data related to aging were not shown.
We performed two-way analysis of variance (ANOVA) followed by Bonferroni’s post hoc test to assess the
eects of the group on aging-related scores. Group [F(2, 855) = 270.6, P < 0.001] and age [F(18, 855) = 76.2,
P < 0.001] signicantly aected the scores of periophthalmic lesions (Fig.2a). ere was a signicant interaction
between group and age [F(36, 855) = 13.2, P < 0.001]. Using the Bonferroni’s post hoc test, the scores of perioph-
thalmic lesions as the sum of right and le eyes in R1 were signicantly low compared to those of the P1_water
group aer the age of 40–42 weeks (40–42 weeks, P = 0.034; from 45–46 to 88 weeks old, P < 0.001) and those of
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the P1_LPP group aer 45–46 weeks (45–46 weeks, P = 0.014; 49–51 weeks, P = 0.001; aer 54 weeks, P < 0.001).
From 69 to 77 weeks, the scores of periophthalmic lesions in the P1_LPP group were signicantly lower than
those in the P1_water group (from 49 to 65 weeks old, P < 0.001; 69 weeks old, P = 0.002; 73 weeks old, P = 0.010;
77 weeks old, P = 0.008).
In terms of hair coarseness, group [F(2, 855) = 40.5, P < 0.001] and age [F(18, 855) = 7.4, P < 0.001] showed
significant effects (Fig.2b). There was a significant interaction between group and age [F(36, 855) = 2.7,
P < 0.001]. Using Bonferroni’s post hoc test, the scores of hair coarseness in R1 were signicantly low compared to
those of P1_water aer 49–51 weeks of age (49–51 weeks, P = 0.008; 54 weeks, P = 0.005; 59 weeks, P = 0.003; 65
and 69 weeks, P < 0.001; 73 weeks, P = 0.003; 77 weeks, P < 0.001; 83 weeks, P = 0.014; 88 weeks, P = 0.003) and
those of P1_LPP aer 73 weeks of age (73 weeks, P = 0.003; from 77 to 88 weeks, P < 0.001). At 65 and 69 weeks
old, the scores of hair coarseness in the P1_LPP group were signicantly lower than those in the P1_water group
(65 weeks, P = 0.003; 69 weeks, P = 0.029).
In terms of hair loss, group [F(2, 855) = 25.7, P < 0.001] and age [F(18, 855) = 6.9, P < 0.001] had signicant
eects on hair loss scores (Fig.2c). ere was a signicant interaction between group and age [F(36, 855) = 1.7,
P = 0.005]. Using Bonferroni’s post hoc test, the scores of hair loss in R1 were signicantly low compared to those
of the P1_water group from 49–51 to 73 weeks of age (49–51 weeks, P = 0.021; 54 weeks, P = 0.035; 59 weeks,
P = 0.002; 65 and 69 weeks, P < 0.001; 73 weeks, P = 0.019), and those of the P1_LPP group at 69 weeks of age
(P = 0.011) and 88 weeks of age (P = 0.008). At 65 weeks, the score of hair loss in the P1_LPP group was signi-
cantly lower than that in the P1_water group (65 weeks, P = 0.014).
In the R1 group, hair coarseness and hair loss were not changed by aging, which retained a youthful appear-
ance (Fig.2b,c). In contrast, hair aging rapidly progressed in the SAMP1 groups from approximately 40 weeks old.
However, aging scores in the P1_LPP group were signicantly lower than those in the P1_water group at 65–69
weeks old. For aging associated with periophthalmic lesions, aging in the R1 group occurred much more slowly
than that in both P1 groups (Fig.2a) and aging scores in the R1 group were one-third lower than those in the P1
groups, even at 88 weeks old. ese results suggest that LPP delays aging, such as periophthalmic lesions, hair
coarseness, and hair loss.
A signicant decrease in the levels of lipid antioxidants and hydrophilic antioxidant carnosine was observed in
the brain of SAMP1, which is characterized by accelerated accumulation of senile features, compared to SAMR123.
Antioxidative compounds delay the increase in aging scores in SAMP18. e main polyphenol component of LPP,
eriocitrin, is metabolized by intestinal bacteria and then absorbed to induce antioxidative activity in the plasma24.
In this experiment, the P1_LPP group may have maintained a higher antioxidative activity by consuming 0.1%
Figure 1. Food consumption, liquid consumption, body weight, and number of surviving mice in P1 mice
drinking water or 0.1% LPP during the lifespan. (a) Food consumption (g/mouse/day). (b) Liquid consumption
(g/mouse/day). (c) Body weight (g/mouse/day). (d) Number of surviving mice. dark blue line: R1 group; green
line: P1_water group; orange line: P1_LPP.
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LPP water rather than tap water throughout life. One possible mechanism by which LPP prevents aging is via
antioxidative activity against oxidative stress in SAMP1.
Changes in locomotor activities in P1 mice drinking water or 0.1% LPP during the lifespan. To
ensure healthy aging, it is necessary to maintain locomotor activity to avoid frailty, sarcopenia, and a bedridden
state, which are caused by not only low physical activity and a lack of protein nutrition but also an inammatory
prole and oxidative stress25,26. We examined the changes in locomotor activity with aging nearly every 3 months
from 13–16 to 92 weeks old (Fig.3). Two-way ANOVA followed by Bonferroni’s post hoc test revealed that loco-
motor activities in the R1 group were signicantly higher than those in the P1_water group at 66 (P < 0.001),
78 (P = 0.028), 86 (P < 0.001), and 92 weeks old (P < 0.001) and in the P1_LPP group at 66 (P = 0.022), 86
(P < 0.001), and 92 weeks old (P < 0.001). Moreover, there was a signicant dierence between the P1_water
and P1_LPP groups at 66 weeks old (P = 0.005). Locomotor activity in the R1 group was nearly unchanged with
aging, while those in both P1 groups were signicantly decreased with aging. Aoyama et al.9 reported that cere-
bellar Purkinje cells in 4-month-old SAMP1 mice persistently express tyrosine hydroxylase, the overexpression of
which is associated with motor dysfunction. In this experiment, locomotor impairment in SAMP1 appeared at 33
weeks old and deteriorated with aging. Interestingly, long-term LPP intake slowly prevented locomotor atrophy
with aging in the P1_LPP group compared to in the P1_water group at 66 weeks old (Fig.3).
We attempted to measure the levels of 8-hydroxydeoxyguanosine (8-OHdG)27, a biomarker of oxidative DNA
damages in the urine at 68–71 weeks in this study. However, this measurement was not possible because the
volumes of urine in SAM mice were too small. In many urine samples, 8-OHdG was below the measurable limit
aer dilution (data not shown). Miyake et al. reported21 that eriocitrin signicantly deceased 8-OHdG in the
urine of diabetic rats aer a 28-day feeding period. Moreover, administration of eriocitrin increased antioxidant
activity in the plasma24. Further, Ferreira et al.28 reported that citrus avanones (hesperidin, eriocitrin, and erio-
dictyol) increase the serum total antioxidant capacity and restrain elevations in inammatory cytokines, such as
interleukin-6 and macrophage chemoattractant protein-1. erefore, the anti-aging eects of LPP may delay not
only increases in aging scores (Fig.2a–c), such as periophthalmic lesions, but also decreases in locomotor activity
(Fig.3) via antioxidative and anti-inammatory activities throughout the body.
Changes in object recognition (long-term object memory) and spatial recognition (short-term
location memory) in P1 mice drinking water or 0.1% LPP during the lifespan. SAMP1 is not
typically used as an early cognitive decit model, similar to SAMP8 and SAMP105,8,29. We investigated whether
Figure 2. Changes in aging-related scores in P1 mice drinking water or 0.1% LPP during the lifespan dark
blue: R1 group; green: P1_water group; orange: P1_LPP. (a) Periophthalmic lesion (Grade 0; no changes, Grade
1; catarrhal changes limited to the periophthalmic area or swelling of palpebra, Grade 2; catarrhal changes
extending to nose, and Grade 3; catarrhal changes extending to further). (b) Hair coarseness (Grade 0; no
coarseness, Grade 1; coarseness of less than an area of the head, Grade 2; coarseness of less than double the area
of the head, Grade 3; coarseness of less than 3 times area of the head, and Grade 4; coarseness of over 3 times
area of the head). (c) Air loss (Grade 0; neither loss or thinning of hair, Grade 1; loss of hair in less than an area
of the head or thinning of hair in less than 1/2 of total area, Grade 2; loss of hair over one area of the head, less
than 1/4 of total area or thinning of hair in more than 1/2 of total area, Grade 3; loss of hair in more than 1/4 in
less than 1/2 of total area, and Grade 4; loss of hair in over 1/2 of total area). Between-group comparison among
R1, P1_water, and P1_LPP groups by two-way ANOVA followed by Bonferroni’s post hoc test for multiple
comparisons. ***P < 0.001, **P < 0.01, *P < 0.05; P1_water group vs. P1_LPP group. †††P < 0.001, ††P < 0.01,
†P < 0.05; R1 group vs. P1_water or P1_LPP groups.
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moderate/lifelong LPP intake protects against age-related impairment of recognition in an object recognition
test (ORT) and object location test (OLT). e recognition indices of familiar and novel objects during the test
phase in ORT for the R1, P1_water, and P1_LPP groups are illustrated in Fig.4a–c, respectively. e recognition
indices of the familiar and novel locations during the test phase in OLT for R1, P1_water, and P1_LPP groups are
illustrated in Fig.4d–f, respectively. During the training phase, the recognition indices were nearly 50% and no
signicant dierence in the preference was observed between the le and right objects (data not shown), as the
two objects were identical.
Figure 3. Changes in locomotor activities in P1 mice drinking water or 0.1% LPP during the lifespan. dark
blue: R1 group; green: P1_water group; orange: P1_LPP. Between-group comparison among R1, P1_water, and
P1_LPP groups by two-way ANOVA followed by Bonferroni’s post hoc test for multiple comparisons. *P < 0.05;
P1_water group vs. P1_LPP groups. †††P < 0.001, †P < 0.05; R1 group vs. P1_water group or P1_LPP group.
Figure 4. Changes in object recognition (long-term object memory) and spatial recognition (short-term
location memory) in P1 mice drinking water or 0.1% LPP during the lifespan. Recognition indexes during the
test session of the ORT: (a) R1 group, (b) P1_water group, (c) P1_LPP group. Recognition indexes during the
test session of the OLT: (d) R1 group, (e) P1_water group, (f) P1_LPP group Δ: familiar object; ▲: novel object
(dark blue: R1 group; green; P1_water group; orange: P1_LPP) ○: familiar location object; ●: novel location
object (dark blue: R1 group; green; P1_water group; orange: P1_LPP). e numbers in parentheses show the
number of surviving mice at the dierent ages. ***P < 0.001, **P < 0.01, *P < 0.05; by unpaired t-test.
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In the ORT during the test phases from 8 to 79 weeks old (i.e., nearly the entire life), the recognition index
for the novel object (lm case) was signicantly higher than that for the familiar object (golf ball) in both the
P1_water and P1_LPP groups (P < 0.05; Fig.4b,c). Although the preference for the novel object in the R1 group
was signicantly higher than that for the familiar object (P < 0.05), except at 34 weeks old, the recognition indices
of the R1 group (novel object: less than 60% except at 8 weeks old) were lower than those of both P1 groups (novel
object: more than 60%), despite that SAMR1 is a senescence-accelerated resistant mouse (Fig.4a). During breed-
ing of these mice, we found dierences in behaviour between SAMR1 and SAMP1 in the home cages. Unlike
SAMP1, SAMR1 always made a deep nest using sliced paper in the cage and hid in the nest. erefore, SAMR1
showed more fear than SAMP1 towards objects in the ORT and OLT. e total access time to objects in the ORT
during the training session in the R1 group was approximately 50% shorter than that in the P1 group (data not
shown). e lower recognition indices in the R1 group may be related to the short approach time to the objects in
the ORT during the training session.
In the OLT during the test phase from 9 to 80 weeks old, regarding the recognition index of familiar and
novel locations, the mice recognized the novel position signicantly more than the familiar location from 13–14
to 35 weeks old in the P1_water group (P < 0.05; Fig.4e) and from 13–14 to 23 weeks old in the P1_LPP group
(P < 0.05; Fig.4f), but not at 80 weeks old. In the R1 group, there were no signicant dierences between the
familiar and novel positions from 13–14 to 35 weeks old, but signicant dierences were identied at 51 and 80
weeks old. e disorder of spatial recognition in the OLT (Fig.4d–f) preceded the impairment of object recogni-
tion in the ORT (Fig.4a–c), as we reported previously30. In the comparison of P1 groups, the recognition indices
for a novel location were signicantly higher than those for a familiar location from 13–14 to 35 weeks old in the
P1_water group, but only from 13–14 to 28 weeks old in the P1_LPP group (Fig.4e,f). However, from 35 to 67
weeks old in the P1_LPP group, the indices for a novel location were higher than those for a familiar location
(no signicance) and tended to be higher at 67 weeks old (P = 0.064), but not in the P1_water group (P = 0.137).
e results of the OLT showed that location-related memory in the P1_LPP group was not worse than that in the
P1_water group. Based on the recognition ability, we examined the temporary changes that occurred during the
lifespan rather than a few time points within a certain period to avoid misleading results.
Compared to the P1_water groups, the recognition indices in the OLT in the R1 group were lower, as observed
in the ORT; however, recognition of the location of a novel object was signicantly better than that for a famil-
iar location at 80 weeks old (Fig.4d). We assumed that the R1 group was not inferior to the P1_groups, as they
approached objects in a short time in the training session, similar to that in the ORT. e cognitive eects of
long-term LPP intake were weaker than those on other phenotypes.
e activities of antioxidant enzymes in the brain of SAMP1 are signicantly lower than those in SAMR122.
Oxidative stress may cause disorder in spatial recognition in SAMP1. Tea polyphenols, epigallocatechin gallate
with antioxidant activity, and its metabolite, 5-(3′,5′-dihydroxyphenyl)-γ-valerolactone can pass the blood–brain
barrier (BBB) and directly aect the memory-retarding activity in aged mice29. Although LPP has strong anti-
oxidant activity21, it has a low ability to pass the BBB because of its glycoside form. Eriocitrin is metabolized to
eriodictyol, methylated eriodictyol, 3,4-dihydroxyhydrocinnamic acid, and their conjugates in the plasma and
urine24. Further studies are needed to determine whether metabolites can pass the BBB and directly aect cogni-
tive functions.
Changes in intestinal microbiome at 19 and 70 weeks old in P1 mice drinking water or 0.1%
LPP. UniFrac analysis is an eective distance metric for microbial species31 and visually expresses the compo-
sition of bacterial species at a specic site. Initially, the overall structure of the intestinal microbiome was evalu-
ated by UniFrac analysis (Fig.5a–d). At 19 weeks old, the structure of the intestinal microbiome in the R1 group
(Fig.5b) diered from that in both P1 groups (Fig.5c,d). Moreover, the microbiome at 19 weeks old diered
from that at 70 weeks old in the P1_water group (Fig.5c), but not in the R1 and P1_LPP groups (Fig.5b,d). ese
results suggest that LPP intake maintains the intestinal environment against aging.
Subsequently, the microbiome composition at the phylum level was evaluated. In between-group compari-
son of SAMR1 with SAMP1, the level of phylum Bacteroidetes in the P1_water group was signicantly higher
than that in the R1 group (Fig.6a) at both 19 (P < 0.001) and 70 weeks old (P = 0.012), while the level of phylum
Firmicutes was signicantly lower (Fig.6b) at 19 (P < 0.001) and 70 weeks old (P = 0.008). Furthermore, the level
of Bacteroidetes/Firmicutes in the R1 group was signicantly lower than that in the P1_water group (Fig.6c) at 19
(P < 0.001) and 70 weeks old (P = 0.018). Recently, links between certain diseases and the intestinal microbiome
have been suggested32. Human gut microbes are associated with obesity; a lower level of Bacteroidetes and higher
level of Firmicutes have been detected in obese subjects33. In the R1 and P1_water groups, the dierence in body
weight between these groups gradually increased, becoming signicant at 33 weeks old onwards (the R1 group
had a higher weight than the SAMP1 group) (Fig.1c), despite similar levels of intake of the same food (Fig.1a).
is may be explained by the composition of the SAMR1 intestinal microbiome.
In between-group comparison of P1_water with P1_LPP, the ratio of the phylum Bacteroidetes in the P1_LPP
group was signicantly higher (P = 0.033; Fig.6a), while that of Firmicutes was signicantly lower (P = 0.031;
Fig.6b). Furthermore, the level of Bacteroidetes/Firmicutes was signicantly higher than that in the P1_water
group (P = 0.023; Fig.6c) at 19 weeks old; this dierence was also observed at 70 weeks old, but was not signi-
cant (P = 0.088). However, the body weight was nearly the same between P1_LPP and P1_water groups, indicat-
ing that the microbiome is inuenced by the feed (high-fat feed or not) and host status (intestinal absorption) in
SAMP1.
Genus-level differences in the microbiome among the 3 groups were evaluated. The level of Bacteroides
(phylum Bacteroidetes) in the P1_water group was signicantly higher than that in the R1_water group at 19
(P < 0.001) and 70 weeks old (P = 0.004), but lower than that in the P1_LPP group at 19 weeks old (P = 0.038), as
shown in Fig.6d.
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Figure 5. Changes in intestinal microbiome by UniFrac analyses at 19 and 70 weeks old in P1 mice drinking
water or 0.1% LPP. All group, (b) R1 group, (c) P1_water group, (d) P1_LPP group. R1 group, purple: 19 weeks
old; yellow: 70 weeks old. P1_water group, orange: 19 weeks old; green: 70 weeks old. P1_LPP group, red: 19
weeks old; blue: 70 weeks old.
Figure 6. Changes in intestinal microbiome phylum and genus families at 19 and 70 weeks old in P1
mice drinking water or 0.1% LPP. (a) Bacteroidetes (phylum), (b) Firmicutes (phylum), (c) Bacteroidetes
(phylum)/Firmicutes (phylum), (d) Bacteroides (genus), (e) Lactobacillus (genus), and (f) Prevotella (genus).
*P < 0.05, **P < 0.01; P1_water group vs. P1_LPP group by unpaired t-test. §P < 0.05, §§P < 0.01, §§§P < 0.001;
19 weeks old vs. 70 weeks old by unpaired t-test. †P < 0.05, ††P < 0.01, †††P < 0.001; R1 group vs. P1_water group
by unpaired t-test.
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An increase in Lactobacillus strains associated with aging has been reported in human microbiota15. In
within-group comparison with aging in each bacterial genus, Lactobacillus (Firmicutes phylum) signicantly
increased with aging in the P1_groups (P1_water group, P < 0.001; P1_LPP group, P = 0.016; Fig.6e), but this
was not observed for the R1_group (P = 0.860; Fig.6e). Interestingly, the level of Lactobacillus at 70 weeks old in
the P1_LPP group was signicantly lower than that in the P1_water group (P = 0.004), indicating that LPP intake
delayed the aging-related increase in Lactobacillus.
Eriocitrin is metabolized by intestinal bacteria, after which the metabolites are absorbed24, increasing
anti-oxidative activities in the plasma24 and decreasing oxidative stress21. We predicted that the anti-aging eects
of LPP are developed through the intestinal microbiome both directly and indirectly. We previously reported that
the intestinal microbiota varies between breeder companies because of their dierent breeding environments,
even in the same strain of mice34. In this aging study, we used the same breeding chamber and same type of cages.
erefore, the changes in the microbiota appear to be related to aging rather than the breeding environment.
Significant decreases with aging were observed in Parabacteroides (P1_water and P1_LPP, P < 0.001),
Prevotella (P1_water, P = 0.001; P1_LPP, P < 0.001, Fig.6f), Oscillospira (P1_water, P = 0.047), and Ruminococcus
(R1, P = 0.037; P1_water, P = 0.020) (data not shown except for Prevotella Fig.6f). Arumugam et al.35 reported
that Prevotella, Bacteroides, and Ruminococcus were dominant in the intestinal microbiome and referred to as the
three “enterotypes.” Enterotypes are linked with long-term diets, particularly protein, fat, and carbohydrates36.
Remarkable changes in key microbiota for the human enterotype35,36 were observed by the LPP intake and aging
in this study.
Our results suggested that lifelong intake of LPP has anti-aging eects not only on the host health status but
also on the intestinal environment. Recently, Henning et al.37 reported that green and black tea polyphenol diets
decrease weight gain and result in a decrease in cecum Firmicutes and increase in Bacteroidetes. Additional stud-
ies are needed to clarify whether intestinal or phenotypic changes occur at rst during the intake of polyphenols.
Conclusions
In the P1_LPP group, the average lifespan was approximately 3 weeks longer, and increases in aging-related
scores (e.g. periophthalmic lesions) and locomotor atrophy were delayed compared to in the P1_water group.
Additionally, possible aging-related changes in the intestinal microbiomes, such as for the genus Lactobacillus,
were restricted by LPP intake. ese results suggest that LPP has anti-aging eects not only on host health but
also on the intestinal environment.
Evaluating lifelong intake of food is important for detecting the eects on human and animals because they
are likely to consume habitual diets throughout their lifespan. Food habits may exert a large inuence on the host.
Materials and Methods
Lemon polyphenol (LPP) extract. Lemon polyphenols (LPP) from lemon peel were obtained as described
in a previous report38. LPP was subdivided into small packages and stored at −30 °C until usage. We freshly
prepared 0.1% (w/v) LPP by dissolution in tap water and ltering through a Stericup™ (0.22 µm, SCGPU02RE;
Merck Millipore, Billerica, MA, USA) twice per week.
Animals. irty-six SAMP1 male mice aged 5 weeks and 18 SAMR1 (Japan SLC, Inc., Hamamatsu, Japan)
were acclimated to the animal facility. e oor of the cage was covered with sliced paper, Palmas μ® (Material
Research Center, Tokyo, Japan), which was changed every week. SAMR1 mice were group-housed (six mice per
cage) with free access to tap water. e SAMP1 mice were also group-housed (six mice per cage) with free access
to tap water (water group, n = 18) or 0.1% (w/v) LPP [LPP group; water until 8 weeks of age, 0.1% (w/v) LPP from
9 weeks of age, n = 18].
All mice were provided ad libitum access to standard chow (CRF-1; Charles River Laboratories, Yokohama,
Japan). e animal facility was maintained at 23 °C ± 1 °C with 55% humidity and a 12-h/12-h light/dark cycle.
We used the same breeding chamber (EBAC-L®, CLEA Japan, Inc., Tokyo, Japan) and same type of cages (Clean
S-PSF®, CLEA Japan Inc., Tokyo, Japan) in this aging study.
All experiments were approved by the Institutional Animal Care and Use Committee of SAPPORO
BREWERIES LTD. (permit number 2014-006) following the Guidelines for the Proper Conduct of Animal
Experiments on the Science Council of Japan. All experimental protocols and animal procedures were conducted
in accordance with the approved guidelines.
Food consumption, liquid consumption, body weight, and survival analysis. Food consumption
and liquid consumption per cage were recorded every week and three times every week, respectively. ese values
were expressed as one mouse’s consumption per day, which was calculated by dividing the total consumption per
cage by the number of mice in that cage. e body weight of each mouse was examined every week.
Grading aging-related scores. During the lifespan of all mice, we examined the changes in grading scores,
such as those for skin and hair conditions (glossiness, hair coarseness, and hair loss), ulcers, eyes (cataract, peri-
ophthalmic lesions, opacity of cornea, and ulcer of cornea), and skeleton (spinal curvature) nearly every month
from 6 weeks to 88 weeks old, as reported previously39.
Object recognition test (ORT) and object location test (OLT). e novel ORT and OLT for spa-
tial cognition are non-invasive and conducted under conditions similar to those used for human cognitive
assessment40.
We used the same boxes [300 × 300 × 350 mm (D × W × H); Brain Science Idea, Inc., Osaka, Japan] covered
with black plastic tetra-laterally for both the ORT and OLT. We measured cognitive functions by ORT and OLT
as previously reported30. For ORT objects, white golf balls (43-mm diameter) were used as training objects and a
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white lm case [29 mm (diameter) × 50 mm (high)] as the novel object. For OLT objects, apple-shaped wooden
blocks without colouring (31-mm width and 50-mm height of main body and 15-mm height of stem end) were
used. In the ORT and OLT training phases, mice were placed in the box and allowed to freely access the two
objects for 10 min, whereas in the test phase, the free access time was 10 min for the ORT and 5 min for the OLT.
e interval between the training and test phases was 24 h for the assessment of long-term memory during the
ORT, but 2 h for short-term memory during the OLT because SAMR1 and SAMP1 mice could not maintain
long-term spatial memory for the OLT (data not shown).
e ORT experiments were conducted at 8, 22, 34, 50, 66, and 79 weeks old and the OLT experiments were
conducted at 13, 14, 23, 35, 51, 67, and 80 weeks old to evaluate cognitive function. In the training phase, the
recognition index of the right and le objects for each mouse was expressed as the ratio of the amount of time
spent exploring object le A (time le A × 100)/(time le A + time right A) and amount of time spent exploring
object right A (time right A × 100)/(time le A + time right A) for both ORT and OLT. During the test phase, the
recognition index for each mouse was expressed as the ratio of the amount of time spent exploring the familiar
object A (time A × 100)/(time A + time B) and amount of time spent exploring the novel object B (time B × 100)/
(time A + time B) for the ORT or familiar object A (time A × 100)/(time A + time A′) and amount of time spent
exploring novel location object A′ (time A′ × 100)/(time A + time A′) for the OLT. Dierences between recogni-
tion indices of le and right (or novel location) objects were assessed by using the unpaired t-test for ORT (OLT)
in each phase.
Locomotor activity. Locomotor activity for 10 min (staying time and locomotor distance in the ORT box)
was quantied using the ANY-maze Video Tracking System (Stoelting Co., Wood Dale, IL, USA). Illumination
was provided at 25 lux. A black patch was attached to the back of each mouse to enable tracking in the ORT box
with a white-coloured oor.
Analysis of intestinal microbiome. Fresh faeces (approximately 100 mg) were collected, stored at −30 °C,
and used to analyse the intestinal microbiome. Bacterial DNA was isolated as described by Matsuki et al.41 with
some modications. Briey, the bacterial suspension was treated with lysis buer at 70 °C for 10 min in a water
bath and vortexed vigorously for 60 s with a Micro Smash MS-100 (Tomy Digital Biology Co., Ltd., Tokyo, Japan)
at 4,000 rpm.
Primers for amplication of the V1 and V2 regions of the 16S rRNA gene reported by Kim et al.42 were used
with some modications34. e following primers were used: forward primer (5′-CCATCTCATCCCTGCGTGT
CTCCGACTCAGNNNNNNNNNNGTagrgtttgatymtggctcag-3′) containing the Ion PGM sequencing primer
A-key, a unique erroneous 10–12-base pair barcode sequence (indicated with N), ‘GT’ spacer, and 27Fmod (agrgtttgaty
mtggctcag); and reverse primer (5′-CCTCTCTATGGGCAGTCGGTGATtgctgcctcccgtaggagt-3′) containing the
Ion PGM primer P1 and 338 R (tgctgcctcccgtaggagt). PCR was performed in a 25-μL reaction volume. Each reac-
tion mixture contained 22.5 μL of platinum PCR mix, 2 μL of template DNA (approximately 4 ng), and 0.5 μL of
10-μM primer mix. e amplication reaction was carried out in the Veriti ermal Cycler (Applied Biosystems,
Foster City, CA, USA) by using the following program: 3 min at 94 °C followed by 25 cycles of 30 s each at 94 °C,
45 s at 55 °C, and 1 min at 68 °C. Aer each reaction, the mixture was puried using a PureLink Quick PCR
Purication Kit (Invitrogen, Carlsbad, CA, USA); the concentration of each puried sample was measured using
a Qubit 2.0 uorometer (Life Technologies, Carlsbad, CA, USA). Puried samples were mixed at equal concentra-
tions. e mixed sample was visualized by electrophoresis on 2% agarose gel and puried by gel extraction using
a FastGene Gel/PCR Extraction Kit (Nippon Gene Co. Ltd., Tokyo, Japan). Emulsion PCR and sequencing were
carried out by Ion PGM sequencing systems (Life Technologies).
Aer sequencing, the obtained reads were analysed by the QIIME pipeline (http://qime.org/) for the assign-
ment of taxonomic classication. e reads that contained precise primer sequences (27Fmod and 338R) were
selected, and those with an average quality value >20 were used for further analysis. e reads were grouped into
operational taxonomic units (OTUs) with a sequence identity threshold of 97%; chimeric OTUs were removed by
using ChimeraSlayer. UniFrac distance analysis was performed, and the proportion of the intestinal microbiome
at the phylum and genus levels was determined by RDP (Ribosomal Database Project) classier using the green-
genes database (gg_13_8_otus/taxonomy/97_otu_taxonomy). e values are presented in the relative abundance
of microbiota.
Statistical analyses. SPSS soware 10.0.7J for Windows (SPSS, Inc., Chicago, IL, USA) was used for all
statistical analyses. Data in the text and gures are presented as the mean ± standard error of the mean. For loco-
motor activity and aging scores, between-group comparisons were performed by two-way analysis of variance
(ANOVA) followed by Bonferroni’s post hoc test for multiple comparisons. Between-group comparisons (between
bacterial species) were performed by unpaired t-tests. In all analyses, a P-value < 0.05 was considered statistically
signicant.
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Acknowledgements
We thank Ms. Izumi Nomura, Frontier Laboratories for Value Creation, SAPPORO HOLDINGS LTD., for
helping with the animal experiments.
Author Contributions
C.S. and Y.W. designed the study and performed experiments. T.I., M.H., M.O., Y.M., S.S. and Y.T. provided
technical support and conceptual advice. T.N. supervised experiments and provided critical revision for the
manuscript.
Additional Information
Competing Interests: C.S., Y.W., T.I., M.H., S.S. and Y.T. are employees of SAPPORO HOLDINGS LTD. Y.M. is
a retiree of SAPPORO HOLDINGS LTD. M.O. is an employee of POKKA SAPPORO Food & Beverage Ltd. T.N.
has consulted for SAPPORO HOLDINGS LTD. and received compensation.
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