Journal of Functional Foods 48 (2018) 1–8

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Journal of Functional Foods

journal homepage: www.elsevier.com/locate/jff

Pancreatic lipase inhibition of strictinin isolated from Pu’er tea (Cammelia T

sinensis) and its anti-obesity effects in C57BL6 mice☆
⁎
Tzu-Yin Chena, Miki M.C. Wanga, Sheng-Kuo Hsieha, Meng-Huang Hsiehb, Wen-Ying Chenc, ,
⁎⁎
Jason T.C. Tzena,
a
Graduate Institute of Biotechnology, National Chung-Hsing University, Taichung 402, Taiwan
b
Goldencrops Corporation, Yun Lin Technology Industrial Park, Yun Lin 640, Taiwan
c
Department of Veterinary Medicine, National Chung-Hsing University, Taichung 402, Taiwan

A R T I C LE I N FO A B S T R A C T

Keywords: Strictinin isolated from Pu’er tea was demonstrated to inhibit pancreatic lipase in vitro. In an olive oil tolerance
Anti-obesity test, the administration of strictinin (100 mg/kg) significantly reduced the blood triglyceride level and increased
Blood triglyceride the fecal oil content of C57BL6 mice. In a long-term animal study, the body weight gained via high-fat diet was
Pancreatic lipase significantly reduced in the mice supplemented with strictinin of 130 mg/kg. Further analysis showed that
Pu’er tea
weight of epididymal fat, but not that of liver or muscle, increased in mice fed with high-fat diet, and the
Strictinin
increase of fat was reduced in the mice supplemented with strictinin. Correspondingly, sizes of adipocytes in
epididymis were substantially enlarged in mice fed with high-fat diet, and the enlargement of adipocytes was
decreased when strictinin was supplemented. Levels of blood triglyceride, cholesterol and glucose were elevated
in mice fed with high-fat diet, and these elevated levels were reduced when strictinin was supplemented.

1. Introduction
Tea is one kind of beverages widely consumed around the world,
and its major soluble compounds, polyphenols have been demonstrated
to provide a variety of beneficial functions to human health (Haslam,
2003; Higdon & Frei, 2003; Lo et al., 2014; Yukihiko, 2012). Pu’er tea is
produced from young leaves of Camellia sinensis var. assamica via a
unique process termed post-fermentation, in which microorganisms
seem to play a key role during the chemical conversion for its specia-
lized aroma and taste (Lee & Foo, 2013; Maeda-Yamamoto et al., 2004).
Besides its distinct aroma and taste, Pu’er tea is assumed to possess a
wide range of therapeutic effects, such as anti-obesity, anti-mutagenic,
antiviral, and antimicrobial activities as well as hyperlipidemia, hy-
poglycemic and free radical scavenging effects (Du et al., 2012; Hou
et al., 2009; Qian, Guan, Yang, & Li, 2008; Saha et al., 2010; Su, Zhang,
Wang, & Li, 2012; Wang, Yu, Chang, Yena, & Duh, 2008; Zhao, Jia,
Tang, Sheng, & Luo, 2011). Empirically, Pu’er teas produced from wild
trees are perceived to possess better biological effects than those pro-
duced from cultivated shrubs, particularly for the healing effects
against viral and microbial infections as well as the anti-obesity and
laxative effects on defecation (Chen, Lin, Hsu, Hsieh, & Tzen, 2015;
Hsieh et al., 2016; Li et al., 2014; Pei, Zhang, Xu, Chen, & Chen, 2011;
Wu et al., 2007).
Strictinin, a hydrolysable tannin belonging to the family of ellagi-
tannin, is frequently identified as a minor phenolic compound in var-
ious teas in comparison with the abundant catechins (Yagi, Goto, &
Nanjo, 2009; Zhao, Chen, Lin, Yu, & Li, 2011). Nevertheless, instead of
catechins, strictinin was identified as the major phenolic compound in
raw Pu’er teas produced from leaves and buds of wild trees (Chen et al.,
2015). Interestingly, the content of strictinin in leaves of wild trees was
found significantly higher than that in leaves of cultivated shrubs.
Several therapeutic activities of strictinin have been reported, such as
antiviral, antibacterial, antiallergic, immunostimulator, and laxative
effects in the past two decades (Chen et al., 2015; Hsieh et al., 2016;
Monobe, Ema, Kato, & Maeda-Yamamoto, 2008; Saha et al., 2010;
Tachibana et al., 2001). Moreover, strictinin was suggested to be an
adequate ingredient for the supplementation in food, cosmetics and
beverages (Tsuji, Yamamoto, Kawamoto, & Tachibana, 2003).
Excessive food consumption prevails over modern diets, and thus
more and more people accumulate excess body fat and suffer in over-
weight. At present, obesity becomes a major public health problem and
is commonly associated with the risk of illness, such as hypertension,

☆
Chemical compounds studied in this article: strictinin (PubChem CID: 73330).
⁎
Corresponding author at: Department of Veterinary Medicine National Chung-Hsing University, Taichung 40227, Taiwan.
⁎⁎
Corresponding author at: Graduate Institute of Biotechnology National Chung-Hsing University, Taichung 40227, Taiwan.
E-mail addresses: jack@goldencrops.com.tw (M.-H. Hsieh), wychen@dragon.nchu.edu.tw (W.-Y. Chen), tctzen@dragon.nchu.edu.tw (J.T.C. Tzen).

https://doi.org/10.1016/j.jff.2018.06.020
Received 28 November 2017; Received in revised form 8 April 2018; Accepted 20 June 2018
Available online 27 June 2018
1756-4646/ © 2018 Published by Elsevier Ltd.
T.-Y. Chen et al. Journal of Functional Foods 48 (2018) 1–8

0.2 A caffeine
Raw Pu’er tea
0.15 5-galloylquinic acid
+ gallic acid
strictinin

0.1
EGCG ECG +
flavonol glycosides
0.05
theobromine

caffeine
0.2 B Ripen Pu’er tea
Relative Absorbance

0.15

0.1

0.05

2 C strictinin
Isolated strictinin
1.5

0.5

0 10 20 30 40 50 60 70 80 90 100
Time (min)
Fig. 1. HPLC profiles of raw Pu’er tea infusion (A), ripen Pu’er tea infusion (B) and isolated strictinin (C) at 254 nm. Chemical constituents in the infusion of Pu’er teas
were separated by HPLC (0–100 min).The peaks of caffeine, strictinin and epigallocatechin-3-gallate (EGCG) were indicated.

heart disease and diabetes. Reasonably, prevention of fat absorption
from diets is an effective way for the treatment of diet-induced obesity.
As direct uptake of dietary triglycerides is unworkable in the digestion
system, pancreatic lipase is secreted into small intestine and hydrolyzes
triglycerides to monoglycerides and free fatty acids prior to absorption
by epithelial cells in the small intestine. In this aspect, orlistat, an au-
thorized anti-obesity drug, is taken to prevent absorption of fat in diets
by acting as an inhibitor of pancreatic lipase in small intestine (Zhi,
Melia, Eggers, Joly, & Patel, 1995).
We wondered if the empirical anti-obesity effects of Pu’er tea might
be resulted from the prevention of fat absorption from diets by in-
hibiting pancreatic lipase in a manner similar to orlistat. To examine
this possibility, raw and ripen Pu’er teas as well as three major com-
pounds, caffeine, strictinin and (−)-epigallocatechin gallate (EGCG) in
raw Pu’er tea were subjected to an in vitro assay of pancreatic lipase
inhibition. The results suggested that strictinin might be an adequate
inhibitor of pancreatic lipase. Subsequently, two animal models, olive
oil tolerance test and reduction of fat accumulation in high-fat diet,
were executed to evaluate the anti-obesity effects of strictinin.
2. Materials and methods
2.1. Chemicals and materials
obtained from J.T. Baker (Mallinckrodt Baker, Inc., Phillipsburg, NJ,
USA). Double-distilled water was afforded by a Millipore clear water
purification system (Direct-Q, Millipore, Billerica, MA, USA). Porcine
pancreatic lipase Type II was purchased from Sigma Co. (Sigma-
Aldrich, St. Louis, MO, USA). Caffeine (≥99%) was obtained from
Merck KGaA (Darmstadt, Germany) and (−)-epigallocatechin gallate
(EGCG) (HPLC ≥ 98%) were purchased from QualiFlex Co. (Taipei,
Taiwan). Raw Pu’er tea prepared from leaves of Yunnan wild trees
(Camellia sinensis var. assamica) in 2008 was purchased from a local
store in Taichung, Taiwan, and used to isolate strictinin. Ripen Pu’er tea
of 2010 was a gift from another local store in Taipei, Taiwan.
2.2. Purification of strictinin
Strictinin was extracted from raw Pu’er tea according to the method
as described previously (Chen et al., 2015). In each preparation, Pu’er
tea leaves of 50 g were ground into fine powder and extracted with
water of 600 mL (×3) at 70 °C for 30 min. The infusion was partitioned
with ethyl acetate and butanol. The butanol layer was concentrated,
and the powder was re-dissolved in water and loaded onto a Sephadex
LH-20 column. Fractions containing strictinin eluted with 30–100%
methanol gradient were pooled and concentrated, and the powder was
dissolved in water for the following experiments.

Ethyl acetate, butanol and methanol were purchased from ECHO 2.3. Preparation of tea infusion and HPLC analysis
Chemical, Co. (Taichung, Taiwan). High performance liquid chroma-
tography (HPLC) grade acetonitrile and methanol were purchased from Tea infusion was prepared by adding 10 mL of water to 0.5 g of raw
Fisher Scientific (Fair Lawn, NJ, USA). Acetic acid (99.7%) was or ripen Pu’er tea at 70 °C for 15 min. After extraction, the brew was

2
T.-Y. Chen et al. Journal of Functional Foods 48 (2018) 1–8

120 350
A Control 100 μg/mL 250 μg/mL 500 μg/mL
A olive oil

Blood triglyceride (mg/dL)

strictinin 10 mg/kg
100 300 strictinin 50 mg/kg
Relative lipase activity (%)

strictinin 100 mg/kg

80 orlistat 10 mg/kg
250
ddw
60
200
40
150
20
100
0
Raw Pu'er Ripen Pu'er Strictinin EGCG Caffeine 50
tea tea 0 1.5 3 4.5 6
Time (h)

Area under the dose-response curve

1400
B
B 1200

1000
Relative lipase activity (%)

800

600

400

200

0
ddw olive oil strictinin strictinin strictinin orlistat
10 mg/kg 50 mg/kg 100 mg/kg 10 mg/kg

0.30 *
ddw
Fecal oil content (mg/6h)

C
Ln concentration of strictinin (μg/mL) olive oil
strictinin 100 mg/kg
Fig. 2. Inhibition of pancreatic lipase by Pu’er teas and its three major com-
0.20
pounds. Relative pancreatic lipase activities were detected in the presence of
raw Pu’er tea infusion, ripen Pu’er tea infusion, strictinin, EGCG, and caffeine of
100, 250 and 500 μg/mL (A). Values are expressed as mean ± SD (n = 6 per
group). *P < 0.05 vs. control. The inhibitory potency on pancreatic lipase was 0.10
detected in the presence of strictinin of different concentrations to calculate its
IC50 value (B).

filtered through a 0.22 μm polyvinylidene difluoride (PVDF) membrane 0.00

filter (PALL Corporation, Glen Cove, NY, USA), and used for the fol-
lowing analysis. Chemical constituents in the tea infusion were ana-
lyzed on a HPLC system coupled to a Model 600E photodiode array
detector (Waters Corporation, Milford, MA, USA) and performed using
a Syncronis T3 column of 250 mm × 4.6 mm inner diameter, 5 μm,
(Waters, USA) as described previously (Lee et al., 2008). The mobile
phase consisted of (A) water and (B) acetonitrile containing 0.5% acetic
acid. The gradient was as following: 0–100 min, linearly gradient from
5 to 30% B; and 100–105 min, linear gradient from 30 to 5% B. The
column was maintained at room temperature and the injection volume
was 20 μL at a flow rate of 1 mL/min. The UV absorbance detection
wavelength was set at 254 nm. Caffeine, strictinin and EGCG shown in
the HPLC profile were identified according to the same procedure as
described previously (Chen et al., 2015).
2.4. Pancreatic lipase activity assay
Pancreatic lipase was dissolved in deionized water at 10 mg/mL,
and centrifuged to collect the supernatant at 9000g for 5 min. The
substrate contained 0.08% w/v p-nitrophenyl laurate dissolved in 5 mM
sodium acetate buffer (pH 5.0) with 1% Triton X-100. The substrate
solution was heated in boiling water for 1 min to aid dissolution, and
then cooled down to room temperature prior to the enzymatic assay.
The assay buffer contained 100 mM of Tris-HCl buffer (pH 8.2). For the
enzymatic assay, tea samples or compounds of 50 μL were firstly mixed
with the assay buffer of 400 μL and pancreatic lipase of 150 μL, and
then incorporated with the substrate solution of 450 μL to initiate the
ddw olive oil strictinin 100 mg/kg
Fig. 3. The profile of blood triglyceride levels in mice after administered with
olive oil. (A) The mice were orally administered with strictinin (10, 50 or
100 mg/kg) or orlistat (10 mg/kg) before administration of olive oil. Plasma
triglyceride levels in mice were measured at 0, 1.5, 3, 4.5 and 6 h after ad-
ministration of olive oil. (B) Area under the dose-response curve was calculated
for each treatment. Each value represents the mean ± S.E.M. (n = 4 per
group). # P < 0.05 vs. double distilled water (ddw) treatment and * P < 0.05
vs. olive oil treatment. (C) Fecal oil contents of mice in three groups, (1) double
distilled water, (2) olive oil and (3) olive oil plus strictinin of 100 mg/kg, were
measured and compared. Values are expressed as mean ± SD (n = 5 per
group). # P < 0.05 vs. ddw group and * P < 0.05 vs. olive oil group.
enzymatic reaction at 37 °C for 30 min. After reaction, samples were
centrifuged at 9000g for 1 min, and the supernatant was read at 400 nm
in the Thermo Scientific Multiskan GO Microplate Spectrophotometer
(Waltham, MA, USA). All samples were assayed in triplicate, and the
relative lipase activity of each tea sample or compound was expressed
as percentage of the original lipase activity by replacing the tea sample
with water as a control.
2.5. Animals
Male C57BL6 mice (25–30 g, 4 weeks old) were purchased from
BioLasco, Taiwan Co., Ltd. (Taipei, Taiwan), and adapted for 1 week
before use. They were housed in a standard controlled environment of
23 ± 2 °C, 60 ± 10% humidity and 12-h light/dark cycle. Mice were

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T.-Y. Chen et al. Journal of Functional Foods 48 (2018) 1–8

45 A 2.5
A

Weight of epididymal fat (g)

40 Str45
HFD # 2.0
Weight (g)

35 Str130 *
1.5

30 NC 1.0

25
0.5

20
0 1 2 3 4 5 6 7 8 0.0
weeks NC HFD str45 str130
20
B 1.2
B
Food intake (kcal/mice/day)

15 1.0

Weight of liver (g)

10 0.8

0.6
5
0.4
0
NC HFD str45 str130 0.2

Fig. 4. Body weight and food intake of mice in a long-term study. The body
weight (A) and average food intake (B) of mice fed with normal (NC) or high fat
0.0
NC HFD str45 str130
diet (HFD) supplemented with strictinin of 45 mg/kg (Str45) or 130 mg/kg
1.8
(Str130) were recorded. Each value represents the mean ± S.E.M. (n = 8 per
group). # P < 0.05 vs. NC group and * P < 0.05 vs. HFD group.
C
1.6

fed with a standard chow diet (calories provided by 29.829% protein, 1.4
13.427% fat, and 56.744% carbohydrate, 5001 Rodent Lab Diet, St.
Weight of muscle (g)

1.2
Louis, MO, USA) or high fat diet (calories provided by 18.1% protein,
61.6% fat, and 20.3% carbohydrate, high-fat 58Y1 Rodent Test Diet, St. 1.0
Louis, MO, USA). During the experimental period, animals were al-
0.8
lowed free access to chow diet and tap water. The animal experiments
were approved by the Institutional Animal Care and Use Committee of 0.6
the National Chung-Hsing University (IACUC Approval No: 105-21).
0.4

2.6. Oral olive oil tolerance test
The intestinal absorption of triglycerides in mice was examined by
an oral olive oil tolerance test. The mice were deprived of food for 18 h
before the test. Strictinin or orlistat (a positively effective control) was
orally administered, and olive oil of 5 mL/kg was administered after-
ward. Six groups of mice (n = 4 per group) were intragastrically ad-
ministered with (1) olive oil as a positive control, (2) tap water as a
negative control, (3) olive oil plus strictinin of 10 mg/kg, (4) olive oil
plus strictinin of 50 mg/kg, (5) olive oil plus strictinin of 100 mg/kg,
and (6) olive oil plus orlistat of 10 mg/kg. Blood samples were collected
at 0, 1.5, 3, 4.5, and 6 h after administration of olive oil, and trigly-
ceride contents were detected by the Accutrend Plus meter (Roche
Diagnostics, Mannheim, Germany).
For the examination of oil content in feces, 15 mice were allocated
into three experimental groups (n = 5 per group). Mice in groups 1 and
2 were intragastrically administrated with tap water and olive oil (5
mL/kg), respectively. In group 3, mice were first orally administrated
with strictinin (100 mg/kg) prior to the administration of olive oil.
After feeding for 6 h, the feces were weighed and dried at 50 °C in an
oven. The dried feces were homogenized in 0.15 M NaCl (Sigma-
Aldrich, St. Louis, MO, USA), and mixed with the extraction buffer of
0.2
0.0
NC HFD str45 str130
Fig. 5. Weights of epididymal fat, liver and muscle of mice at the end of the
long-term study. Weights of epididymal fat (A), liver (B) and muscle (C) of mice
fed with normal (NC) or high fat diet (HFD) supplemented with strictinin of
45 mg/kg (Str45) or 130 mg/kg (Str130) were recorded. Values are expressed
as mean ± S.E.M. (n = 8 per group). # P < 0.05 vs. NC group and *
P < 0.05 vs. HFD group.
chloroform/methanol (2:1, v/v) (Sigma-Aldrich) for 10 min. The sus-
pensions were centrifuged at 400g for 5 min, and the extracts in the
lower phase were withdrawn, dried under nitrogen gas, and dissolved
in methanol. Oil contents in the feces were measured by a commercial
kit (Human, Wiesbaden, Germany) according to the manufacturer’s
instruction.
2.7. Reduction of fat accumulation in mice fed with high-fat diet
For a long-term (eight weeks) animal study, mice were randomly
assigned to four groups (n = 8 per group): (1) normal diet control (NC),

4
T.-Y. Chen et al. Journal of Functional Foods 48 (2018) 1–8

180
A
160

Blood triglyceride (mg/dL)

140
120
100
80
60
40

NC 50 μm
20
0
NC HFD str45 str130

180
B
160

Blood cholesterol (mg/dL)

140

120

100

80

60
HFD 50 μm 40

20

0
NC HFD str45 str130

140
C
Fasting blood glucose (mg/dL)

120

100

80

60
Str45 50 μm 40

20

0
NC HFD str45 str130
Fig. 7. Measurement of blood triglyceride, cholesterol and glucose at the end of
the long-term study. Blood triglyceride (A), blood cholesterol (B) and blood
glucose (C) of mice fed with normal (NC) or high fat diet (HFD) supplemented
with strictinin of 45 mg/kg (Str45) or 130 mg/kg (Str130) were detected. All
values are expressed as mean ± S.E.M. (n = 8 per group). # P < 0.05 vs. NC
group and * P < 0.05 vs. HFD group.

Str130 50 μm
Fig. 6. Representative photomicrographs of H&E staining in epididymal fat
tissues. Adipocytes in epididymis of mice fed with normal (NC) or high fat diet
(HFD) supplemented with strictinin of 45 mg/kg (Str45) or 130 mg/kg (Str130)
were photographed at a magnification of 400×.
(2) high fat diet (HFD), (3) high fat diet supplemented with strictinin of
45 mg/kg (Str45), and (4) high fat diet supplemented with strictinin of
130 mg/kg (Str130). Food intake was recorded daily and weights of
mice were recorded weekly. Eight weeks after treatments, the blood
samples were collected from overnight fasted mice. The plasma levels of
triglyceride and cholesterol were assayed enzymatically using com-
mercial kits (Human, Wiesbaden, Germany) and performed in ac-
cordance with the manufacturer’s instructions. The levels of blood
glucose were measured by the ACCU-CHEK® Active glucose meter with
test strips (Roche, Germany).

5
T.-Y. Chen et al.
2.8. Histological examination
At the end of the long-term animal study, the mice were anesthe-
tized with Zoletil 50 (Virbac Laboratories, Carros, France), and then
epididymis fats, gastrocnemius muscle and livers were removed quickly
and weighted. The epididymis fats were fixed in 10% formalin, and
then stained with hematoxylin and eosin (H&E) for histological ex-
amination. Histological images were captured by a light microscope
(Olympus, BX43, Tokyo, Japan) equipped with a digital camera
(Cannon EOS 600D, Tokyo, Japan).
2.9. Statistical analysis
All the data are presented as mean ± standard error of mean
(S.E.M.). The differences were analyzed by one-way analysis of variance
(ANOVA) followed by post-hoc analysis of Duncan’s test to determine
significant differences between each groups. Statistical calculation was
performed by SigmaStat (version 3.5). A level of P < 0.05 was con-
sidered to be statistically significant.
3. Results
3.1. Isolation of strictinin from raw Pu’er tea
Infusion of raw Pu’er tea produced from young leaves of wild trees
was analyzed on a HPLC system and found to contain abundant stric-
tinin (Fig. 1A). In accord with an earlier report (Chen et al., 2015),
strictinin and EGCG were detected as two abundant phenolic com-
pounds in the raw Pu’er tea but not in ripen Pu’er tea that contained low
contents of phenolic compounds (Fig. 1B). Strictinin was isolated to
homogeneity from the raw Pu’er tea, and its purity of approximately
95% was examined in the same HPLC system (Fig. 1C).
3.2. Inhibition of pancreatic lipase by Pu’er tea and its major compounds
Inhibitory potency on pancreatic lipase activity in vitro was ex-
amined for raw Pu’er tea and ripen Pu’er tea as well as three major
compounds found in raw Pu’er tea, strictinin, EGCG and caffeine. The
results showed that both raw Pu’er tea and ripen Pu’er tea possessed
basal inhibitory potency on pancreatic lipase activity (Fig. 2A). While
caffeine showed no apparent inhibition, both strictinin and EGCG ex-
hibited inhibitory potency on pancreatic lipase in a dose-dependent
manner. Relatively, strictinin possessed higher inhibitory potency on
pancreatic lipase than ECGC. The IC50 value of strictinin for inhibiting
pancreatic lipase was determined to be approximately 90 μg/mL
(Fig. 2B).
3.3. Effect of strictinin on the reduction of fat absorption
To evaluate the effect of strictinin on the reduction of fat absorption,
three dosages (10, 50 and 100 mg/kg) of strictinin as well as 10 mg/kg
of orlistat were administrated to mice prior to the administration of
olive oil (5 mL/kg). The blood triglyceride contents of mice were de-
tected at 0, 1.5, 3, 4.5, and 6 h after administration of olive oil. The
results showed that the blood triglyceride levels of mice in each group
peaked at 1.5 h after administration of olive oil, and were significantly
reduced by the preceding administration of strictinin in a dose-depen-
dent manner (Fig. 3A). Quantitatively, the effect on the reduction of fat
absorption in mice administrated with strictinin of 100 mg/kg was
found to be similar to that in mice administrated with orlistat of 10 mg/
kg (Fig. 3B). To examine if administration of strictinin affected the oil
content in fecal excretion in olive oil tolerance test, feces of the mice
orally administered with or without strictinin (100 mg/kg) were col-
lected for 6 h after feeding. The result showed that administration of
strictinin really increased the oil content in fecal excretion of mice
(Fig. 3C).
6
Journal of Functional Foods 48 (2018) 1–8
3.4. Effects of strictinin on fat accumulation in mice fed with high-fat diet
To examine the long-term effects of strictinin on fat accumulation,
mice were fed with normal diet or high-fat diet supplemented with
strictinin of 0, 45 and 130 mg/kg for eight weeks. As expected, body
weights of mice fed with high-fat diet increased much faster than those
fed with normal diet (Fig. 4A). However, the body weight gained by
high-fat diet was significantly reduced in the mice supplemented with
strictinin of 130 mg/kg. On the topic of food intake (by calories), no
significant difference was observed among mice in different groups
(Fig. 4B).
Further analysis showed that weight of epididymal fat, but not that
of liver or muscle, obviously increased in mice fed with high-fat diet
(Fig. 5). Nonetheless, the accumulation of epididymal fat in mice fed
with high-fat diet was reduced when strictinin of 130 mg/kg was sup-
plemented. In accord with the above examination, sizes of adipocytes in
epididymis were substantially enlarged in mice fed with high-fat diet in
comparison with those fed with normal diet, and the enlargement of
adipocytes was decreased when strictinin of 130 mg/kg was supple-
mented (Fig. 6). Moreover, levels of blood triglyceride, cholesterol and
glucose were evidently elevated in mice fed with high-fat diet in
comparison with those fed with normal diet, and these elevated levels
were also significantly reduced when strictinin of 130 mg/kg was sup-
plemented (Fig. 7).
4. Discussion
Empirically, drinking a variety of teas, particularly Pu’er tea, is re-
garded to result in an anti-obesity effect (Lee & Foo, 2013; Yukihiko,
2012). It has been shown that extracts of white and green teas, parti-
cularly those fractions enriched in strictinin, possessed inhibitory po-
tency on pancreatic lipase in vitro (Gondoin, Grussu, Stewart, &
McDougall, 2010). In this study, we first demonstrated that strictinin
isolated from Pu’er tea possessed inhibitory potency on pancreatic li-
pase in vitro, and then showed that administration of strictinin was able
to reduce fat absorption of mice in the olive oil tolerance test and to
lower fat accumulation in mice fed with high-fat diet in a long-term
study. The results were in good agreement with a previous animal study
showing that strictinin possessed a laxative activity on rat defecation,
presumably caused by accelerating small intestinal transit, instead of
enhancing gastric emptying or inducing diarrhea in the rats (Hsieh
et al., 2016). On the basis of this study, we propose that strictinin may
be a major component responsible for the empirical anti-obesity effects
of Pu’er tea, possibly resulted from the prevention of fat absorption
from diets by inhibiting pancreatic lipase.
The excess dietary fat uptake is generally regarded as one of the
major factors for obesity. Orlistat, an anti-obesity drug, reduces the
intestinal absorption of dietary fat by acting as an effective inhibitor of
pancreatic lipase (Zhi et al., 1995). However, gastrointestinal adverse
effects, such as oily stools, diarrhea, abdominal pain, and faecal spot-
ting as well as severe hepatic adverse effects, such as cholelithiasis,
cholesteric hepatitis, and subacute liver failure have been reported for
administration of orlistat on clinical treatments (Filippatos et al., 2008;
Sall et al., 2014). Therefore, orlistat is not recommended to be daily
used for a long time. In this study, we found that the effect on the
reduction of fat absorption in mice administrated with strictinin of
100 mg/kg was similar to that in mice administrated with orlistat of
10 mg/kg in the olive oil tolerance test (Fig. 3). Being a natural tannin
with several therapeutic activities, strictinin is regarded as a safe in-
gredient for the supplementation in food (Tsuji et al., 2003). It seems
that strictinin possesses great potential to be developed as a mild nat-
ural substitute for orlistat provided it undergoes necessary clinical
evaluation.
Besides the levels of blood triglyceride and cholesterol, the level of
glucose was also found to be elevated in C57BL6 mice fed with high-fat
diet in comparison with those fed with normal diet in this study (Fig. 7).
T.-Y. Chen et al.
It has been reported that the elevation of glucose level in C57BL6J mice
was attributed to impaired glucose tolerance (insulin resistance) in-
duced by high-fat diet (Winzell & Ahrén, 2004). Indeed, this high-fat
diet-fed mouse with elevated glucose level was designed as a model
system to study mechanisms and treatment of impaired glucose toler-
ance and type 2 diabetes. Similarly, elevated effects on the levels of
triglyceride, cholesterol and glucose were also observed in Sprague-
Dawley rats in a rodent model of metabolic syndrome induced by high-
fat diet (Chen et al, 2015).
Significant anti-obesity effects of strictinin (130 mg/kg) were ob-
served in the fat accumulation animal model (Figs. 4–7). According to
the calculation of human dose equivalent (HED) by the formula
(Reagan-Shaw, Nihal, & Ahmad, 2008): HED (mg/kg) = Animal dose
(mg/kg) × Animal Km/Human Km (HED = 130 mg/kg × 3/
37 = 10.54 mg/kg) where Km = Body Weight (kg)/Body Surface Area
(m2), the equivalent dosage of strictinin is approximately 0.63 g for an
adult of 60 kg weight (60 × 10.54 = 632.4 mg = 0.6324 g). The tea
infusion prepared from 1 g of raw Pu'er tea in this study contained
approximately 0.09 g of strictinin. Thus, 7 g (0.63–0.09 = 7 g) of raw
Pu'er tea is assumed to evidently result in the anti-obesity effects in
humans. Furthermore, other constituents in raw Pu'er tea, such as
EGCG, may additionally enhance the anti-obesity effects. Commonly,
5–10 g of raw Pu’er tea is used in one preparation of tea infusion, and
several tea preparations were consumed for tea drinkers daily. Thus, the
results observed in the animal model are in good agreement with the
empirical sensation of anti-obesity effects by drinking raw Pu’er tea.
One of the efficient strategies for the treatment of diet-induced
obesity is to develop or identify new effective inhibitors of metabolic
enzymes responsible for fat digestion and absorption (Bray, 2000). As
medicinal plants might provide adequate sources for safe, natural and
cost-effective alternatives to synthetic drugs, some plant extracts, such
as polyphenols and saponins, were examined and shown to be adequate
inhibitors of pancreatic lipase, and thus were assumed to be applicable
for the treatment of obesity (Birari & Bhutani, 2007; Sumantran, 2007).
Among plant extracts, aqueous extracts of various teas including oolong
tea, black tea and Pu’er tea have been shown to possess inhibitory
potency on pancreatic lipase, and the active ingredients were proposed
to be catechins, theaflavins, quercetin glycosides, quinic acid, gallic
acid, caffeine, free amino acids, and soluble sugar (Han, Takaku, Li,
Kimura, & Okuda, 1999; Yuda et al., 2012; Oi, Hou, Fujita, & Yazawa,
2012; Zeng, Yan, Luo, & Zhang, 2015). Here, we showed that strictinin
should be also an active ingredient in raw Pu’er tea as an effective in-
hibitor of pancreatic lipase. Moreover, administration of strictinin ef-
fectively reduced the levels of blood triglyceride, cholesterol and glu-
cose in mice fed with high-fat diet (Fig. 7), and should be also a useful
strategy to alleviate metabolic syndrome. It is expectable that more and
more effective inhibitors of pancreatic lipase or other metabolic en-
zymes will be identified from natural compounds and utilized in the
prevention and treatment of obesity.
5. Conclusion
Strictinin, a major phenolic compound in Pu’er teas produced from
young leaves of wild trees, was demonstrated to possess inhibitory
potency on pancreatic lipase in vitro. Furthermore, the administration of
strictinin was shown to reduce the blood triglyceride level of mice in
olive oil tolerance test and to lower fat accumulation in mice fed with
high-fat diet in a long-term study. Taken together, strictinin seems to be
a major component responsible for the empirical anti-obesity effects of
Pu’er tea, possibly resulted from the prevention of fat absorption from
diets by inhibiting pancreatic lipase.
6. Conflict of interest
The authors have no conflict of interest/financial disclosure.
7
Journal of Functional Foods 48 (2018) 1–8
Acknowledgements
The work was supported by a grant to Jason TC Tzen of National
Chung-Hsing University (NCHU-106D605).
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