Plant Foods Hum Nutr (2009) 64:116–121

DOI 10.1007/s11130-009-0112-5

ORIGINAL PAPER

Antihyperglycemic, Antioxidant and Antiglycation Activities

of Mulberry Leaf Extract in Streptozotocin-Induced Chronic
Diabetic Rats
Jarinyaporn Naowaboot & Patchareewan Pannangpetch & Veerapol Kukongviriyapan &
Bunkerd Kongyingyoes & Upa kukongviriyapan

Published online: 12 May 2009

# Springer Science + Business Media, LLC 2009

Abstract In Thailand, beverages containing mulberry leaf Abbreviation

(Morus alba L.) are believed to promote good health, AGEs Advanced glycation endproducts
especially in people with diabetes. We examined the effects MA Morus alba-ethanolic extract
of long-term administration of an ethanolic extract of MDA Malondialdehyde
mulberry leaf (MA) on blood glucose, oxidative damage, STZ Streptozotocin
and glycation in streptozotocin-induced diabetic rats. Daily
administration of 1 g/kg MA for six weeks decreased blood
glucose by 22%, which was comparable to the effect of
4 U/kg insulin. Lipid peroxidation, measured as malondial- Introduction
dehyde and lipid hydroperoxide concentrations (3.50±0.33
and 3.76±0.18μM, respectively) decreased significantly (P< Sustained hyperglycemia is a central element in the
0.05) compared to nontreated control diabetic rats (8.19± pathogenesis of macrovascular and microvascular compli-
0.45 and 7.50±0.46μM, respectively). Hemoglobin A1C, a cations in diabetics [1]. Nonenzymatic products of glucose
biomarker for chronic exposure to high concentration of (advanced glycation endproducts [AGEs]) and free radical
glucose, was also significantly decreased in the MA-treated formation have been implicated in vascular complications
group (6.78±0.30%) in comparison to untreated group (9.02 [2]. AGEs are formed from covalent reactions between free
±0.30%). The IC50 of in vitro antiglycation and free radical amino groups of amino acids and the oxo groups of sugars;
scavenging activities of MA were 16.4±5.6μg/ml and 61.7± glucose, fructose, ribose. One of the most well-known
2.1μg/ml, respectively. These findings support that long- AGEs is hemoglobin A1C (HbA1C). It has been shown that
term administration of MA has antihyperglycemic, antioxi- chronic diabetic complications are caused by the cross-
dant and antiglycation effects in chronic diabetic rats, which linking of AGEs with long-lived proteins such as collagen,
may be beneficial as food supplement for diabetics. so that protein structure and function are altered [3].
Oxidative stress in diabetes is caused by hyperglycemia
Keywords Antiglycation . Antioxidant . Diabetes mellitus . inducing increased free radical formation via interruption of
Morus alba . Mulberry leaf the electron transport chain and glucose auto-oxidation. It
also occurs during AGE formation [4, 5]. A rise in the
J. Naowaboot : P. Pannangpetch (*) : V. Kukongviriyapan : oxidant level has two main effects: damage to various cell
B. Kongyingyoes components such as lipid peroxidation, and trigger the
Department of Pharmacology, Faculty of Medicine,
specific signaling pathway such as nuclear factor-κB and
Khon Kaen University,
Khon Kaen 40002, Thailand protein kinase C [6] leading to the release of proinflamma-
e-mail: patc_pan@kku.ac.th tory cytokines.
Mulberry (Morus alba L., family of Moraceae) is a native
U. kukongviriyapan
plant in Thailand. Its leaves are used for making herbal tea
Department of Physiology, Faculty of Medicine,
Khon Kaen University, that is used to promote good health and control diabetes.
Khon Kaen 40002, Thailand Intriguingly, Egyptian mulberry root bark extract adminis-
Plant Foods Hum Nutr (2009) 64:116–121 117

tered to rats with streptozotocin (STZ)-induced diabetes for blood was collected to determine fasting blood glucose
10 days at a dose of 600 mg/kg/day reduced blood glucose, level. Only rats with fasting blood glucose over 200 mg/dl
increased insulin levels, and reduced lipid peroxidation [7]. were considered diabetic and included in the experiments.
Our recent preliminary study found that short term admin-
istration (5 days) of an ethanolic extract of mulberry leaves Experimental Design
also possesses hypoglycemic activity in STZ-induced dia-
betic rats. Since diabetes is a chronic disease with severe The rats were divided into six groups with six rats in each
complications, the aim of the present study was to evaluate group and treated as follows: Group I: Non-diabetic control
the antihyperglycemic, antioxidant and antiglycation activi- rats with distilled water; Group II: Diabetic rats with
ties of the long term administrations of mulberry leaf extract distilled water; Group III–V: Diabetic rats with MA at
in chronic diabetic rats. doses of 0.25, 0.5 and 1.0 g/kg/day, respectively and Group
VI: Diabetic rats with 4 U/kg/day insulin (Monotard).
Treatments were continued for 6 weeks.
Materials and Methods Fasting blood glucose levels of all rats were determined
before and after 6 weeks of treatment. Blood glucose level
Chemicals was determined using a glucometer (Accu-Chek Advantage
II, Roche Diagnostics, Mannheim, Germany). During
All chemicals used in this study were of analytical grade fasting, rats were deprived of food for 12 h but had free
and obtained from Sigma (St. Louis, MO, USA). access to water.
At the baseline stage (before induction of diabetes) and
Plant Extraction after the 6-weeks treatment period, blood was collected
from tail vein for malondialdehyde, lipid hydroperoxide
Leaves of Morus alba Linn. were collected from a and HbA1C assays.
demonstration plot at the Faculty of Agriculture, Khon Kaen
University, Khon Kaen, Thailand. This plant was identified Measuring the Effect of MA on Oxidative Stress Markers
by Sericulture Research Institute, Ministry of Agriculture,
Thailand. Dried leaves were extracted with 50% ethanol. The Determination of malondialdehyde (MDA) by TBARS
mixture was filtered, evaporated in vacuum evaporator and assays: plasma MDA reacted with thiobarbituric acid
lyophilized. Using this procedure, the yield was 23.70% of (TBA) to form a colored complex so called thiobarbituric
the starting dry weight of the leaves. The obtained Morus acid-reactive substance (TBARS), according to the method
alba leaf extract (MA) was kept at −20 °C until it was used. described by Draper et al. [8]. The absorbance was
The extract was standardized using HPLC examining the measured at 532 nm by spectrophotometer. The amount of
amount of gallic acid and quercetin, which were 65±7 and MDA in plasma was calculated using a standard curve of
79±1μg/g, respectively. 1,1,3,3-tetra-ethoxypropane.
Determination of lipid hydroperoxide by ferrous ion
Animals oxidation xylenol orange (FOX) assay: plasma lipid
hydroperoxide oxidized Fe2+ to Fe3+ which bound to
Male Sprague-Dawley rats (200–250 g) were obtained from xylenol orange to form a color complex according to the
the Experimental Animal Unit, Faculty of Medicine, Khon method described by Nourooz-Zadeh et al. [9]. The
Kaen University, Thailand. They were maintained in an air- absorbance was measured at 560 nm by spectrophotometer.
conditioned room (25±1 °C), a 12 h light:12 h dark cycle The amount of lipid hydroperoxide in plasma was calcu-
and fed with standard diet (C.P. mouse feed, Bangkok, lated using a standard curve of hydrogen peroxide.
Thailand) and water ad libitum. All procedures complied
with National standards for the care and use of experimen- Measuring the Effect of MA on Glycation
tal animals and were approved by the Animal Ethics
Committee of Khon Kaen University, Khon Kaen, Thailand Degree of in vivo glycation was determined by assay of
(Rec. No: AEKKU0019/05). hemoglobin A1C (HbA1C). Whole blood with anticoagulant
was hemolysed and used for determining hemoglobin (Hb)
Induction of Diabetes in Rats and HbA1C concentrations. Total Hb was measured colori-
metrically and HbA1C was immunoturbidimetrically deter-
Diabetes was induced by a single intraperitoneal injection mined using COBAS INTEGRA 700 (Roche Diagnostics,
of 45 mg/kg body weight STZ dissolved in 0.1 M citrate Basel, Switzerland). The amount of HbA1C was expressed as
buffer (pH 4.5). After 7 days of STZ injection, the tail vein %Hb, which was calculated from the HbA1C/Hb ratio [10].
118 Plant Foods Hum Nutr (2009) 64:116–121

In vitro antiglycation activity of MA was examined by were performed using Student’s paired t-test. Comparisons
measuring the amount of fluorescent AGEs produced by among several groups were performed by analysis of
glycation reaction between bovine serum albumin and variance (ANOVA) followed by Student Newman-Keuls
sugar. Equal volume of bovine serum albumin (10 mg/ml test to show specific group differences. P values less than
in 50 mM phosphate buffer pH 7.4 with 0.02% sodium 0.05 were considered to be statistically significant. The IC50
azide to prevent bacterial growth), glucose (25 mM) and was calculated by the non linear curve fitting program
fructose (25 mM) were mixed together. Various concen- (Sigmaplot version 9, Systat software Inc.). The data were
trations of MA extract were added to the mixture. After fitted into the Hill’s equation, Y = Emax.Cn/[(IC50)n + Cn],
incubating at 37 °C for 2 weeks, the fluorescent AGEs were where Emax is maximal effect, C is concentration, n is Hill
measured by a fluorescence spectrophotometer (Gemini coefficient and IC50 is concentration that produced a half of
XPS, Molecular Devices, CA, USA) with an excitation maximal response.
wavelength of 350 nm and an emission wavelength of
450 nm [11]. All experiments were repeated four times. The
results were expressed in terms of IC50 value (concentration Results
in μg/ml required to inhibit AGE formation by 50%). For
the positive control group, aminoguanidine was used as a Effect of MA on Fasting Blood Glucose Level
standard antiglycation substance.
As shown in Table 1, the long-term antihyperglycemic effect
Measuring DPPH Radical Scavenging Activity of MA was examined. The fasting blood glucose levels of
the control diabetic group were still high after 6 weeks
The 1,1-diphenyl-2-picrylhydrazyl (DPPH) is a stable free of treatment. Interestingly, 0.5 and 1.0 g/kg MA, but not
radical which has maximum optical absorbance at 510 nm. 0.25 g/kg MA, significantly (P<0.05) reduced blood glucose
The reaction of DPPH with free radicals scavenger causes a levels by 16±7% and 23±10%, respectively. The oral
decline in the absorbance value [12]. MA extract at various administration of 1.0 g/kg MA reduced blood glucose levels
concentrations (1–1,000μg/ml; final concentration) was in diabetic rats to the same degree as 4 U/kg insulin.
added to equal volume of 0.1 mM DPPH solution in ethanol After all of the treatments had been discontinued for 1
(0.05 mM; final concentration) and left for 20 min at room week, the blood glucose of all of the diabetic rats returned
temperature. Then, the absorbance of the DPPH was to the high levels resembled the before treatment period
measured at 510 nm using a microplate ELISA reader (Table 1). This indicated that the diabetic condition existed
(TECAN, New York, USA). All experiments were repeated throughout the period of the experiment, or at least for
four times. The IC50 was calculated. For the positive control 7 weeks following the STZ injection.
group, vitamin C was used as a standard antioxidant.
Effects of MA on Plasma MDA and Lipid Hydroperoxide
Statistical Analysis Concentrations

All results are expressed as mean ± SEM. Comparisons of The basal levels of MDA and lipid hydroperoxide in all
blood glucose levels between before and after treatments of the studied animals were similar. After 6 weeks of

Table 1 Effect of mulberry leaf extract (MA) administration on fasting blood glucose in chronic diabetic rats

Groups Mean fasting blood glucose

Before treatment (mg/dl) After 6 weeks of treatment 1 week after treatment withdrawal (mg/dl)

mg/dl % Change

Control normal 73±3 75±3 +2 ±2 73±2

Control diabetic 297±18 326±16 +11±4 363±18
Diabetic + Insulin 4 U/kg 342±27 252±11a −24±7b 327±13
Diabetic + MA 0.25 g/kg 333±12 290±20 −12±6 353±19
Diabetic + MA 0.5 g/kg 296±15 243±19a −16±7b 315±14
Diabetic + MA 1.0 g/kg 328±15 237±22a −23±10b 318±17

+: increase, −: decrease, a: P<0.05 by paired t-test when compared to before treatment, b: P<0.05 by ANOVA followed by Student Newman-
Keuls test when compared to the diabetic control group
Plant Foods Hum Nutr (2009) 64:116–121 119

treatments, as shown in Fig. 1a and b, the amounts of

plasma MDA and lipid hydroperoxide of control normal
rats were only slightly increased, whereas the control
chronic diabetic rats showed a significant increase (P<
0.05) in both markers compared with the baseline level
(before induction of diabetes). Long-term administration
of 0.5 or 1.0 g/kg MA or 4 U/kg insulin significantly
decreased the amount of plasma MDA and lipid hydro-
peroxide in diabetic rats. The effects of MA on MDA and
lipid hydroperoxide formations were apparent in a dose-
dependent manner.

Effect of MA on HbA1C Levels

As shown in Fig. 2, the baseline HbA1C was similar in all

experimental groups. The control chronic diabetic rats had Fig. 2 Effect of mulberry leaf extract (MA) on plasma levels of
HbA1C in chronic diabetic rats. a P<0.05 by ANOVA followed by
an approximately 2-fold increase in HbA1C levels (P<0.05) Student Newman-Keuls test when compared to after treatment of
control normal rats, b P<0.05 by ANOVA followed by Student
Newman-Keuls test when compared to after treatment of control
diabetic rats

over the baseline measurement. However, HbA1C level

increases were significantly reduced in chronic diabetic rats
administered either 0.25–1.0 g/kg MA or 4 U/kg insulin for
6 weeks.

Effect of MA on Glycation In Vitro

MA inhibited in vitro AGEs formation with IC50 of 16.4±

5.6μg/ml (Fig. 3). The IC50 of aminoguanidine, a well
known AGE formation inhibitor, was 2.8±1.2μg/ml (Fig. 3).
MA showed lesser glycation inhibitory activity than that of
aminoguanidine.

DPPH Free Radical Scavenging Activity of MA

MA was a potent DPPH free radical scavenger with IC50 of

61.7±2.1μg/ml (Fig. 4). It scavenged DPPH free radicals in
a dose-dependent manner (3–1,000μg/ml). However, vita-
min C, a standard free radical scavenger, was approximately
6 times more potent than MA. The IC50 of vitamin C was
10.2±2.1μg/ml (Fig. 4).

Discussion

This present results showed that long-term daily admin-

Fig. 1 Effects of mulberry leaf extract (MA) on plasma MDA (a) istration of ethanolic extract of Morus alba leaves
and lipid hydroperoxide (b) concentrations in chronic diabetic rats. a
significantly decreased fasting blood glucose levels,
P< 0.05 by paired t-test compared to before induction of diabetes, b
P< 0.05 by ANOVA followed by Student Newman-Keuls test when oxidative stress, and glycation in STZ-induced diabetic
compared to after treatment of control diabetic rats rats. The intraperitoneal injection of STZ at doses of 50–
120 Plant Foods Hum Nutr (2009) 64:116–121

utes to the reduction in HbA1C levels. It should be noted

that although HbA1C levels were decreased in all of the
treatment groups, including the insulin-treated group, the
levels were still not normalized to the level observed in
the control groups.
The increase in free radicals observed in diabetics may
be derived from changes in mitochondrial respiration,
cytochrome P450, xanthine oxidase, protein kinase C,
glucose autoxidation, and AGE formation [4]. The present
data also demonstrated that hyperglycemia produced
marked oxidant effects, as revealed by the significant
increase in lipid peroxidation products, including MDA
and lipid hydroperoxide (Fig. 1). Interestingly, the long-
term administration of MA to STZ-induced diabetic rats
diminished the formation of MDA and lipid hydroperoxide
(Fig. 1). These effects may be caused by antioxidant
activity possessed by MA, because we found that MA
Fig. 3 The log dose-response curves representing the inhibitory
activities of mulberry leaf extract (MA) and aminogunidine on the scavenged the free radical DPPH, which corresponds with a
advanced glycation endproducts (AGEs) formation report of MA extract from other investigators [17] as well
as reports of fenugreek seed [18] and Lepidium meyenii root
[19]. Another possible cause may be the direct inhibitory
60 mg/kg into rats damages the pancreas, and insulin activity of MA on glycation, resulting in a decrease in free
levels typically fall to 10–30% of normal levels leading to radical release.
hyperglycemia. However, severe ketosis does not develop, In conclusion, mulberry leaf extract possesses antihy-
and the animal can survive for some weeks or months perglycemic, antioxidant and antiglycation activities in
without insulin replacement [13]. The glycemic reduction STZ-induced chronic diabetic rats, which promisingly
activity of 1 g/kg MA in diabetic rats was comparable with support the use of MA as food supplement or adjunct
the activity of 4 U/kg/d insulin (−22%, Table 1). The treatment for diabetics.
ability of MA to lower fasting blood glucose levels in
STZ-induced diabetic rats is probably mediated both by
the stimulation of insulin release [7] and by the intestinal
glucosidase inhibitory activity of the known mulberry-leaf
component 1-deoxynojirimycin (DNJ) [14]. Singab et al.
[7] demonstrated that an oral administration of Egyptian
Morus alba root bark extract for 3–10 days increased
serum insulin levels in STZ-induced diabetic rat. Howev-
er, an investigation using isolated cells could not show
insulin secretagogue activity [15].
The formation and accumulation of AGEs in various
tissues increases rapidly in chronic diabetes [2]. We
found that MA decreased in vitro fluorescent AGEs
formation and in vivo HbA1C, a kind of AGEs. In patients
with type 2 diabetes, the incidence of myocardial
infarction doubles as HbA1C levels increase from 5.5%
to 10.5% [16]. Interestingly, daily administration of MA
for 6 weeks significantly reduced the level of plasma
HbA1C in STZ-induced chronic diabetic rats. Most likely,
the hypoglycemic activity of MA contributed substantially
to the decrease in HbA1C levels. However, although a dose
of 0.25 g/kg MA did not reduce blood glucose levels, it Fig. 4 The log dose-response curves representing the DPPH (1,1-
still decreased HbA1C levels in the diabetic rats. Thus, MA diphenyl-2-picrylhydrazyl) scavenging activities of mulberry leaf
may possess specific antiglycation property that contrib- extract (MA) and vitamin C
Plant Foods Hum Nutr (2009) 64:116–121 121

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