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

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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 host1113.
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 mouses 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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... Further, anti-SASP effects of dietary flavonoids apigenin and kaempferol in bleomycin-induced senescence in fibroblasts were also reported that involved inhibition of the NF-κB pathway via IRAK1/IκBα signaling (Lim et al., 2015). In addition, cellular senescence suppressive attributes of polyphenol-rich fractions isolated from fruits such as lemons (Shimizu et al., 2019), grape seed extract Xu et al., 2021), as well as red wine (Botden et al., 2012) have also been documented. In addition, a growing interest amongst polyphenols is the identification of novel senolytics that may selectively induce apoptosis in SC and thus alleviate SC burden in tissues with age Wang et al., 2021). ...
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The process of cellular senescence is rapidly emerging as a modulator of organismal aging and disease. Targeting the development and removal of senescent cells is considered a viable approach to achieving improved organismal healthspan and lifespan. Nutrition and health are intimately linked and an appropriate dietary regimen can greatly impact organismal response to stress and diseases including during aging. With a renewed focus on cellular senescence, emerging studies demonstrate that both primary and secondary nutritional elements such as carbohydrates, proteins, fatty acids, vitamins, minerals, polyphenols, and probiotics can influence multiple aspects of cellular senescence. The present review describes the recent molecular aspects of cellular senescence-mediated understanding of aging and then studies available evidence of the cellular senescence modulatory attributes of major and minor dietary elements. Underlying pathways and future research directions are deliberated to promote a nutrition-centric approach for targeting cellular senescence and thus improving human health and longevity.
... The gut microbiota plays a central role in the conversion of dietary polyphenols into phenolic acids [165] and these were shown to increase gut microbiota diversity, decrease the Firmicutes/Bacteroidetes ratio, and modulate metabolic activity, leading the gut microbiota to a healthier profile [166,167]. The administration of lemon polyphenols in drinking water improved aging-related scores and locomotor activity, and increased the ratio of Bacteroidetes/Firmicutes senescenceaccelerated mouse prone 1 (SAMP1) [168]. Curcumin, a polyphenolic substance, improved cognition and reduced amyloid plaque in APP/PS1 mice while modulating the relative abundance of bacteria involved in AD development [169]. ...
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Recent research has been uncovering the role of the gut microbiota for brain health and disease. These studies highlight the role of gut microbiota on regulating brain function and behavior through immune, metabolic, and neuronal pathways. In this review we provide an overview of the gut microbiota axis pathways to lay the groundwork for upcoming sessions on the links between the gut microbiota and neurogenerative disorders. We also discuss how the gut microbiota may act as an intermediate factor between the host and the environment to mediate disease onset and neuropathology. Based on the current literature, we further examine the potential for different microbiota-based therapeutic strategies to prevent, to modify, or to halt the progress of neurodegeneration.
... Chlorogenic acid was reported to have a good scavenging rate for DPPH free radicals and ABTS+ free radicals (Xu et al., 2012). Studies have shown that eriocitrin is the main lemon polyphenol, and by consuming eriocitrin daily, the antioxidant capacity of rats was improved (Shimizu et al., 2019). Vitexin can inhibit the release of proinflammatory factor and up-regulate the level of anti-inflammatory factor (Borghi et al., 2013). ...
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Inflammation is a characteristic of obesity. The rich compounds in lemon peel have anti‐inflammatory effects. This study examined whether fermented lemon peel can have an anti‐obesity effect on obese mice induced by a high‐fat diet (HFD) by regulating inflammation. The lemon peel fermentation supernatant (LPFS) could inhibit the weight gain of mice and improve the lesions of the liver and epididymal adipose tissue. In addition, LPFS regulates blood lipids, liver function, and inflammation‐related indicators in the serum of obese mice. LPFS plays a positive role in regulating the inflammation and obesity‐related genes in liver tissue and adipose tissue of obese mice. High‐performance liquid chromatography showed an increase in the contents of compounds with antioxidant or/and anti‐inflammatory effects and compounds with anti‐obesity effects. These results suggest that the LPFS could help reduce obesity in obese mice induced by an HFD by adjusting the balance of the inflammatory response. Practical applications Obesity often increases the risk of chronic diseases, and mild inflammation is a feature of obesity. Therefore, timely suppression of inflammation in the body can help control the occurrence of obesity. This study clarified the anti‐obesity effect of fermented lemon peel on a high‐fat diet (HFD)‐induced obese mice by regulating the body's inflammatory response and confirmed that fermentation improves the anti‐inflammatory activity of lemon peel. This study provides important references for future investigation, prophylaxis, and treatment of inflammation and obesity‐related diseases, as well as the advances in functional foods and fermented foods with anti‐inflammatory and anti‐obesity activities.
... The activities of Cu/Zn-superoxide dismutase [18] and uncoupling protein 1 (Ucp1) are decreased in SAMP1 [18,19]. Due to these defects in the mitochondrial function, SAMP1 exhibits increased oxidative stress in various organs [18,20,21] and has been used to test antioxidants [22,23]. ...
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Objective Age is a risk factor for type 2 diabetes (T2D). We aimed to elucidate whether β cell glucose metabolism is altered with aging and contributes to T2D. Methods As a model of aging, we used senescence-accelerated mouse (SAM), C57BL/6J (B6), and ob/ob mice. As a diabetes model, we used db/db mice. Glucose responsiveness of insulin secretion and [U-¹³C]-glucose metabolic flux were examined in isolated islets. As molecular signatures of β cell identity, we analyzed expression of β-cell-specific genes in isolated islets and pancreatic sections. β cells defective in the malate-aspartate (MA) shuttle were previously generated from MIN6-K8 cells by knockout of Got1, a component of the shuttle. We analyzed Got1 KO β cells as a model of increased glycolysis. Results We identified hyperresponsiveness to glucose and compromised cellular identity as dysfunctional phenotypes shared in common between aged and diabetic mouse β cells. We also reveal a metabolic commonality between aged and diabetic β cells: hyperactive glycolysis through increased expression of Nmnat2, a cytosolic NAD-synthesizing enzyme. Got1 KO β cells showed increased glycolysis, β cell dysfunction, and impaired cellular identity, phenocopying aging and diabetes. Using Got1 KO β cells, we show that attenuation of glycolysis or Nmnat2 activity can restore β cell function and identity. Conclusion Our study demonstrates that hyperactive glycolysis is a metabolic signature of aged and diabetic β cells, which may underlie age-related β cell dysfunction and loss of cellular identity. We suggest Nmnat2 suppression as an approach to counteract age-related T2D.
... The results also revealed a very low compliance with those practices that are associated with boosting the immune system. Although there are some controversies on the efficacy of some these food products in enhancing the immune system, some studies have shown some antimicrobial, antioxidant and health-promoting properties of many of them [79][80][81][82]. Specifically, the intake of warm water has been found to enhance the management of fluids in patients with upper respiratory tract infections [79], while garlic possesses some antimicrobial properties [80]. ...
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... 54 For example, the abundance of the intestinal microbiota that may be associated with aging was limited by the intake of lemon polyphenols. 55 Dietary consumption of anthocyanins increased Bacteroidetes and short-chain fatty acids (SCFAs) and decreased Firmicutes. 56 Red wine polyphenols significantly increased the number of Bifidobacteria and Lactobacillus and butyrateproducing bacteria (Faecalibacterium prausnitzii and Roseburia) but decreased undesirable bacterial groups such as Escherichia coli and Enterobacter cloacae. ...
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Identifying ways to deal with the challenges presented by aging is an urgent task, as we are facing an aging society. External factors such as diet, exercise and drug therapy have proven to be major elements in controlling healthy aging and prolonging life expectancy. More recently, the intestinal microbiota has also become a key factor in the anti-aging process. As the intestinal microbiota changes with aging, an imbalance in intestinal microorganisms can lead to many age-related degenerative diseases and unhealthy aging. This paper reviews recent research progress on the relationship between intestinal microorganisms and anti-aging effects, focusing on the changes and beneficial effects of intestinal microorganisms under dietary intervention, exercise and drug intervention. In addition, bacteriotherapy has been used to prevent frailty and unhealthy aging. Most of these anti-aging approaches improve the aging process and age-related diseases by regulating the homeostasis of intestinal flora and promoting a healthy intestinal environment. Intervention practices based on intestinal microorganisms show great potential in the field of anti-aging medicine.
... Despite the advances in drug discovery and development during the last decades, herbal medicine continues to be used as primary therapy in many developing countries (nearly 4 billion persons) (Ekor, 2014). Regular consumption of polyphenols has been related to beneficial health effects, including regulation of the intestinal microbiota and antiaging effects (Shimizu et al., 2019), a risk reduction of atherosclerosis (Nie et al., 2019), a decrease in the risk of colorectal cancer development (Bahrami et al., 2019), and the modulation of antioxidant enzymes through Nrf2 regulation . One of the major challenges for the therapeutic use of polyphenols is their low oral bioavailability. ...
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Polyphenols constitute an important group of natural products that are traditionally associated with a wide range of bioactivities. These are usually found in low concentrations in natural products and are now available in nutraceuticals or dietary supplements. A group of polyphenols that include apigenin, quercetin, curcumin, resveratrol, EGCG, and kaempferol have been shown to regulate signaling pathways that are central for cancer development, progression, and metastasis. Here, we describe novel mechanistic insights on the effect of this group of polyphenols on key elements of the signaling pathways impacting cancer. We describe the protein modifications induced by these polyphenols and their effect on the central elements of several signaling pathways including PI3K, Akt, mTOR, RAS, and MAPK and particularly those affecting the tumor suppressor p53 protein. Modifications of p53 induced by these polyphenols regulate p53 gene expression and protein levels and posttranslational modifications such as phosphorylation, acetylation, and ubiquitination that influence stability, subcellular location, activation of new transcriptional targets, and the role of p53 in response to DNA damage, apoptosis control, cell- cycle regulation, senescence, and cell fate. Thus, deep understanding of the effects that polyphenols have on these key players in cancer-driving signaling pathways will certainly lead to better designed targeted therapies, with less toxicity for cancer treatment. The scope of this review centers on the regulation of key elements of cancer signaling pathways by the most studied polyphenols and highlights the importance of a profound understanding of these regulations in order to improve cancer treatment and control with natural products.
... The prebiotic function of polyphenols has been widely recognized as a health-related issue, where some specific dietary polyphenols not only enhance the growth of probiotics such as Lactobacillus and Bifidobacterium but also inhibit the reproduction of pathogenic bacteria. For example, several polyphenols identified in mango [139] and lemon [140] and non-extractable polyphenols [141] can promote the growth of some bacteria that are beneficial to human health, such as Ackermann, Christensenellaceae, Verrucomicrobia Bifidobacterium, and Lactobacillus (Fig. 7). ...
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Tobacco is grown in large quantities worldwide as a widely distributed commercial crop. From the harvest of the field to the process into the final product, a series of procedures generate enormous amount of waste materials that are rarely recycled. In recent years, numerous potential bioactive compounds have been isolated from tobacco, and the molecular regulatory mechanisms related to the performance of some functionalities have been identified. This review describes the source of tobacco waste and expounds a large amount of biomass during the tobacco processing, and the necessity of exploring the reuse of tobacco waste. In addition, the review summarizes the bioactive compounds from tobacco that have been discovered so far, and links them to various functions from tobacco extracts, including anti-inflammatory, antitumor, antibacterial, and antioxidant, thus proving the po-tential value from tobacco waste reuse. In this regard, nornicotine in tobacco is the culprit of many health issues, while the polyphenols and polysaccharides often contribute to the health benefits of tobacco extract. In addition, it is hard to ignore that realization of these functions of tobacco extracts require the involvement of intestinal flora metabolism, which should be considered in the development of new product dosage forms.
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In the food and beverage industries, implementing novel methods using digital technologies such as artificial intelligence (AI), sensors, robotics, computer vision, machine learning (ML), and sensory analysis using augmented reality (AR) has become critical to maintaining and increasing the products’ quality traits and international competitiveness, especially within the past five years. Fermented beverages have been one of the most researched industries to implement these technologies to assess product composition and improve production processes and product quality. This Special Issue (SI) focused on the latest research on the application of digital technologies on beverage fermentation monitoring and the improvement of processing performance, product quality and sensory acceptability.
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Aging induces significant shifts in the composition of gut microbiota associated with decreased microbial diversity. Age‐related changes in gut microbiota include a loss of commensals and an increase in disease‐associated pathobionts. These alterations are accelerated by lifestyle factors, such as poor nutritional habits, physical inactivity, and medications. Given that diet is one of the main drivers shaping the gut microbiota, nutritional interventions for restoring gut homeostasis are of great importance to the overall health of older adults. Polyphenols, ubiquitously present in fruits and vegetables, have emerged as promising anti‐aging candidates because of their ability to modulate some of the common denominators of aging, including gut dysbiosis. These compounds can influence the composition of the gut microbiota, and gut bacteria metabolize polyphenols into bioactive compounds that produce relevant health effects. Although the role of polyphenols on the aging gut has not been fully characterized, accumulating evidence suggests that these compounds exert selective effects on the gut microbial community. Here, we discuss the reciprocal interactions between polyphenols and gut microbiota and summarize the latest findings on the effects of polyphenols on modulating intestinal bacteria during aging.
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Background 16S rRNA gene amplicon sequencing analysis (16S amplicon sequencing) has provided considerable information regarding the ecology of the intestinal microbiome. Recently, metabolomics has been used for investigating the crosstalk between the intestinal microbiome and the host via metabolites. In the present study, we determined the accuracy with which 16S rRNA gene data at different classification levels correspond to the metabolome data for an in-depth understanding of the intestinal environment. Results Over 200 metabolites were identified using capillary electrophoresis and time-of-flight mass spectrometry (CE-TOFMS)-based metabolomics in the feces of antibiotic-treated and untreated mice. 16S amplicon sequencing, followed by principal component analysis (PCA) of the intestinal microbiome at each taxonomic rank, revealed differences between the antibiotic-treated and untreated groups in the first principal component in the family-, genus, and species-level analyses. These differences were similar to those observed in the PCA of the metabolome. Furthermore, a strong correlation between principal component (PC) scores of the metabolome and microbiome was observed in family-, genus-, and species-level analyses. Conclusions Lower taxonomic ranks such as family, genus, or species are preferable for 16S amplicon sequencing to investigate the correlation between the microbiome and metabolome. The correlation of PC scores between the microbiome and metabolome at lower taxonomic levels yield a simple method of integrating different “-omics” data, which provides insights regarding crosstalk between the intestinal microbiome and the host. Electronic supplementary material The online version of this article (10.1186/s12866-018-1311-8) contains supplementary material, which is available to authorized users.
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Purpose: Decaffeinated green tea (GT) and black tea (BT) polyphenols inhibit weight gain in mice fed an obesogenic diet. Since the intestinal microflora is an important contributor to obesity, it was the objective of this study to determine whether the intestinal microflora plays a role in the anti-obesogenic effect of GT and BT. Methods: C57BL/6J mice were fed a high-fat/high-sucrose diet (HF/HS, 32% energy from fat; 25% energy from sucrose) or the same diet supplemented with 0.25% GTP or BTP or a low-fat/high-sucrose (LF/HS, 10.6% energy from fat, 25% energy from sucrose) diet for 4 weeks. Bacterial composition was assessed by MiSeq sequencing of the 16S rRNA gene. Results: GTP and BTP diets resulted in a decrease of cecum Firmicutes and increase in Bacteroidetes. The relative proportions of Blautia, Bryantella, Collinsella, Lactobacillus, Marvinbryantia, Turicibacter, Barnesiella, and Parabacteroides were significantly correlated with weight loss induced by tea extracts. BTP increased the relative proportion of Pseudobutyrivibrio and intestinal formation of short-chain fatty acids (SCFA) analyzed by gas chromatography. Cecum propionic acid content was significantly correlated with the relative proportion of Pseudobutyrivibrio. GTP and BTP induced a significant increase in hepatic 5'adenosylmonophosphate-activated protein kinase (AMPK) phosphorylation by 70 and 289%, respectively (P < 0.05) determined by Western blot. Conclusion: In summary, both BTP and GTP induced weight loss in association with alteration of the microbiota and increased hepatic AMPK phosphorylation. We hypothesize that BTP increased pAMPK through increased intestinal SCFA production, while GTPs increased hepatic AMPK through GTP present in the liver.
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Scope: To understand the mechanism by which green tea lowers the risk of dementia, focus was placed on the metabolites of epigallocatechin gallate (EGCG), the most abundant catechin in green tea. Much of orally ingested EGCG is hydrolyzed to epigallocatechin (EGC) and gallic acid. In rats, EGC is then metabolized mainly to 5-(3', 5'-dihydroxyphenyl)-γ-valerolactone (EGC-M5) and its conjugated forms, which are distributed to various tissues. Therefore, we examined the permeability of these metabolites into the blood-brain barrier (BBB), and nerve cell proliferation/differentiation in vitro. Methods and results: The permeability of EGC-M5, glucuronide and the sulfate of EGC-M5, pyrogallol, as well as its glucuronide into the BBB were examined using a BBB model kit. Each brain- and blood-side sample was subjected to liquid chromatography tandem-mass spectrometry analysis. BBB permeability (%, in 0.5 h) was 1.9-3.7 %. In human neuroblastoma SH-SY5Y cells, neurite length was significantly prolonged by EGC-M5, and the number of neurites was increased significantly by all metabolites examined. Conclusion: The permeability of EGC-M5 and its conjugated forms into the BBB suggests that they reached the brain parenchyma. In addition, the ability of EGC-M5 to affect nerve cell proliferation and neuritogenesis suggests that EGC-M5 may promote neurogenesis in the brain. This article is protected by copyright. All rights reserved.
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Purpose: Heavy and long-term alcohol consumption increase the risk of alcohol-related diseases. Epidemiological studies show moderate drinking reduces the risk of mortality, cardiovascular diseases, and brain infarction in the J-shaped or U-shaped curve effect. However, why moderate drinkers may be healthy and non-drinkers may be ill in diverse populations remains controversial. Herein, we examined the relationship between moderate/lifelong alcohol intake and aging, especially aging-related cognitive functions in senescence-accelerated mouse prone 8 (SAMP8) model. Methods: SAMP8 model (5-week-old, male, n = 36), a model of age-related cognitive deficit, were group-housed (n = 6/cage) and provided free access to water (water group, n = 18) or 1% ethanol (EtOH group, n = 18, intake started when mice were 9 weeks old). The object recognition test (ORT) and object location test (OLT) were used to evaluate cognitive functions. The intestinal flora at the age of 87 weeks was analyzed by terminal restriction fragment length polymorphism (T-RFLP). Results: The lifespan of the EtOH-group mice was about 4 weeks longer than that of the water-group mice. In the EtOH group, spatial recognition impairment, assessed by OLT, was observed later (age, 73 weeks) than that in the water group (age, 52 weeks). The spinal curvature and skin conditions progressed significantly slower in the EtOH group than in the water group. Moreover, diarrhea symptoms only appeared in the water group, at the age of 82 weeks. The T-RFLP analysis of the intestinal flora indicated higher Lactobacillales order and lower Clostridium cluster XI in the EtOH group than in the water group, although those were extremely high in some mice close to death in both groups. Water-group mice with diarrhea presented significantly higher Clostridium cluster XI than did those without diarrhea (P = 0.017). Conclusion: Moderate alcohol intake changes intestinal flora and positively affects aging of SAMP8 model.
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Background It has been reported that the composition of human gut microbiota changes with age; however, few studies have used molecular techniques to investigate the long-term, sequential changes in gut microbiota composition. In this study, we investigated the sequential changes in gut microbiota composition in newborn to centenarian Japanese subjects. Results Fecal samples from 367 healthy Japanese subjects between the ages of 0 and 104 years were analyzed by high-throughput sequencing of amplicons derived from the V3-V4 region of the 16S rRNA gene. Analysis based on bacterial co-abundance groups (CAGs) defined by Kendall correlations between genera revealed that certain transition types of microbiota were enriched in infants, adults, elderly individuals and both infant and elderly subjects. More positive correlations between the relative abundances of genera were observed in the elderly-associated CAGs compared with the infant- and adult-associated CAGs. Hierarchical Ward’s linkage clustering based on the abundance of genera indicated five clusters, with median (interquartile range) ages of 3 (0–35), 33 (24–45), 42 (32–62), 77 (36–84) and 94 (86–98) years. Subjects were predominantly clustered with their matched age; however, some of them fell into mismatched age clusters. Furthermore, clustering based on the proportion of transporters predicted by phylogenetic investigation of communities by reconstruction of unobserved states (PICRUSt) showed that subjects were divided into two age-related groups, the adult-enriched and infant/elderly-enriched clusters. Notably, all the drug transporters based on Kyoto Encyclopedia of Genes and Genomes (KEGG) Orthology groups were found in the infant/elderly-enriched cluster. Conclusion Our results indicate some patterns and transition points in the compositional changes in gut microbiota with age. In addition, the transporter property prediction results suggest that nutrients in the gut might play an important role in changing the gut microbiota composition with age. Electronic supplementary material The online version of this article (doi:10.1186/s12866-016-0708-5) contains supplementary material, which is available to authorized users.
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The flavanones hesperidin, eriocitrin and eriodictyol were investigated for their prevention of the oxidative stress and systemic inflammation caused by high-fat diet in C57BL/6J mice. The mice received a standard diet (9.5% kcal from fat), high-fat diet (45% kcal from fat) or high-fat diet supplemented with hesperidin, eriocitrin or eriodictyol for a period of four weeks. Hesperidin, eriocitrin and eriodictyol increased the serum total antioxidant capacity, and restrained the elevation of interleukin-6 (IL-6), macrophage chemoattractant protein-1 (MCP-1), and C-reactive protein (hs-CRP). In addition, the liver TBARS levels and spleen mass (g per kg body weight) were lower for the flavanone-treated mice than in the unsupplemented mice. Eriocitrin and eriodictyol reduced TBARS levels in the blood serum, and hesperidin and eriodictyol also reduced fat accumulation and liver damage. The results showed that hesperidin, eriocitrin and eriodictyol had protective effects against inflammation and oxidative stress caused by high-fat diet in mice, and may therefore prevent metabolic alterations associated with the development of cardiovascular diseases in other animals.
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In a present internet investigation, a randomized double-blind, placebo-controlled, parallel group investigation, 625 non-patient subjects feeling fatigue were randomized to oral citric acid or placebo administration for 28 days so that there were no statistical differences in age and gender. Subjects took a can of drink containing lemon citric acid (2700mg) or placebo drink per day for 28 days. As a result, administration of lemon citric acid attenuated the fatigue feeling in visual analogue scale (VAS). Therefore, it was turned out that the drink containing lemon citric acid was useful for people frequently feeling fatigue.
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Abnormal composition of intestinal bacteria-"dysbiosis"-is characteristic of Crohn's disease. Disease treatments include dietary changes and immunosuppressive anti-TNFα antibodies as well as ancillary antibiotic therapy, but their effects on microbiota composition are undetermined. Using shotgun metagenomic sequencing, we analyzed fecal samples from a prospective cohort of pediatric Crohn's disease patients starting therapy with enteral nutrition or anti-TNFα antibodies and reveal the full complement and dynamics of bacteria, fungi, archaea, and viruses during treatment. Bacterial community membership was associated independently with intestinal inflammation, antibiotic use, and therapy. Antibiotic exposure was associated with increased dysbiosis, whereas dysbiosis decreased with reduced intestinal inflammation. Fungal proportions increased with disease and antibiotic use. Dietary therapy had independent and rapid effects on microbiota composition distinct from other stressor-induced changes and effectively reduced inflammation. These findings reveal that dysbiosis results from independent effects of inflammation, diet, and antibiotics and shed light on Crohn disease treatments.