International Journal of Food Science and Technology 2018 1

Original article
Effects of polysaccharides and polyphenolics fractions of Zijuan
tea (Camellia sinensis var. kitamura) on a-glucosidase activity and
blood glucose level and glucose tolerance of hyperglycaemic mice

Dejing Chen,1,2* Jingyuan Sun,2 Weixue Dong,2 Yixiao Shen3 & Zhimin Xu3*
1 Shaanxi Key Laboratory of Bio-Resources, Shaanxi University of Technology, Hanzhong, Shaanxi, China
2 School of Biological Science and Engineering, Shaanxi University of Technology, Hanzhong, Shaanxi, China
3 School of Nutrition and Food Sciences, Louisiana State University Agricultural Center, Baton Rouge, LA, USA
(Received 5 March 2018; Accepted in revised form 29 April 2018)

Summary The effects of polysaccharides, theaflavins, thearubigins and theabrownin fractions of Zijuan tea on a-glu- cosidase and blood
glucose level and intolerance of hyperglycaemic mice were evaluated. The polysaccha- rides or theaflavins fraction exhibited
greater inhibition rate of a-glucosidase than acarbose positive control, thearubigins fraction or theabrownin fraction. The
four fractions were delivered to the treatment mice through oval gavage each day for 15 days. The mice in polysaccharides
and theaflavins high- and low-dose and theabrownin high-dose treatments significantly lowered their blood glucose levels
while all the treatment mice gained body weight. The mice in polysaccharides, theaflavins and theabrownin high- and low-
dose treatments had greater glucose tolerance as well. Thus, the theaflavins and polysaccharides fractions of Zijuan tea
effectively moderated the complications of hyperglycaemic mice. The lower effec- tiveness of thearubigins and theabrownin
fractions may be caused by the highly polymerised polypheno- lics which decreased their accessibility to a-glucosidase
and digestibility in mice.
Keywords Diabetes, hyperglycaemia, polyphenols, polysaccharides, tea, a-glucosidase.

blood glucose level (Srinivasan, 2006). Gymnema syl- vestre is

Introduction
The changes of living style and environment in the modernised
society have been triggering the increase in diabetes population
around the world and the trend of developing the chronic
disease at younger age. The diabetes generally results in the
hyperglycaemia and glucose intolerance and the serious
complications related to the high level of blood glucose and due
to pancreatic insulin secretion deficiency (Stote & Baer, 2008).
To prevent the hyperglycaemia and the compli- cations, many
chemotherapeutics including insulin analogues, biguanides,
sulfonylureas, meglitinides, thi- azolidinedione and a-
glucosidase inhibitors are often used in the diabetes treatment
(Chaudhury et al., 2017). These drugs offer a fast and instant cure
effect; however, they could have some severe side-effects to the
patient with a long-term treatment.
A few of herbal plants have been reported to possess anti-
diabetic potentials. For example, fenugreek was found to have
the capability of decreasing postprandial
*Correspondent: Fax: 225-578-5300; e-mail: zxu@agcenter.lsu.edu (ZX);
cdjslg@126.com (DC)
an herb species that is abundant in gymnemic acids,
gymnemosides and polypeptide gurmarin and it is able to improve
the glucose uptake in peripheral tis- sues and insulin secretion
(Dey et al., 2002). The extract of Russian Tarragon favourably
affected insulin signalling to enhance the whole-body insulin
sensitivity (Wang et al., 2011). Recently, tea theaflavins, thearubi-
gins, theabrownin and polysaccharides have been recognised
as important bioactive compounds in tea leaves. Theaflavins, a
group of the benzotropolone ring compounds, are regarded as the
‘golden molecules’ in tea due to their anti-oxidation, anti-
mutagenicity, hypolipidaemic and anti-microbial effects (He,
2017). Thearubigins, a group of high molecular weight poly-
mers consisting of condensed flavan-3-ols, were reported to
be able to counteract the effects of botuli- num neurotoxins
(Satoh et al., 2001). Tea polysaccha- rides were reported to
associate with antioxidant, anti-bacterial and anti-skin-aging
properties, as well as improvement in the immunity and
alleviation of hepa- totoxicity (Du et al.,2016).
Although Zijuan tea exhibited the health benefit of
regulating lipid metabolism in a in vivo rat model (Peng

doi:10.1111/ijfs.13825
© 2018 Institute of Food Science and Technology
2 Anti-diabetic properties of Zijuan fractions D. Chen et al.
et al., 2013), most studies on the health function of Zijuan tea
were still limited in determining its antioxi- dant capability
using in vitro models, such as DPPH (2,2-diphenyl-1-
picrylhydrazyl) radical scavenging activity and ferric reducing
antioxidant power (FRAP) assays (Jiang et al., 2013). The
effectiveness of Zijuan tea, especially different bioactives in the
tea, on dia- betes treatment has not been well documented. In this
study, inhibitory effects of the important Zijuan tea bioactives,
polysaccharides, theaflavins, thearubigins, and theabrownin on a-
glucosidase were investigated. Additionally, the capabilities of
these bioactive frac- tions in lowering blood glucose level and
increasing glucose tolerance in diabetic state were evaluated using
a hyperglycaemic stress mouse model. The results of this study
could be useful to well understand the health benefits of Zijuan tea
and developing a moderate treat- ment for diabetic patient
without side-effect.
Materials and methods
Materials and chemicals
Acarbose, a-glucosidase, 4-Nitrophenyl b-D-glucopyr- anoside
(PNPG) and 96-well plates were purchased from Yuanye
Biotech Co Ltd. (Shanghai, China). Alloxan and glucose were
purchased from Sigma- Aldrich Company (St Louis, MO,
USA) and Youji Pharmaceutical and Chemical Co Ltd.
(Shanghai, China), respectively. Glucose meter and blood glucose
test strips were supplied by Sonicare Company (Changsha,
China). Zijuan tea (Pengxiang Tea Indus- try Company, Shannxi,
China) was purchased from a local grocery (Hanzhong,
China).
Preparation of Zijuan tea fractions
The polysaccharides fraction of tea was prepared based on the
method described in the study of Qiao et al. (2009). Zijuan tea
infusion was concentrated by a rotary evaporator and then
mixed with three times volume of dehydrated ethanol. The
mixture was kept at room temperature for 24 h before
centrifugation. The supernatant was collected for extractions
of the theaflavins, thearubigins and theabrownin fractions. The
precipitate was collected and dissolved in distilled water. The
proteins in the dissolved precipitate were removed using
trichloroacetic acid precipitation method. Then, the clear
solution was mixed with dehy- drated ethanol to a final ethanol
concentration of 90% to precipitate polysaccharides. The
precipitated polysaccharides were collected after centrifugation
and then lyophilised. The lyophilised polysaccharides frac- tion
was stored at 20 °C before use.
The theaflavins, thearubigins and theabrownin frac- tions
were extracted by the — methods described in
InternationalJournalof Food Scienceand Technology2018
previous studies (Gong et al., 2010; Murad & Abdallah, 2014).
The supernatant obtained in the above-men- tioned previous
step was extracted with chloroform (1:1, v/v) to remove caffeine.
The aqueous layer was collected and extracted with ethyl acetate.
The theaflavins frac- tion was obtained after ethyl acetate organic
layer was totally dried by a rotary evaporator. Then, the left aque-
ous residue was extracted with n-butanol. The thearubi- gins
fraction was obtained after the n-butanol solvent was completely
evaporated. The left aqueous layer was precipitated by anhydrous
ethanol (1:4, v/v) for 12 h at room temperature. The precipitate
was collected and dried to obtain the theabrownin fraction.
Determination of inhibition rate of a-glucosidase
The determination was based on the method described in Stanely
et al. (2000). 4-Nitrophenyl b-D-glucopyra- noside (PNPG),
used as a substrate, was hydrolysed by a-glucosidase to generate
p-nitrophenol at 37 °C. Thus, the inhibition rate of a-glucosidase
of each Zijuan tea fraction was calculated using the amount of p-
nitrophe- nol generated in 15 min after the reaction (Liu et al.,
2016). a-Glucosidase solution (0.2 U mL—1) or PNPG substrate
(2.5 mmol L—1) was prepared in phosphate- buffered saline
(PBS) buffer (pH 6.8). An aliquot of 110 lL of the PBS buffer
and 20 lL of a-glucosidase solution (0.2 U mL—1) were mixed
and added ineach well on a 96-well plate. Then, an aliquot of
10 lL of different concentration solution of each Zijuan tea frac-
tion was added in the wells as a treatment. An aliquot of 10 lL of
the PBS buffer instead of the fraction solu- tion was used as a
blank. Ten microliters of acarbose solutions (0.25, 0.5, 1.0, 1.5
or 2.0 mg mL—1) were added in the wells as a positive control.
The well with 110 lL of the PBS buffer and 10 lL of different
con- centration solution of each Zijuan tea fraction and
without a-glucosidase was used as a treatment blank. After
adding another 10 lL of the PBS buffer, the 96- well plate was
shaken at 37°C for 15 min. Then, 20 lL of PNPG solution (2.5
mmol L—1) was added to the treatments, positive controls and
treatment blanks, except the blank which was added with 20
uL of the PBS buffer instead of the PNPG solution. The plate
was shaken and incubated at 37°C for 15 min. In total, 100 lL of
Na2CO3 (0.2 mol L—1) solution was added in each well. The
absorbance of each well was recorded by a multi-mode
microplate reader at 405 nm. The inhibition rate of a-
glucosidase of each fraction treat- ment was calculated using
thefollowing formula:
Atreatment — Atreatmentblank
Inhibitionrate % 1
Acontrol — Ablank
× 100;
Σ Σ
where Atreatment, Atreatment blank, Acontrol and Ablank are the
ð Þ¼
absorbance rates of each treatment and its
—
© 2018 Institute of Food Science and Technology

corresponding treatment blank, control and
respectively.
Determination of blood glucose level, glucose tolerance
and body weight of hyperglycaemic mouse
Adult male Kuming mice (3-week old and 20–24 g of body
weight) were used in the animal experiment. The mice were
housed in a room at temperature of 22 °C and relative
humidity of 50–60% with a 12-h light/ dark cycle. All the mice
had free access to water and food for 2 weeks of
acclimatisation period. All the procedures of the animal
experiment were in accor- dance with the State Code of
Practice for the Care and Use of Animals for Scientific
Purposes.
Hyperglycaemic stress was induced to the mouse by a single
intraperitoneal injection of 2% alloxan mono- hydrate in PBS
solution (pH = 7.4) whose level was based on 200 mg kg—1 of
body weight. The normal control mice were injected with the
same amount of saline solution instead of the alloxan
monohydrate solution. After 72 h, a blood sample of each
hypergly- caemic stress-induced mouse was collected by tail inci-
sion after 12 h of fasting to measure the glucose level. The mouse
having the blood glucose level higher than
16.0 mmol L—1 was considered to successfully have
hyperglycaemic stress and then selected for the animal study. In
addition, the normal control (NC), the hyperglycaemic stress
mice were randomly divided into 10 experimental groups,
including the diabetic control (DC), positive control (PC), and
eight treatments such as polysaccharides (PS) fraction high dose
(PSH) and low dose (PSL), theaflavins (TF) fraction high dose
(TFH) and low dose (TFL), thearubigins (TR) fraction high dose
(TRH) and low dose (TRL), and theabrow- nin (TB) fraction
high dose (TBH) and low dose (TBL). Each control or
treatment group had 10 hyper- glycaemic mice. The mouse in the
low-dose treatment group was fed 1 mL of distilled water
solution con- taining the level of each fraction based on
200 mg kg—1 of body weight through oval gavage each day, while
the mouse in the high-dose treatment received the amount
of the fraction according to 600 mg kg—1 of body weight. The
mouse in positive control (PC) was fed 1 mL of distilled water
solution containing 5 mg of acarbose through oval gavage each
day. The mouse in normal control (NC) and diabetic control
(DC) received 1 mL of distilled water at the same time. On the
15th day of the experiment, a blood sample of each mouse was
collected by tail incision after 12 h of fasting to measure the
blood glucose level as a final blood glucose level of the mouse.
The body weight of each mouse was recorded every day during
the experimental period.
The mice of each group still received the same treat- ment for
another 2 days. On the last day, all the mice
© 2018 Institute of Food Science and Technology
Anti-diabetic properties of Zijuan fractions D. Chen et al. 3
blank, were fasted for 10 h and then received the same treat- ment as
before. After 15 min, all the mice were given an intraperitoneal
injection of glucose solution whose level was based on 2.0 g kg—1
of body weight. Three blood samples of each mouse were
collected at 0, 0.5 and 2 h by tail incision to measure their blood
glucose levels and plot an area under the curve (AUC). The AUC
was cal- culated to indicate glucose tolerance according to the
formula below.
Glucose AUCðmmol L—1Þ ¼ ðA + 4B + 3CÞ=4;
where A, B and C represent the blood glucose levels at 0, 1.5 and 2
h, respectively.
Data analysis
Each determination was replicated in three times. Value was
expressed as mean standard deviation.
T Statistical analysis
was performed by ANOVA using the General Linear Model
procedure (SAS system, SAS Institute Inc., Cary, NC, USA) with
significant differ- ences between means at P < 0.05.
Result and discussion
Inhibitory effects of Zijuan tea fractions on a-glucosidase
activity
The biological function of a-glucosidase is to degrade starch and
other compounds with glucoside sugar moi- ety and release free
glucose in the digestion system. Then, the released glucose is
absorbed by the blood circulation and contributes to the blood
glucose level. However, the carbohydrates hydrolysis and
degrada- tion of a-glucosidase can be inhibited to stop supply-
ing glucose to the blood circulation and block the increase in
blood glucose level (Yilmazer-Musa et al., 2012). As shown in
Table 1, all of the fractions exhib- ited inhibitory effect on a-
glucosidase activity with a dose-dependent manner. The
inhibition rates of the fractions were in a range of 10.38% for
thearubigins (TR) fraction (0.5 mg mL—1) to 78.31% for
theaflavins (TF) fraction (1.0 mg mL—1; Table 1). The
theaflavins (TF) fraction had the lowest IC50 (half maximal
inhibi- tory concentration; 0.24 mg mL—1) followed by the
polysaccharides (PS) fraction (0.92 mg mL—1) and pos- itive
control (1.05 mg mL—1), whereas the IC50 of theabrownin
(TB) fraction was 1.39 mg mL—1 (Table 1). The theaflavins
(TF) fraction in Zijuan tea was the most efficient fraction in
inhibiting a-glucosi- dase activity among the four fractions.
Tea theaflavins usually consist of a group of free and double
or triple polymerised flavan-3-ols including epicatechin,
epigallocatechin, epicatechin-3-gallate and epigallocatechin-3-
gallate (Kanwar et al., 2012). In the study of Yilmazer-Musa et
al. (2012), it was found
InternationalJournal of Food Science and Technology2018
4 Anti-diabetic properties of Zijuan fractions D. Chen et al.

that the presence of a gallate group esterified at the 3- position of active site of a-glucosidase. Compared with thearubi- gins, tea
the C-ring of flavan-3-ols contributes to the interaction of the theabrownin has a relatively high polarity which can make it
polyphenol with a-glucosidase (Yilmazer-Musa et al., 2012). readily and uniformly disturb in the reaction solution of the a-
Besides, gallic acid, p- coumaric acid and quinic acid glucosidase assay and increased its accessibility to a-
derivatives, caf- feoylquinic acid isomers, and caffeoyl glucose glucosidase. A pervious study also found that tea theabrownin
were also the essential phenolics in tea theaflavins (Lorenzo & extracts from 18 different black teas had good capability in
Munekata, 2016). The number of phenolic hydroxyl groups of inhibit- ing a-glucosidase activity (Wang et al., 2017). In the
polyphenols are mainly responsible for their inhibition rates of present study, the thearubigins fraction had the lowest inhibition
a-glucosidase activity (Yin et al., 2014). Tea polysaccharides rate of a-glucosidase among the four frac- tions. It was not able
usually contain arabinose, galactose, rhamnose and relatively to reach 50% of inhibition rate when the concentration
lower amount of xylose, galacturonic acid, mannose, ribose and increased to 3 mg mL—1. The higher molecular size and low
glu- curonic acid (Scoparo et al., 2013). According to the study polarity of thearubigins may result in the lower rate of
of Deng et al. (2015), a group of carbohydrates found in Pu-erh inhibiting a-glucosi- dase.
tea polysaccharides can inhibit a-glu- cosidase as well. The
inhibition rate of tea polysaccha- rides was also found to be Effects of Zijuan tea fractions on blood glucose level,
dependent upon the proportion of low molecular weight glucose tolerance and body weight of hyperglycaemic
polysaccharides which had higher inhibition efficiency than mice
the high molecular weight polysaccharides in green and Oolong
teas (Jian et al., 2017). Alloxan can specifically inhibit insulin secretion by suppressing
The chemical structures of tea thearubigins and the beta cell glucose sensor glucokinase (Lenzen, 2008). It was
theabrownin are more complicated than that of tea theaflavins. applied to induce hypergly- caemic stress to the experimental
They have a large number of polymerised chemical units and can mice in this study. The initial blood glucose levels of all the
reach 2–100 kDa of molecular weight (Kuhnert, 2010; Gong et groups are listed in Table 2. The initial blood glucose level in the
al., 2012). The higher molecular size may affect their mice of normal control (NC) was 4.9 mmol L —1, whereas all
accessibility to the other groups with the induced hypergly- caemic stress mice
had an initial blood glucose level between 21.7 and 23.6 mmol
L—1 without significant difference. In the treatment groups, two
Table 1 Inhibition rates of Zijuan tea fractions against a-glucosi- dase doses, low dose (200 mg) and high dose (600 mg) per kg of body
activity weight of each mouse, were delivered to the mice. The low and
high doses were approximately equivalent to the levels of daily
Concentration Inhibition IC50 intake of the tea infusions brewed by 10–30 g of dried Zijuan
Treatment (mg mL —1) rate (%) (mg mL —1) tea leaves, respectively.
Polysaccharides (PS) 0.5 20.51 T 0.84 0.92 Although the initial blood glucose levels of the
1.0 55.32 T 1.11 hyperglycaemic mice in different groups were not sig- nificantly
1.5 63.07 T 0.91 different, a reduction of blood glucose level at the end of study
2.0 73.33 T 0.96 was observed in the positive con- trol (PC; 3.28 mmol L—1),
Theaflavins (TF) 0.05 23.14 T 0.98 0.24 polysaccharides
— high dose (PSH; 4.1 mmol L—1),
0.1 38.49 T 1.5 polysaccharides
— low dose (PSL; 1.8 mmol L —1), theaflavins
0.25 54.75 T 0.72 high dose— (TFH;
0.5 65.79 T 0.4
—6.2 mmol L—1), theaflavins low dose (TFL; 2.4 mmol— L —1)
1.0 78.31 T 0.96
and theabrownin high dose (TBH; 3.0 mmol L ) — —1 mice
Thearubigins (TR) 0.5 10.38 T 0.83 >3.0
1.0 33.62 T 0.66
(Table 2). Among the treatment groups, the reduction of
2.0 42.65 T 0.78 blood glucose level in polysaccharides or theaflavins high-
3.0 45.40 T 1.14 dose treatment mice was greater and even slightly higher than the
Theabrownin (TB) 0.5 18.73 T 0.49 1.39 pos- itive control mice (Table 2). However, the change of blood
1.0 43.26 T 0.96 glucose level of thearubigins high (TRH) or low
1.5 52.48 T 0.86
2.0 59.11 T 0.84

Acarbose 0.25 12.03 T 0.91 1.05 dose (TRL) was not significantly different from that of diabetic
(positive control) 0.5 30.17 T 1.52
control (DC; Table 2). It may be caused by the lower
1.0 49.02 T 1.47
inhibition rate of a-glucosidase of the
1.5 58.76 T 0.50 thearubigins fraction which was found in the present
2.0 69.73 T 1.08
study.

InternationalJournalof Food Scienceand Technology2018 © 2018 Institute of Food Science and Technology
 Anti-diabetic properties of Zijuan fractions D. Chen et al. 5

Table 2 Effects of Zijuan tea fractions on blood glucose level of mice in each group

Initial glucose level End glucose Increased

Group name (mmol L —1) level (mmol L—1) value (mmol L—1)

Normal control (NC) 5.1 T 0.3 4.9 T 0.2 —0.2

Diabetic control (DC) 22.1 T 1.2n 29.9 T 1.9np 7.8
Positive control (PC) 22.4 T 2.3n 19.1 T 2.6nd —3.3
Polysaccharides high dose (PSH) 22.6 T 2.3n 18.5 T 2.9nd —4.1
Polysaccharides low dose (PSL) 22.9 T 1.3n 21.1 T 2.7nd —1.8
Theaflavins high dose (TFH) 22.3 T 1.6n 16.1 T 1.9nd —6.2
Theaflavins low dose (TFL) 23.1 T 1.9n 20.7 T 2.5nd —2.4
Thearubigins high dose (TRH) 21.7 T 1.5n 28.6 T 3.1np 6.9
Thearubigins low dose (TRL) 22.5 T 1.2n 29.6 T 2.7np 7.1
Theabrownin high dose (TBH) 23.6 T 2.0n 20.6 T 2.7nd —3.0
Theabrownin low dose (TBL) 21.8 T 1.4n 23.8 T 4.3ndp 2.0

n indicates value significantly different from NC at P < 0.05. d

indicates value significantly different from DC at P < 0.05. p
indicates value significantly different from PC at P < 0.05.

The glucose tolerance of each fraction was assessed by the area The greater glucose tolerance of the theaflavins fraction
under curve (AUC) overa period of2 h (Fig. 1). The glucose level had treatment mice may be contributed byits abundant phenolic
a trend of increase before 0.5 h and then decrease in all the groups compounds, especially flavonoids, polyphenols and tannins. It
(Table 3). Figure 1 shows was reported that green tea extract could enhance the insulin
that the mice in theaflavins high-dose (TFH) treatment (44.57 sensitivity and improve the glucose absorption into peripheral
1.59 mmolT L—1 9 2 h) exhibited the greatest glucose tolerance and glucose homeostasis (Yu et al., 2017). Some polyphe- nolic
(Fig. 1). It was followed by polysaccha- rides high-dose (PSH; compounds are also able to regulate the glucose intolerance by a
46.47 2.57 mmol L—1 9 2 h), T facilitated insulin response (Bahado- ran et al., 2013). The
theabrownin high-dose (TBH; 47.40 T 2.15 mmol interaction of phenolics, espe- cially catechins, epicatechins,
L—1 9 2), theaflavins low-dose (TFL; 49.84 1.59 mmol T L— chlorogenic acids, ferulic acids, caffeic and tannic acids,
1 9 2 h), polysaccharides low-dose (PSL; quercetin in tea extracts may result in the absorption of glucose
50.20 T3.82 mmol L—1 9 2 h) and theabrownin low- dose from intestine by suppression of Na+-dependent glucose
(TBL; 52.82 2.31 mmol T L—1 9 2 h) treatments. Except the transporters as well (Kobayashi et al., 2000).
theabrownin high- (TRH) and low- (TRL) The mice in the polysaccharides fraction treatments also had
dose treatments, all other treatment mice had signifi- cantly higher glucose tolerance. Zijuan tea
lower AUC than the diabetic control mice (DC;
59.76 T 2.35 mmol L—1 9 2 h; Fig. 1).

70
np np np
60
nd
nd nd
Figure 1 Area under the curve (AUC) of glucose nd nd nd
AUC (mmol L–1*2 h)

50 ndp
tolerance of mice in each group. NC, normal control;
DC, diabetic control (DC); PC, positive control; PSH,
polysaccharides high dose; PSL, polysaccharides 40
low dose; TFH, theaflavins high dose; TFL,
theaflavins low dose; TRH, thearubigins high 30
dose; TRL, thearubigins low dose; TBH, theab-
rownin high dose; TBL, theabrownin low dose. n 20
indicates value significantly different from NC at P <
0.05. d indicates value sig- nificantly different
from DC at P < 0.05. p indicates value significantly 10
different from PC at P < 0.05.
0
NC DC PC PSH PSL TFH TFL TRH TRL TBH TBL

© 2018 Institute of Food Science and Technology InternationalJournal of Food Science and Technology2018
6 Anti-diabetic properties of Zijuan fractions D. Chen et al.

Table 3 Effects of Zijuan tea fractions on glucose tolerance of mice in each group

Blood glucose concentration (mol L—1)

Group name 0h 0.5 h 2h

Normal control (NC) 5.1 T 0.25 7.14 T 0.26 5.6 T 0.41

Diabetic control (DC) 28.6 T 1.06np 30.5 T 1.19np 29.5 T 1.21np
Positive control (PC) 18.8 T 1.23nd 28.9 T 1.08nd 19.7 T 1.04nd
Polysaccharides high dose (PSH) 19.5 T 1.46nd 25.9 T 1.27ndp 20.9 T 1.44ndp
Polysaccharides low dose (PSL) 22.2 T 2.36ndp 26.7 T 1.81ndp 23.9 T 2.09ndp
Theaflavins high dose (TFH) 17.9 T 1.33nd 25.2 T 0.84ndp 19.9 T 0.92nd
Theaflavins low dose (TFL) 21.8 T 1.02ndp 26.3 T 1.27ndp 24.1 T 1.22ndp
Thearubigins high dose (TRH) 28.0 T 0.83np 30.1 T 0.72np 29.1 T 0.81np
Thearubigins low dose (TRL) 29.3 T 1.05np 31.1 T 1.04np 29.8 T 0.94np
Theabrownin high dose (TBH) 20.9 T 1.18ndp 25.6 T 0.90ndp 22.1 T 1.27ndp
Theabrownin low dose (TBL) 24.2 T 0.85ndp 27.3 T 1.01ndp 25.8 T 1.18ndp

n indicates value significantly different from NC at P < 0.05. d

indicates value significantly different from DC at P < 0.05. p
indicates value significantly different from PC at P < 0.05.

20
Increased body weight

15

10
(g)

–5
NC DC PC PSH PSL TFH TFL TRH TRL TBH TBL

Figure 2 Change of body weight of mice in each group. NC, normal control; DC, diabetic control (DC); PC, positive control; PSH, polysac- charides high dose; PSL,
polysaccharides low dose; TFH, theaflavins high dose; TFL, theaflavins low dose; TRH, thearubigins high dose; TRL, thearubigins low dose; TBH, theabrownin high
dose; TBL, theabrownin low dose. n indicates value significantly different from NC at P < 0.05. d indicates value significantly different from DC at P < 0.05. p
indicates value significantly different from PC at P < 0.05.

polysaccharides may be similar to Pu-erh tea polysac- charides
and contain a specific protein and uronic acid which have
inhibitory effect on a-glucosidase activity and preventive
effects on the glucose home- ostasis in diabetic mice (Du et al.,
2016). The effects of plant polysaccharides extracts on glucose
modula- tion were reported in several previous studies. For
example, the lyceum barbarum L. polysaccharides extract
inhibited the glucose absorption through down-regulation of
the expression of sodium–glucose linked transporter to limit
glucose uptake in entero- cytes (Cai et al., 2017).
The loss of body weight is another important physi- ological
index associated with diabetes due to the per- sistence of
catabolism and degradation of structural proteins which is
caused by the insulin deficiency that affects both glucose
metabolism and protein metabo- lism (Al-Attar & Zari, 2010).
In this study, the body
weight of the diabetic control mice decreased by
1.49 T0.72 g at the end of experiment, while all the treatment
mice had an increase in body weight (Fig. 2). Especially, the
body weight of the theaflavins high-dose (TFH) treatment
mice increased by
5.98 1.40
T g and was significantly higher than the positive
control mice (3.94 1.01 g; Fig.T2). The mice in the theaflavins
low-dose (TFL) and polysaccharides high-dose (PSH) and low-
dose (PSL) treatments also gained their body weights by 4.03
1.03, 3.59 0.47 and 3.07 0.48 g, respectively
T (Fig. 2). It indicated
T
that the Zijuan
T tea fractions were able to prevent the body
weight loss caused by diabetes. The protective effect of these
tea fractions might be contributed by the modulation of insulin
secretion. It was reported that phenolics-rich Tinospora
cordifolia plant extract could assist in the body weight gain in
diabetic rats as well (Stanely et al., 2000).

InternationalJournalof Food Scienceand Technology2018 © 2018 Institute of Food Science and Technology

Conclusion
In summary, Zijuan tea fractions, especially theaflavins and
polysaccharides, had great inhibitory effect against a-glucosidase
activity. They also significantly decreased the blood glucose levels
and increased glucose toler- ance in hyperglycaemic stress
mouse. Compared with the highly polymerised thearubigins and
theabrownin, the theaflavins and polysaccharides in Zijuan tea
have higher effectiveness in preventing hyperglycaemic stress and
reducing the complications of diabetes.
Acknowledgments
This study was supported by the Innovation Program of
Agriculture (NYKJ-2015-031) and the 2016 Talents Program of
Shaanxi Province.
References
Al-Attar, A.M. & Zari, T.A. (2010). Influences of crude extract of tea leaves,
Camellia sinensis, on streptozotocin diabetic male albino mice. Saudi Journal
of Biological Sciences, 17, 295–301.
Bahadoran, Z., Mirmiran, P. & Azizi, F. (2013). Dietary polyphenols as potential
nutraceuticals in management of diabetes: a review. Journal of Diabetes &
Metabolic Disorders, 12, 43.
Cai, H., Yang, X., Cai, Q., Ren, B., Qiu, H. & Yao, Z. (2017).
Lycium barbarum L. Polysaccharide (LBP) reduces glucose uptake via down-
regulation of SGLT-1 in Caco2 cell. Molecules, 22, 1–12. Chaudhury, A.,
Duvoor, C., Reddy Dendi, V.S. et al. (2017). Clini- cal review of antidiabetic
drugs: implications for type 2 diabetes
mellitus management. Frontiers in Endocrinology, 8, 6.
Deng, Y.T., Lin-Shiau, S.Y., Shyur, L.F. & Lin, J.K. (2015). Pu-erh tea polysaccharides
decrease blood sugar by inhibition of alpha-glucosi- dase activity in vitro and in
mice. Food & Function,6, 1539–1546.
Dey, L., Attele, A.S. & Yuan, C.S. (2002). Alternative therapies for type 2
diabetes. Alternative Medicine Review, 7, 45–58.
Du, L.L., Fu, Q.Y., Xiang, L.P. et al. (2016). Tea polysaccharides and their
bioactivities. Molecules, 21, 1449.
Gong, J., Peng, C., Chen, T., Gao, B. & Zhou, H. (2010). Effects of theabrownin
from Pu-erh tea on the metabolism of serum lipids in rats: mechanism of
action. Journal of Food Science, 75, H182– H189.
Gong, J., Tang, C. & Peng, C.X. (2012). Characterization of the chemical
differences between solvent extracts from Pu-erh tea and Dian Hong black tea
by CP–Py–GC/MS. Journal of Analytical and Applied Pyrolysis, 95, 189–
197.
He, H.F. (2017). Research progress on theaflavins: efficacy, forma- tion, and
preparation. Food & Nutrition Research, 61, 1344521.
Jian, H., Qiao, F., Chen, J. & He, N. (2017). Physicochemical char- acterisation of
polysaccharides from the seeds and leaves of miracle fruit (Synsepalum
dulcificum) and their antioxidant and a-glucosi- dase inhibitory activities
in vitro. Journal of Chemistry, 2017, 9.
Jiang, L., Shen, X., Shoji, T., Kanda, T., Zhou, J. & Zhao, L. (2013).
Characterization and activity of anthocyanins in Zijuan tea (Camellia sinensis
var. kitamura). Journal of Agricultural and Food Chemistry, 61, 3306–
3310.
Kanwar, J., Taskeen, M., Mohammad, I., Huo, C., Chan, T.H. & Dou, Q.P. (2012).
Recent advances on tea polyphenols. Frontiers in Bioscience, 4, 111–131.
© 2018 Institute of Food Science and Technology
Anti-diabetic properties of Zijuan fractions D. Chen et al. 7
Kobayashi, Y., Suzuki, M., Satsu, H. et al. (2000). Green tea polyphenols
inhibit the sodium-dependent glucose transporter of intestinal epithelial cells
by a competitive mechanism. Journal of Agricultural and Food Chemistry,
48, 5618–5623.
Kuhnert, N. (2010). Unraveling the structure of the black tea thearu- bigins.
Archives of Biochemistry and Biophysics, 501, 37–51.
Lenzen, S. (2008). The mechanisms of alloxan- and streptozotocin- induced
diabetes. Diabetologia, 51, 216–226.
Liu, S., Yu, Z., Zhu, H., Zhang, W. & Chen, Y. (2016). In vitro a- glucosidase
inhibitory activity of isolated fractions from water extract of Qingzhuan
dark tea. BMC Complementary and Alterna- tive Medicine, 16, 378.
Lorenzo, J.M. & Munekata, P.E.S. (2016). Phenolic compounds of green tea:
health benefits and technological application in food. Asian Pacific Journal
of Tropical Biomedicine, 6, 709–719.
Murad, H.A. & Abdallah, H.M. (2014). Black tea extract and its thearubigins
relieve the sildenafil-induced delayed gut motility in mice: a possible role of
nitric oxide. Phytotherapy Research, 28, 1687–1691.
Peng, C., Wang, Q., Liu, H., Gao, B., Sheng, J. & Gong, J. (2013). Effects of Zijuan
pu-erh tea theabrownin on metabolites in hyper- lipidemic rat feces by Py-
GC/MS.Journal of Analytical and Applied Pyrolysis, 104(Supplement C),
226–233.
Qiao, D., Hu, B., Gan, D., Sun, Y., Ye, H. & Zeng, X. (2009).
Extraction optimized by using response surface methodology, purification
and preliminary characterization of polysaccharides from Hyriopsis
cumingii. Carbohydrate Polymers, 76, 422–429.
Satoh, E., Ishii, T., Shimizu, Y., Sawamura, S. & Nishimura, M. (2001). Black tea
extract, thearubigin fraction, counteract the effects of botulinum
neurotoxins in mice. British Journal of Phar- macology, 132, 797–798.
Scoparo, C.T., de Souza, L.M., Rattmann, Y.D. et al. (2013).
Polysaccharides from green and black teas and their protective effect against
murine sepsis. Food Research International, 53, 780– 785.
Srinivasan, K. (2006). Fenugreek (Trigonella foenum-graecum): a review of
health beneficial physiological effects. Food Reviews Inter- national, 22, 203–
224.
Stanely, P., Prince, M. & Menon, V.P. (2000). Hypoglycaemic and other related
actions of Tinospora cordifolia roots in alloxan- induced diabetic rats.
Journal of Ethnopharmacology, 70, 9–15.
Stote, K.S. & Baer, D.J. (2008). Tea consumption may improve biomarkers of
insulin sensitivity and risk factors for diabetes. The Journal of Nutrition, 138,
1584s–1588s.
Wang, Z.Q., Ribnicky, D., Zhang, X.H. et al. (2011). An extract of Artemisia
dracunculus L. enhances insulin receptor signaling and modulates gene
expression in skeletal muscle in KK-A(y) mice. The Journal of Nutritional
Biochemistry, 22, 71–78.
Wang, Y., Zhang, M., Zhang, Z., Lu, H., Gao, X. & Yue, P. (2017). High-
theabrownins instant dark tea product by Aspergillus niger via submerged
fermentation: alpha-glucosidase and pancreatic lipase inhibition and
antioxidant activity. Journal of the Science of Food and Agriculture, 97,
5100–5106.
Yilmazer-Musa, M., Griffith, A.M., Michels, A.J., Schneider, E. & Frei, B. (2012).
Inhibition of a-amylase and a-glucosidase activity by tea and grape seed
extracts and their constituent catechins. Journal of Agricultural and Food
Chemistry, 60, 8924–8929.
Yin, Z., Zhang, W., Feng, F., Zhang, Y. & Kang, W. (2014). a-Glu- cosidase
inhibitors isolated from medicinal plants. Food Science and Human
Wellness, 3, 136–174.
Yu, J., Song, P., Perry, R., Penfold, C. & Cooper, A.R. (2017). The effectiveness of
green tea or green tea extract on insulin resistance and glycemic control in type
2 diabetes mellitus: a meta-analysis. Diabetes & Metabolism Journal, 41,
251–262.
InternationalJournal of Food Science and Technology2018