Journal of Ethnopharmacology 138 (2011) 142–149

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Journal of Ethnopharmacology
journal homepage: www.elsevier.com/locate/jethpharm

The hypoglycemic activity of Lithocarpus polystachyus Rehd. leaves in the

experimental hyperglycemic rats
Shao-zhen Hou a , Shu-xian Chen b , Song Huang c,∗ , Dong-xu Jiang c , Cai-Jie Zhou a ,
Chang-qing Chen c , Ying-min Liang a , Xiao-ping Lai a,∗∗
a
School of Chinese Pharmaceutical Science, Guangzhou University of Chinese Medicine, University Town, Guangzhou 510006, China
b
The Third Affiliated Hospital of Sun Yat-Sen University, Guangzhou, China
c
Dongguan Mathematical Engineering Academy of Chinese Medicine and Guangzhou University of Traditional Chinese Medicine, Dongguan 523808, China

a r t i c l e i n f o a b s t r a c t

Article history: Ethnopharmacological relevance: Leaves of Lithocarpus polystachyus Rehd. are used for the treatment of
Received 27 April 2011 disorders such as diabetes, hypertension, and epilepsy in folk medicine of South China. The possible
Received in revised form 14 August 2011 antidiabetic effects of the leaves were investigated in experimental type 2 and type 1 diabetic rats.
Accepted 29 August 2011
Materials and methods: Type 2 diabetic rats received orally three different extracts of Lithocarpus
Available online 7 September 2011
polystachyus Rehd. leaves for 4 weeks (aqueous extract [ST-1], ethanol extract [ST-2], flavonoid-rich frac-
tion [ST-3]). At the end of the experiment biochemical parameters were tested and livers and pancreases
Keywords:
were excised for histological study. After the comparison of the pharmacological test results of the three
Lithocarpus polystachyus Rehd. leaves
Sweet Tea
extracts, the one which showed the best bioactivity was further studied to confirm its antidiabetes effect
Flavonoid-rich fraction on both type 2 and type 1 diabetic rats.
Type 2 diabetic rats Results: Compared to ST-1 and ST-2, ST-3 had better effects on regulation of blood glucose, glycosylated
Type 1 diabetic rats serum protein, cholesterol, triglyceride, malondialdehyde, superoxide dismutase and attenuation of liver
injury in type 2 diabetic rats (p < 0.01 or p < 0.05). ST-3 administration for four weeks also significantly
reduced the fasting serum insulin and C-peptide level and improved the insulin tolerance (p < 0.05).
In type 1 diabetic rats, ST-3 supplement for three weeks caused significant reduction in fasting blood
glucose, total cholesterol, triglyceride, urea nitrogen, creatinine and liver mass, along with significantly
inhibiting the decline of insulin level compared to diabetic control (p < 0.05 or p < 0.01).
Conclusion: The flavonoid-rich fraction of Lithocarpus polystachyus Rehd. leaves (ST-3) had better bene-
ficial effect than that of the ethanol or aqueous extract in experimental diabetic rats, which means that
the bioactivity of the herbal leaves is probably due to the presence of flavonoids. The results also strongly
suggest that the antidiabetic effect of ST-3 was possibly through multiple mechanisms of action includ-
ing blood lipid and antioxidant mediation. The results indicated that the aqueous flavonoid-rich fraction
of Lithocarpus polystachyus Rehd. leaves possessed significant protective activity in type 2 and type 1
diabetes.
© 2011 Elsevier Ireland Ltd. All rights reserved.

1. Introduction

According to the WHO definition the term diabetes mellitus

Abbreviations: AUC, area under the curve; BUN, urea nitrogen; C-P, C-peptide;
CREA, creatinine; DM, diabetes mellitus; DMC, Diamicron; FSG, fasting serum glu-
cose; FSN, fasting serum insulin; GSP, glycosylated serum protein; ITT, insulin
tolerance test; Lep, leptin; MDA, malondialdehyde; MFM, Metformin; OGTT, oral
glucose tolerance test; S.E.M., standard error of mean; SOD, superoxide dismutase;
ST, Sweet Tea; STZ, streptozotocin; TC, total cholesterol; TG, triglyceride; T1DM,
type 1 diabetes; T2DM, type 2 diabetes.
∗ Corresponding author. Tel.: +86 20 39358103; fax: +86 20 39358103.
∗∗ Corresponding author. Tel.: +86 20 39358045; fax: +86 20 39358390.
E-mail addresses: hsl318@yahoo.com.cn (S. Huang), lxp88@gzhtcm.edu.cn
(X.-p. Lai).
(DM) covers a group of metabolic diseases characterized by ele-
vated blood glucose levels. The two main representatives of this
group are type 1 and type 2 diabetes. DM is currently one of the
most costly and burdensome chronic diseases (American Diabetes
Association, 2011). It is caused by inherited or acquired deficiency
in insulin secretion and by decreased responsiveness of the organs
to secreted insulin (Lebovitz, 2001; Janka and Michaelis, 2002).
Although several drugs are available or the treatment of diabetes,
adverse effects and drug resistance are of great concern. As an
alternative, more and more people are seeking natural products
to prevent or treat diabetes.

0378-8741/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.jep.2011.08.067
 S.-z. Hou et al. / Journal of Ethnopharmacology 138 (2011) 142–149 143

Fig. 1. Liver tissue slice from a normal control rat (A) showed the normal tissue structure. The tissue slice from high fat feeding and streptozotocin-induced diabetic rats
(T2DM, B) showed abnormal retention of lipids as steatosis and edema within cells. Lipid accumulation displaced the cytoplasm and the nuclei were distorted. The tissue
sections from type 2 diabetic rats that received the Sweet Tea extracts (ST-1 of 1.5 g/kg, ST-2 of 1.5 g/kg, ST-3 of 0.3 g/kg; C, D, and E, respectively) demonstrated reduced lipid
deposition and cellular edema. One representative microphotograph from each of the five experimental groups was shown. Original magnification 100× and H&E staining.

Lithocarpus polystachyus Rehd. (family Fagaceae) locates mainly
as a wild plant in the mountain area in southern China with
a great drug resource (Institute of Botany, Chinese Academy of
Sciences, 1972; Wang et al., 1999). It is popularly named “Sweet
Tea” (ST) in folk and its leaves have been used as traditional herbal
medicine against a variety of diseases for hundreds of years. ST
is claimed to have a wide range of biological activities, such as
anti-oxidative, anti-bacterial, hypoglycemic, anti-obese and anti-
inflammatory (Liao and Zhou, 1994; Wang et al., 1999; Yang et al.,
2007). However, very few studies have confirmed the information
and measured its antidiabetic activity. Till now, there are no indica-
tions of this herbal medicine’s mechanism of hypoglycemic action
or whether it can improve glycemic control of type 1, type 2 or
both types of diabetes. In order to further validate the beneficial
effects of Lithocarpus polystachyus Rehd. leaves as an antidiabetic
natural product, and to better understand its action, we investi-
gated the effect of the leaves’ extracts in experimental type 2 and
type 1 diabetic animals.
2. Materials and methods
2.1. Kits, chemicals, and reagents
The kits of blood glucose, total cholesterol (TC), triglyceride
(TG), urea nitrogen (BUN) and creatinine (CREA) were purchased
from Beijing Zhongsheng Chemical Factory (Beijing, China). The
144 S.-z. Hou et al. / Journal of Ethnopharmacology 138 (2011) 142–149

Fig. 2. Pancreatic tissue slice from a normal control rat (A) showed the normal tissue structure. The tissue slice from high fat feeding and streptozotocin-induced diabetic
rats (T2DM, B) showed abnormal reduced size of pancreatic acini (arrow). The tissue sections from type 2 diabetic rats that received the Sweet Tea extracts (ST-1 of 1.5 g/kg,
ST-2 of 1.5 g/kg, ST-3 of 0.3 g/kg; C, D, and E, respectively) exhibited the attenuated atrophy of pancreatic acini. One representative microphotograph from each of the five
experimental groups was shown. Original magnification 100× and H&E staining.

kits of malondialdehyde (MDA), superoxide dismutase (SOD),
glycosylated serum protein (GSP) were purchased from Nan-
jing Jiancheng Chemical Factory (Nanjing, China). ELISA kits of
insulin (FSN), leptin (Lep) and C-peptide (C-P) were obtained
from R&D Systems (Minneapolis, MN, USA). Streptozotocin (STZ,
Sigma Chemicals, St. Louis, MO, USA), Diamicron (Huajin Phar-
maceutical Factory, Tianjin, China), Metformin hydrochloride
(Tian-shi-li Pharmaceutical Ltd., Guangzhou, China), Novolin (Novo
Nordisk Ltd., Danish), portable blood glucose meter (Roche Phar-
maceutical Ltd., Basel, Switzerland) were used in the study.
The other chemicals used were reagent grade from commercial
source.
2.2. Plant material
The fresh leaves of Lithocarpus polystachyus Rehd. were locally
purchased in summer in Jiangxi Province, identified and authenti-
cated by Prof. Lai Xiao-ping in our institute, Guangzhou University
of Chinese Medicine. The leaves were dried in the shade. A voucher
 S.-z. Hou et al. / Journal of Ethnopharmacology 138 (2011) 142–149
specimen is deposited in the herbarium of the Guangzhou Univer-
sity of Chinese Medicine.
2.3. Different extract preparation
The dry leaves (1 kg) were powdered and extracted twice by
using Soxhlet equipment with 5 L water for 2 h each time. The aque-
ous extract solution was filtered and concentrated by using a rotary
evaporator. Then extract was dried to a powder in vacuo at 105 ◦ C
(ST-1, yield 20.7% [w/w]) and stored in labeled sterile screw-capped
bottles at −20 ◦ C until it was used. Ethanol extract of Sweet Tea
(ST-2) was prepared similarly to the aqueous extract. Briefly, the
dry powdered leaves (1 kg) were extracted twice with 4 L of 70%
ethanol for 2 h each time and concentrated with a final yield of
22.3% (w/w). The flavonoid-rich fraction of Sweet Tea leaves was
prepared by low temperature equipment with different extract-
ing pressure (ZLUPD-S-350A, Guangzhou Zeli Pharmtech Co., Ltd.,
Guangzhou, China). Then the aqueous extract solution was pro-
cessed by the technique of ultrafiltration membrane concentration
and purified by using AB-8 macroporous resin column chromatog-
raphy. Finally, the extract was dried to a powder on an atomizing
drier to yield a yellow coloured powder extract (ST-3, yield 8.0%;
total flavonoids purity is about 90% determined by colorimetric
method). The extracts were freshly diluted with distilled water just
before ad ministration to animals during experiment.
2.4. Animals
Male Sprague-Dawley rats were obtained from central animal
house facility of Guangzhou University of Chinese Medicine. They
were acclimatized in an air conditioned room at 22 ± 2 ◦ C for 7
days with a 12 h light and 12 h darkness cycle. All the animals
were free to standard laboratory feed and tap water before experi-
ment. The studies were approved by the Animal Ethics Committee
of Guangzhou University of Chinese Medicine.
2.5. Evaluation of Sweet Tea in type 2 diabetic rats
2.5.1. Effect of different extracts of Sweet Tea on type 2 diabetic
animals
Type 2 diabetic rats (140–180 g) were induced by five weeks’
feeding with a high-cholesterol diet (HCD, 74.3% standard labo-
ratory feed, 5% lard, 10% yolk powder, 0.2% cholesterol, 0.5% bile
salt, 10% sucrose) and a final single injection of STZ (30 mg/kg body
weight) by intraperitoneal route after 12-h fasting. The STZ was
freshly dissolved in ice cold citrate buffer (0.1 M, pH 4.0). Diabetes
was confirmed by the determination of fasting blood glucose con-
centration with the help of a glucose meter on the sixth day after
administration of STZ. Rats that exhibited blood glucose levels of
11.1 mM/L or more were segregated and kept into cages marked
with groups.
Normal rats served as non-diabetic control which were fed with
standard laboratory feed and free to water (n = 6). Type 2 diabetic
rats were randomly divided into four groups (groups 2–5) of nine
animals each. Group 2 represented type 2 diabetic control rats
which received distilled water daily (T2DM). Group 3 received 1.5 g
of ST-1/kg body weight (ST-1). Group 4 (ST-2) and group 5 (ST-
3) were given ST-2 (1.5 g/kg body weight) and ST-3 (0.3 g/kg body
weight), respectively. Before drugs’ administration, content of total
flavonoids in the three ST extracts was determined by spectropho-
tometry to make sure groups 3–5 received the same amount of
flavonoids. The test drugs were fed orally for 4 weeks. Groups 1
and 2 rats received the same amount of distilled water. On 28th
day, fasting blood samples were collected from tail vein of all the
groups of rats. Blood was allowed to clot and serum separated
by centrifugation (3500 rpm × 10 min, RT) for the estimation of
145
glucose (FSG), total cholesterol (TC), triglyceride (TG), malondialde-
hyde (MDA), superoxide dismutase (SOD) and glycosylated serum
protein (GSP) using kits and assayed according to the manufac-
turer’s protocol. All animals were overdosed with pentobarbital
sodium (200 mg/kg, i.p.) and immediately sacrificed. Livers and
pancreases were removed promptly and weighed. All the tissues
from all groups were obtained and fixed in 10% neutral-buffered
formaldehyde for 48 h, embedded in paraffin and sectioned at
5 ␮m for histological study. The sections were stained with haema-
toxylin and eosin, and examined by light microscopy. Degree of
lipid deposits and cellular edema in liver tissues were scored as
0: absent; 1: slight; 2: moderate; and 3: severe (Bekheet, 2010).
Similar method was used to calculate the grades of islets atrophy
in pancreas tissues. Histopathological evaluation was performed
twice in one section from all animals in each group.
2.5.2. Further evaluation of ST-3 in type 2 diabetic rats
Animals were allocated into four groups with food and water
freely available. The first group was normal rats which received a
standard diet (Normal, n = 6), while the other groups (groups 2–4)
were type 2 diabetic rats fed with the high-fat diet: group 2, the type
2 diabetic control group received distilled water (T2DM); group 3
was given Diamicron (55 mg/kg, DMC); group 4 was treated with
ST-3 (0.3 g/kg, ST-3). The test and standard drug, distilled water
were fed orally once daily for four weeks. All of the rats were sub-
jected to an oral glucose tolerance test (OGTT) on the 15th day.
Blood samples of all rats were collected from the tail vein after fast-
ing for 12 h. Then, the rats were given a glucose solution by gavage
(2 g/kg) and tail blood was collected 30, 60, and 120 min later. On
the 22nd day, insulin tolerance test (ITT) was performed with sim-
ilar process of OGTT. Blood samples were collected before and 40,
80, 120 min after rats were injected subcutaneously with 0.5 U/kg
of insulin. Glucose responses during the OGTT and ITT were com-
puted from the area under the curve (AUC), using the trapezoidal
method (Prakasam et al., 2003; Kishino et al., 2009). At the end of
the experiment, fasting blood samples of all rats were collected to
determine the insulin (FSN), leptin (Lep) and C-peptide (C-P) in the
serum. The three biochemical parameters were tested with ELISA
kits according to the manufacturer’s recommendations.
2.6. Evaluation of ST-3 in type 1 diabetes rats
Type 1 diabetic rats (200–240 g) were induced with a single
injection of STZ (60 mg/kg) by intraperitoneal route. Rats with
blood glucose level of 16.7 mM/L or above were considered diabetic
and divided randomly into three experimental groups (n = 6–8):
group 2 was served as the type 1 diabetic control group (T1DM);
group 3 received 150 mg/kg of Metformin (MFM); group 4 was
treated with ST-3 (0.3 g/kg, ST-3). Sex- and age-matched control
animals were injected with the citrate buffer vehicle and served as
normal control group (Normal, n = 6). The test and standard drugs
were fed orally once daily for three weeks. Fasting blood glucose
was determined every week for all the rats. At the end of the exper-
iment, rats were fasted for 12 h and blood samples were collected
under anesthesia. FSG, FSN, TC, TG, urea nitrogen (BUN) and crea-
tinine (CREA) levels were determined as described in Section 2.5.
2.7. Statistical analysis
All the data were expressed as mean ± S.E.M. Within group com-
parisons were performed using analysis of variance (ANOVA) test.
Significant difference between control and experimental groups
were assessed by Student’s t-test. A probability level of less than
5% (p < 0.05) was considered significant.
146 S.-z. Hou et al. / Journal of Ethnopharmacology 138 (2011) 142–149
Fig. 3. Semiquantitative analysis of the atrophy of pancreatic tissues in different
treated type 2 diabetic rats. Data are expressed as mean ± S.E.M.; number of rats per
group n = 6. ## p < 0.01, compared with the normal control. **p < 0.01, compared with
the diabetic control using Mann–Whitney U-test.
3. Results
3.1. Different effect of ST-1, ST-2 and ST-3 on type 2 diabetic rats
There was a significant elevation in fasting blood glucose (FSG)
and glycosylated serum protein (GPS) in the diabetic control rats
as compared with non-diabetic control group (p < 0.05 or p < 0.01,
Table 1). The abnormal changes in FSG and GSP significantly were
inhibited in the ST-3 treated group as compared to the diabetic
control group after 4 weeks’ administration (p < 0.05). But no sig-
nificant decrease was detected in FSG or GSP in the ST-1 treated
and ST-2 treated diabetic rats (Table 1). The type 2 diabetic rats
showed a significant elevation in MDA and decrease in SOD in
serum (p < 0.01). Oral administration of ST-1 and ST-3 significantly
restored both MDA and SOD to normal status (p < 0.05 or p < 0.01)
while no obvious effect was shown by ST-2 treatment.
In Table 1, the results also showed that total cholesterol (TC)
and triglyceride (TG) levels were significantly elevated in the
type 2 diabetic control group as compared to the normal control
group (p < 0.05 or p < 0.01). After 4 weeks’ treatment of Sweet Tea
extracts, the alteration in lipid metabolism was attenuated by ST-
3 as evidenced by decreased serum TG and TC levels in diabetic
rats (p < 0.05 or p < 0.01). Similar response was found in the ST-1
treated group while ST-2 seemed no significant effect. After feed-
ing high-fat diet for total 8 weeks, liver mass significantly increased
in diabetic control rats compared to the normal control group
(p < 0.01). ST-3 had better effect on reducing the increasing of liver
mass than ST-1 and ST-2 but the liver mass in these three groups
was not significantly different from diabetic control group. Livers of
each group were also studied by histological examination for lipid
accumulation. Lipid deposits as macrovesicular and microvesicu-
lar steatosis were abundant in the liver of diabetic control group
along with severe hepatocellular edema. Treatment with all three
extracts of Sweet Tea reduced the lipid accumulation very notice-
ably with less fatty degenerations and cytoplasm displacements
(Table 2 and Fig. 1A–D; p < 0.01).
Pancreatic tissues were diffusely atrophied and characterized
by reduced size along with severe atrophy or loss of islets as a
direct effect of STZ treatment. The degree of atrophy of pancre-
atic acini in ST-1, ST-2 and ST-3 treated groups was significantly
attenuated than the diabetic control (Figs. 2A–E and 3; p < 0.05
or p < 0.01). Among the three extracts of Sweet Tea, ST-3 showed
better inhibition in hyperglycemia, hyperlipidemia, generation of
peroxide and tissue protection than other extracts in type 2 diabetic
models.
Fig. 4. Fasting blood glucose levels of normal and type 2 diabetic rats in each week.
Results were presented as mean ± S.E.M.; number of rats per group n = 6–9. Groups:
Normal = normal control; T2DM = type 2 diabetic control; DMC = Diamicron-treated
group; ST-3 = group treated by the flavonoid-rich fraction of Sweet Tea.**p < 0.01,
*p < 0.05 compared with the diabetic control.
3.2. Further study on the effect of ST-3 in type 2 diabetic rats
The blood glucose elevated every week (Fig. 4), along with sig-
nificant increase of insulin, leptin and C-peptide (C-P) in the type 2
diabetic control group compared to the non-diabetic control group
(p < 0.01, Table 3). Compared to the diabetic control group, adminis-
tration of DMC or ST-3 inhibited significantly the increase in glucose
level compared to the diabetic control group (p < 0.05 or p < 0.01,
Fig. 4). DMC also reduced C-P level significantly (p < 0.05) but had no
significant suppressing effect on the elevation of FNS and Lep. ST-3
administration significantly reduced the FNS, C-P and Lep (p < 0.05).
In the OGTT study, glucose concentration in normal group
reached peak level 30 min after glucose administration (2 g/kg) and
then began to decrease. We observed a different response in the
diabetic control group wherein the peak of plasma glucose level
significant increased within 30 min compared to the one in nor-
mal group (p < 0.01) and the glucose concentration began to decline
until 60 min after oral glucose administration (Fig. 5A). In DMC-
treated group, glucose concentration peak also began to decline
at 60 min. While in ST-3 treated group, the plasma glucose con-
centration peaked at 30 min after glucose load and subsequently
decreased. The area under the curve (AUC) for glucose was less
in the ST-3 group compared to the diabetic control group with
significant difference (p < 0.05, Fig. 5B).
The amount of insulin injected was reduced to 0.5 U/kg in order
to minimize hypoglycemia in ITT study. Blood glucose time profiles
in each group were shown in Fig. 6. In normal control group and
ST-3 treated group, blood glucose concentration reached the low-
est level within 40 min after insulin injection (Fig. 6A). While in the
diabetic control and DMC-treated group, the lowest glucose level
appeared 80 min after insulin injection. The AUC for glucose after
insulin supplement was less in the ST-3 treated group with signif-
icant difference from the one in diabetic control group (p < 0.05,
Fig. 6B).
3.3. Effects of ST-3 in type 1 diabetic rats
The single high-dose STZ-induced type 1 diabetic rats were con-
firmed by the significant hyperglycemia and weight loss compared
to the normal control rats (p < 0.01). These diabetic rats also had sig-
nificant alternations in FNS, TC, CREA, BUN and liver mass (p < 0.01
or p < 0.05, Table 4). In the Metformin-treated group, body weight
increased significantly and the elevation of FSG, TC and TG was sig-
nificantly suppressed as compared to diabetic control rats (p < 0.05
 S.-z. Hou et al. / Journal of Ethnopharmacology 138 (2011) 142–149 147

Table 1
Effect of Sweet Tea extracts on biochemical parameters and liver weight in type 2 diabetic rats.

Normal T2DM ST-1 ST-2 ST-3

FSG (mM/L) 5.9 ± 0.74 25.2 ± 1.73## 20.0 ± 2.39 23.1 ± 0.98 17.8 ± 1.76*
GSP (mg/mL) 1.3 ± 0.04 3.2 ± 0.39## 2.5 ± 0.14 2.7 ± 0.24 2.2 ± 0.07*
TC (mM/L) 0.9 ± 0.05 6.8 ± 1.71# 2.5 ± 0.88* 3.9 ± 0.93 2.5 ± 0.73*
TG (mM/L) 1.3 ± 0.08 11.8 ± 2.95## 3.0 ± 0.32* 5.7 ± 0.7 3.4 ± 0.31**
MDA (U/L) 4.4 ± 0.69 10.2 ± 1.10## 5.5 ± 1.15* 8.2 ± 0.79 5.8 ± 0.69**
SOD (U/L) 160 ± 4.41 132 ± 3.90## 149 ± 2.68** 139 ± 6.73 157 ± 2.52**
Liver weight (% of BW) 5.4 ± 0.19 9.6 ± 0.46## 8.6 ± 1.33 8.8 ± 0.87 8.0 ± 0.59

Data are expressed as mean ± S.E.M.; number of rats per group n = 6–9. ## p < 0.01, # p < 0.05 compared with the normal control. **p < 0.01, *p < 0.05 compared with the diabetic
control using ordinary ANOVA test. Groups: Normal = normal control; T2DM = type 2 diabetic control; ST-1 = group treated by aqueous extract of Sweet Tea; ST-2 = group
treated by ethanol extract of Sweet Tea; ST-3 = group treated by the flavonoid-rich fraction of Sweet Tea. FSG = fasting blood glucose; GSP = glycosylated serum protein;
TC = total cholesterol; TG = triglyceride; MDA = malondialdehyde; SOD = superoxide dismutase.

Table 2
Semiquantitative analysis of the hepatic lipid deposits and cellular edema in different treated type 2 diabetic rats.

Normal T2DM ST-1 ST-2 ST-3

Lipid deposits 0.0 ± 0.00 2.4 ± 0.18 ##

1.3 ± 0.14** 1.2 ± 0.12** 0.7 ± 0.12**
Hepatocellular edema 0.0 ± 0.00 2.8 ± 0.08## 1.7 ± 0.16** 1.5 ± 0.14** 1.0 ± 0.08**

Data are expressed as mean ± S.E.M.; number of rats per group n = 9. Groups: Normal = normal control; T2DM = type 2 diabetic control; ST-1 = group treated by aqueous extract
of Sweet Tea; ST-2 = group treated by ethanol extract of Sweet Tea; ST-3 = group treated by group treated by the flavonoid-rich fraction of Sweet Tea. ## p < 0.01, compared
with the normal control. **p < 0.01, compared with the diabetic control using Mann–Whitney U-test.

Table 3
Effect of ST-3 on serum insulin, C-peptide and leptin in type 2 diabetic rats.

Normal T2DM DMC ST-3

FSN (pM/L) 52.8 ± 7.95 88.3 ± 5.87 ##

74.7 ± 3.53 65.2 ± 2.29*
C-P (ng/mL) 0.47 ± 0.05 0.80 ± 0.04## 0.63 ± 0.04* 0.61 ± 0.03**
Lep (ng/mL) 0.30 ± 0.04 0.69 ± 0.08## 0.87 ± 0.11 0.48 ± 0.07*

Data are expressed as mean ± S.E.M.; number of rats per group n = 6–9. ## p < 0.01, compared with the normal control. **p < 0.01, *p < 0.05 compared with the diabetic control.
FSN = fasting serum insulin; C-P = C-peptide; Lep = leptin.

Fig. 5. Effects of ST-3 on glucose tolerance in experimental type 2 diabetic rats. (A) Establishment of oral glucose-intolerant state after three weeks’ treatment. Results
were presented as mean ± S.E.M.; number of rats per group n = 5. (B) Area under the serum glucose curve obtained through OGTT at the end of the treatment. Results were
presented as mean ± S.E.M.; number of rats per group n = 5. ## p < 0.01, compared with the normal control; *p < 0.05 compared with the diabetic control.

Table 4
Effects of ST-3 on biochemical parameters and liver weight in type 1 diabetic rats.

Normal T1DM MFM ST-3

Initial bw (g) 234 ± 1.9 222 ± 5.08 238 ± 4.8 227 ± 4.5
Final bw (g) 310 ± 5.3 223 ± 5.3## 256 ± 4.7* 243 ± 4.6
FSG (mM/L) 5.9 ± 0.32 26.1 ± 2.16## 16.8 ± 2.70* 19.1 ± 1.84*
FNS (mM/L) 81 ± 6.99 35 ± 3.96## 45 ± 3.17 48 ± 3.56*
TG (mM/L) 0.48 ± 0.03 0.68 ± 0.10 0.28 ± 0.05** 0.38 ± 0.02*
TC (mM/L) 0.90 ± 0.05 1.18 ± 0.06## 0.96 ± 0.06* 1.18 ± 0.07
CREA (mM/L) 30.3 ± 1.15 37.6 ± 2.04# 34.7 ± 1.44 35.9 ± 1.37
BUN (mM/L) 4.9 ± 1.05 15.3 ± 1.07## 15.4 ± 1.31 10.9 ± 0.96**
BUN/Cr 129 ± 11.1 358 ± 10.04## 305 ± 27.87 286 ± 21.79*
Liver weight (% of BW) 3.4 ± 0.08 4.5 ± 0.48## 3.9 ± 0.52 3.6 ± 0.11**

Data are expressed as mean ± S.E.M.; number of rats per group n = 6–9. ## p < 0.01, # p < 0.05 compared with the normal control. **p < 0.01, *p < 0.05 compared with the diabetic
control using ordinary ANOVA test. Groups: Normal = normal control; T2DM = type 2 diabetic control; MFM = Metformin-treated group; ST-3 = group treated by the flavonoid-
rich fraction of Sweet Tea. FSG = fasting blood glucose; GSP = glycosylated serum protein; TC = total cholesterol; TG = triglyceride; MDA = malondialdehyde; SOD = superoxide
dismutase.
148 S.-z. Hou et al. / Journal of Ethnopharmacology 138 (2011) 142–149
Fig. 6. Effects of ST-3 on insulin tolerance in experimental type 2 diabetic rats. (A) Establishment of insulin-intolerant state after four weeks’ treatment. Results were
presented as mean ± S.E.M.; number of rats per group n = 5. (B) Area under the serum glucose curve obtained through ITT at the end of the treatment. Results were presented
as mean ± S.E.M.; number of rats per group n = 5. ## p < 0.01, compared with the normal control; *p < 0.05 compared with the diabetic control.
or p < 0.01; Table 4). But Metformin did not regulate the level of FNS,
BUN, CREA, BUN/Cr or liver mass significantly. The administration
of ST-3 tended to bring down the level of FSG, TG, BUN, BUN/Cr and
liver mass, along with significantly increasing insulin level com-
pared to diabetic control rats (p < 0.05 or p < 0.01; Table 4). But ST-3
did not show obvious effect on animal body weight, CREA and TC.
4. Discussion
Lithocarpus polystachyus Rehd. (Sweet Tea) is well-known
folk herbal medicine in south China. Phytochemical studies
have revealed the presence of several classes of compounds.
These include flavonoids, polyphenols, free amino acid, vitamins,
pigments and microelements. Its pharmacological effects are con-
sidered due to the higher content of flavonoids and polyphenols
(Yang et al., 2007). The flavonoids in Sweet Tea leave include phlo-
ridzin, phloretin, quercetin, luteolin (Li et al., 2010). Phloridzin is
the most abundant component and it can improve hyperglycemia
in diabetic animals (Zhang et al., 1998; Zhao et al., 2004; Gosch et al.,
2010). Phloretin, quercetin and luteolin also have been reported to
possess antidiabetes effect. These studies suggest that Sweet Tea
leaves could be an antidiabetic agent. Different extraction methods
had a great impact on the yield of the flavonoids. We found that
aqueous extraction, low temperature with high extracting pressure
was the important extracting factors (data not shown). In our previ-
ous study, among different extracts of Sweet Tea, the flavonoid-rich
fraction (ST-3) exerted better biological activity including anti-
inflammation, antalgic, improving blood circulation, anti-fatty liver
(data not shown). In this study, ST-3 also exhibited more noticeable
effect on type 2 and type 1 diabetic rat models.
In all population, the overwhelming majority of diabetics are
subjects with type 2 diabetes (T2DM). T2DM is a polygenic disorder
characterized by defects in insulin secretion and insulin sensitiv-
ity, which lead to dysregulation of glucose and lipid metabolism
(LeRoith, 2002; Buchanan, 2003). Hyperglycemia (glucotoxicity)
and dyslipidemia (lipotoxicity) are common in T2DM patients
(Robertson et al., 2003). So any product with therapeutic effects on
hyperglycemia and hyperlipidemia would have a bright prospect
for treating T2DM.
In the present study, T2DM rat models were induced by long-
term high-fat feeding and the administration of small amount of
STZ. As expected, the rats resulted in hyperglycemia, increased
glycosylated serum protein (GSP), C-peptide (C-P) and insulin.
Supplementation of ST-3 inhibited hyperglycemia significantly. As
measurement of the level of GSP has been suggested as an indica-
tor of time-averaged glucose control in diabetic subjects (Nelson
et al., 1985), the decline of GSP in ST-3 treating group further con-
firmed the efficiency of ST-3. ST-3 also reduced the elevation of
insulin and C-P levels, which implied that ST-3 could improve the
insulin resistance in the T2DM rats. Furthermore, administration
of ST-3 could accelerate the restoration of blood glucose level after
glucose load or insulin injection. These results suggested that ST-3
could improve glucose metabolism and insulin sensitivity in T2DM
subjects.
Progression of T2DM can be exacerbated by consumption of
high-calorie foods and preventing lipotoxicity is an important ther-
apeutic target for T2DM (Lee et al., 1994; Unger, 1995; Kusminski
et al., 2009; Chavez and Summers, 2010). Our results also demon-
strated that the high level of serum cholesterol (TC), triglycerides
(TG) and lipid deposits in type 2 diabetic rats was caused by high
cholesterol diet. All of the Sweet Tea extracts could attenuate the
hypercholesterolemia, hypertriglyceridemia and liver lipid accu-
mulation in T2DM rats. Similar hypolipidemic effect was found
in fatty-liver rat models which were also fed by high-fat diet for
at least two months (data not shown). Though we did not know
the exact mechanism of Lithocarpus polystachyus Rehd. leaves’
hypoglycemic and hypolipidemic effects, our results provide a
good explanation of its usage as a traditional diabetes and obesity
medicine.
Hyperglycemia can produce reactive oxygen species (ROS)
which play a central role in complications of diabetes (Bonnefont-
Rousselot, 2002). Antioxidants play a major role in protection
against molecular oxidative damage (Evans, 2007; Evans et al.,
2008). ST-3 supplementation restored the decreased SOD activity
and increased MDA in T2DM animals, which indicated the efficacy
of plant extract in antioxidant status in type 2 diabetic serum.
The single high-dose STZ-induced diabetic rat is one of the ani-
mal models of human type I diabetes mellitus (T1DM). In this
model, diabetes arises from irreversible destruction of the ␤-islet
cells of the pancreas, causing marked reduction of insulin secre-
tion (Pushparaj et al., 2001). Three weeks of ST-3 administration
reduced the hyperglycemia significantly as well as inhibited the
decline of serum insulin in the T1DM rats. It has long been known
that in the STZ-induced T1DM rats, the rise in blood glucose is
accompanied by an increase in TG, TC, BUN and CREA (Cam et al.,
1993; Pushparaj et al., 2001). Treatment with ST-3 reversed some
of these parameters, such as TG, BUN and BUN/Cr to near normal.
5. Conclusion
In the present study, supplementation of purified aqueous
extract of Lithocarpus polystachyus Rehd. leaves such as the
 S.-z. Hou et al. / Journal of Ethnopharmacology 138 (2011) 142–149
flavonoid-rich fraction (ST-3) could suppress the hyperglycemia,
increase glycosylated serum protein, cholesterol, triglycerides, per-
oxide, urea nitrogen and liver mass in experimental T2DM or T1DM
rats. Interestingly, ST-3 showed desirable management of hyperin-
sulinemia in T2DM models and hypoinsulinemia in T1DM models
in this study. These findings suggested that ST-3 might attenuate
the development of obesity-related type 2 diabetes mellitus with
insulin resistance, as well as might be effective in type 1 diabetes
mellitus in humans. The mechanisms underlying the effect of ST-
3 on both type 2 and type 1 diabetes mellitus deserved further
investigation.
Conflicts of interest
There are no conflicts of interest of authors or consortium mem-
bers since the project was funded by public funds. This project is
dedicated to basic research and there are no commercial interests.
Acknowledgment
The financial support for this research from the National Natural
Science Foundation of China (No. u0972004) is gratefully acknowl-
edged.
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