Combretum lanceolatum flowers extract shows
antidiabetic activity through activation of
AMPK by quercetin
Carlos Roberto Porto Dechandt, Juliany Torres Siqueira,
Damiana Luiza Pereira de Souza, Lais Cobianchi Junqueira
Araujo, Virginia Claudia da Silva, Paulo Teixeira de Sousa
Junior, Claudia Marlise Balbinotti Andrade, Nair Honda
Kawashita, Amanda Martins Baviera*
Departamento de Química, Universidade Federal de Mato Grosso, Brazil.
Abstract: The present study evaluated the antidiabetic activity of the Combretum
lanceolatum Pohl ex Eichler, Combretaceae, flowers extract (ClEtOH) in diabetic
rats. Streptozotocin-diabetic rats were divided into four groups: diabetic control,
diabetic treated with 500 mg/kg of metformin and diabetic treated with 250 or 500
mg/kg of ClEtOH for 21 days. The treatment of diabetic rats with 500 mg/kg of
ClEtOH promoted an increase in the weight of liver, white adipose tissues and
skeletal muscles, improving body weight gain. Diabetic rats treated with 500 mg/kg
of ClEtOH also presented reduction in glycemia, glycosuria and urinary urea levels,
and increase in liver glycogen content. HPLC chromatogram showed that quercetin
is the major compound in the extract. The phosphorylation levels of adenosine
monophosphate-activated protein kinase were increased in liver slices incubated in
vitro with 50 µg/mL of ClEtOH, similarly to the incubation with metformin (50 µg/
mL) or quercetin (10 µg/mL). The antihyperglycemic effect of ClEtOH was similar
to that of metformin and appears to be through inhibition of gluconeogenesis,
since urinary urea was reduced and skeletal muscle mass was increased. These
data indicate that the antidiabetic activity of the Combretum lanceolatum extract
could be mediated, at least in part, through activation of adenosine monophosphateactivated protein kinase by quercetin.
Introduction
Diabetes mellitus, a chronic metabolic disease
characterized by a deiciency in the pancreas insulin
production and/or by peripheral insulin resistance, can
be referred as a global epidemic disease; data from the
World Health Organization (WHO) estimate that this
disorder affected 285 million people worldwide in 2010
and projections rise to 439 million in 2030 (Shaw et al.,
2010). In Brazil, diabetes mellitus appeared as one of
the ten main causes of deaths in Brazil in 2002 (WHO,
2006). Schmidt and collaborators (2011) have shown that
the prevalence of diabetes in Brazil has been rising in
association to obesity and to increased western diet and
physical inactivity, affecting 5.3 % of Brazilians aged 20
years or older in 2008, in comparison to 3.3 % in 1998.
The adverse effects of hypoglycemic drugs
and insulin and the excessive cost of these medications
can be mentioned as some disadvantages regarding the
diabetes treatment, which stimulate the search for new
therapeutic agents that present safety, effectiveness and
Revista Brasileira de Farmacognosia
Brazilian Journal of Pharmacognosy
23(2): 291-300, Mar./Apr. 2013
Article
Received 1 Sep 2012
Accepted 1 Nov 2012
Available online 4 Dec 2012
Keywords:
AMPK
antidiabetic activity
Combretum lanceolatum
metformin
quercetin
streptozotocin-diabetic rats
ISSN 0102-695X
DOI 10.1590/S0102-695X2012005000140
low cost. Nowadays, there is growing trend towards using
herbal preparations and/or derivatives in traditional and
complementary medicine to treat diabetes symptoms (Yeh
et al., 2003). In this way, it has been crescent the interest of
current ethnopharmacological research to investigate the
plants species with antihyperglycemic effect, focusing in the
evaluation of the eficacy and safety of plant preparations
for diabetes treatment, as well as the mechanisms of action
that explain their antidiabetic activities (Kawashita &
Baviera, 2010; Prabhakar & Doble, 2011).
Combretum lanceolatum Pohl ex Eichler,
Combretaceae, commonly known as “pombeiro-vermelho”,
is distributed from northern to southeastern of Brazil and
is found in several phytogeographic domains including
Amazon, Pantanal, Caatinga, Cerrado and Atlantic Forest
(Marquete & Valente, 2010). Ethnopharmacological
studies have demonstrated that plants from Combretum
genus presented antidiabetic activity. The six weeks
treatment of streptozotocin-diabetic rats with ethanolic
extract of Combretum decandrum Jacq. leaves decreased
fasting blood glucose levels (Pannangpetch et al.,
291
Antidiabetic effect of Combretum lanceolatum
Carlos Roberto Porto Dechandt et al.
2008). Chika & Bello (2010) showed that Combretum
micranthum G. Don leaves aqueous extract treatment
improved glucose tolerance and reduced fasting glycemia
in both normal and alloxan-diabetic rats. The treatment of
streptozotocin-diabetic rats with mollic acid glucoside, a
hydroxycycloartenoid isolated from leaves of Combretum
molle R. Br. ex G. Don, reduced glycemia levels in a dosedependent manner (Ojewole & Adewole, 2009). Phenolic
compounds, especially lavonoids, are widely reported in
Combretum genus, and they are probably the responsible
for its antihyperglycemic activity (Eloff et al., 2004;
Lopes et al., 2010). These indings stimulate the present
study, since there is no systematic study attempting to the
investigation of the antidiabetic activity of C. lanceolatum,
one of the most important species of Combretaceae in
the Brazilian Pantanal (Pott et al., 2011). In this way,
our laboratory initiated the evaluation of the antidiabetic
properties of several parts (lowers, fruits, leaves) of this
plant, attempting to its further indication in phytotherapic
formulations. The present study was undertaken to assess
the subchronic antidiabetic activity of the C. lanceolatum
lowers ethanolic extract (ClEtOH) in streptozotocindiabetic rats.
Materials and Methods
Plant material collection and preparation of the ClEtOH
extract
Flowers of Combretum lanceolatum Pohl ex
Eichler, Combretaceae, were collected in Poconé Porto
Cercado road (km 10), Poconé-MT, Brazil (S 16º18'56.4";
W 056°32'21.5"; 126 m of elevation) in July 2010. The
access to plant samples was authorized by Conselho de
Gestão do Patrimônio Genético of Ministério do Meio
Ambiente (license numbers 010457/2010-0). The plant
material was identiied by Dr. Germano Guarim Neto,
Central Herbarium, Universidade Federal de Mato Grosso,
where a voucher specimen (numbers 39,149) was deposited
for future reference. The lowers of C. lanceolatum were
dried at room temperature and grounded in electric grinder.
Later, 5,960 kg of the botanical material was placed
in maceration with ethanol at room temperature under
occasional shaking, in seven cycles of seven days. The
mixture was then iltered and concentrated on the rotary
evaporator at reduced pressure and 38 ºC approximately,
obtaining the crude ethanol extract of C. lanceolatum ClEtOH (2,350 kg; 39.43 % w/w).
Preparation of sample solutions
Quercetin (Riedel-de Haën, Germany) standard
stock solution and ClEtOH stock solution were prepared
in methanol (1.0 mg/mL and 5.0 mg/ml respectively). For
the sample fortiication quercetin and ClEtOH methanol
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Rev. Bras. Farmacogn. Braz. J. Pharmacogn. 23(2): Mar./Apr. 2013
solutions were mixed in the ratio of 2:1. All sample
solutions were iltered through 0.45 µm membrane ilter
(Millipore, USA), and injected directly into the HPLC-UV
for analysis.
Chromatographic analysis
High performance liquid chromatographic
analyses were carried out on a Varian ProStar liquid
chromatograph, equipped with binary gradient pumping
and a ProStar 325 Dual Wavelength UV-Vis Detector
(Varian). A HICHROM 5C18 reversed-phase column (ø
4.6 mm×250 mm) packed with 5 µm diameter particles
with a Kromasil® 100-5C18 guard column (4.6 mm×10
mm i.d., 5 μm) was employed. As the mobile phase, a
gradient of methanol-water containing 0.1% triluoroacetic
acid (50-80% in 35 min) was employed. The detections
were performed at 368 nm. Flow rate and injection volume
were 0.5 mL/min and 10 µL, respectively. The presence of
quercetin was conirmed by comparing its retention time
and by fortiication of the extract with standard quercetin.
All chromatographic operations were carried out at ambient
temperature.
Classical column chromatography on silica gel
and preparative thin layer chromatography were performed
in ClEtOH, affording the isolation of quercetin, among
other lavonoids, as a majoritary compound. The details
will be published elsewhere (Araujo, 2012).
NMR analyses
NMR analyses were recorded in CD3OD, using
TMS as internal standard in a Bruker spectrometer (500
MHz for 1H and 125 MHz for 13C).
Animals
Male Swiss-Webster mice weighing 25-30 g
(acute toxicity study) and male Wistar rats weighing 180210 g (subchronic antidiabetic activity) were housed in
a room under standard laboratory conditions (12:12 h
light-dark cycle, 24±1 °C) and had free access to water
and commercial lab chow diet (Purina®Labina). During
the experiments of the antidiabetic activity, rats were
housed in individual metabolic cages. All experiments
took place between 8 and 10 am. Experimental procedures
were made according to the Brazilian College of Animal
Experimentation and received prior institutional approval
by the Committee for Ethics in Animal Experimental from
UFMT (protocol number 23108.029613/09-3).
Acute oral toxicity study (Hippocratic test)
Groups of male mice received orally by gavage
a single administration of ClEtOH at crescent doses
Antidiabetic effect of Combretum lanceolatum
Carlos Roberto Porto Dechandt et al.
(100; 250; 500; 1,000; 2,500 and 5,000 mg/kg). The
control group received vehicle (water). The animals were
individually observed at 0, 5, 10, 15, 30 min; 1, 2, 4 and 8
h and after four and ifteen days (once a day) following the
extract administration. General behavioral observations
were noted in the table described by Malone (1977).
Subchronic evaluation of extract in streptozotocin-induced
diabetic rats
Streptozotocin (STZ, 40 mg/kg) dissolved in 0.01
mol/L citrate buffer (pH 4.5) was administered through a
single intravenous injection in 15 h fasted rats. Five days
after STZ administration, rats with glycemia levels of
approximately 400 mg/dL were selected to the experiments
and randomly assigned into four groups: DC, diabetic
control rats treated with vehicle (water); DMet, diabetic rats
treated with 500 mg/kg of metformin; DT250, diabetic rats
treated with 250 mg/kg of ClEtOH; DT500, diabetic rats
treated with 500 mg/kg of ClEtOH. The groups received
vehicle, metformin or freshly prepared extract by oral
gavage for 21 days. Body weight, food and water intake
and urinary volume were daily monitored. At every ive
days, plasma glucose levels were determined (Bergmeyer
et al., 1974), as well as the urea (Bernt & Bergmeyer,
1965) and glucose urinary levels (Summerson et al., 1947)
in 24 h urine. At the end of the treatment, white adipose
tissues (retroperitoneal, perirenal and epididymal), skeletal
muscles (soleus and extensor digitorum longus - EDL)
and liver were removed and weighted. Liver glycogen was
extracted with 30% KOH and precipitated with ethanol
(Sjörgren et al., 1938) and the quantity recovered was
measured by colorimetric phenol-sulfuric acid method
(Montgomery, 1957).
Incubation procedure and Western blotting analysis
Measurement of the adenosine monophosphateactivated protein kinase (AMPK) activation was
performed in liver slices incubated in vitro in the presence
of ClEtOH, quercetin or metformin. Rats were killed, liver
was rapidly dissected and uniform-shaped liver slices
(1 mm thick) were obtained from the right lobe with a
tissue chopper (Dogterom, 1993; Bach et al., 1996). Liver
slices were incubated during 1 h at 37 oC in Krebs-Ringer
bicarbonate buffer (pH 7.4) equilibrated with 95% O2/5%
CO2 and containing glucose (5 mM) in the absence (basal
sample) or in the presence of ClEtOH (50 and 100 µg/
mL), quercetin (10 and 50 µg/mL) or metformin (10 and
50 µg/mL). After incubation, tissues were immediately
frozen in liquid nitrogen before Western blotting analysis.
Following, tissues were homogenized in 50 mM Tris-HCl
buffer (pH 7.4) containing 150 mM NaCl, 1 mM EDTA,
1% Triton X-100, 0.1% sodium dodecyl sulfate (SDS), 10
mM sodium pyrophosphate, 100 mM sodium luoride, 10
mM sodium orthovanadate, 5 µg/mL aprotinin and 1 mM
phenylmethylsulfonyl luoride; supernatants samples were
used for protein levels determination (Bradford, 1976) and
for electrophoresis separation. Samples (100 μg of protein)
were subjected to SDS-PAGE analysis on 10% acrylamide
gels (Laemmli, 1970). Gels were electroblotted onto
nitrocellulose membranes (Towbin et al., 1979) and blotted
with anti-AMPKα (1:1,000, Cell Signaling Technology,
USA) and anti-phospho-[Thr172]-AMPKα (1:1,000,
Cell Signaling Technology, USA). Primary antibody was
detected by peroxidase-conjugated secondary antibody
(1:7,500, Santa Cruz Biotechnology, USA) and visualized
with SuperSignal West Pico chemiluminescent substrate
(Pierce Biotechnology, USA). Band intensities were
quantiied using the ImageJ Program (Version 1.38,
National Institutes of Health, USA, 2004).
Statistical analysis
Data were expressed as mean±SEM. Oneway analysis of variance followed by Tukey’s multiple
comparison tests was used to analyze the differences
between treated and control groups. Unpaired t test was
used to compare the means of AMPK phosphorylation
values from liver slices incubated under different conditions
with basal sample. Differences were considered signiicant
at p<0.05 and p<0.01.
Results
Isolation and identiication of quercetin in ClEtOH
The HPLC chromatogram (Figure 1A-C) has
shown that the lavonoid quercetin seems to be the major
compound in ClEtOH, and its presence was evidenced
by fortiication with standard quercetin solution, through
the increase in peak area at 18.365 min. Furthermore,
quercetin (1) has been isolated from ClEtOH through
classical column chromatographic fractionation (Araujo,
2012) and identiied by 1H and 13C NMR experiments and
comparison with literature data (Adeyemi et al., 2010) and
an authentic sample.
Quercetin (1): (256 mg; 0.039 %) yellow crystals, mp.
318-319 °C (315°C; [29]), 1H NMR (500 MHz, CD3OD):
7.75 (d, J2’,6’ 2.05 Hz, H-2’), 7.65 (dd, J6’,2’ 2.05 Hz and J6’,5’
8.5 Hz, H-6’), 6.90 (d, J5’,6’ 8.5 Hz, H-5’), 6.40 (d, J8,6 1.95
Hz, H-8), 6.20 (d, J6,8 1.95 Hz, H-6). 13C NMR (125 MHz,
CD3OD): 147.4 (C-2), 135.8 (C-3), 175.9 (C-4), 161.1
(C-5), 97.8 (C-6), 164.2 (C-7), 93.0 (C-8), 156.8 (C-4a),
103.1 (C-8a), 122.7 (C-1’), 114.6 (C-2’), 144.8 (C-3’),
146.6 (C-4’), 114.8 (C-5’), 120.3 (C-6’).
Rev. Bras. Farmacogn. Braz. J. Pharmacogn. 23(2): Mar./Apr. 2013
293
Antidiabetic effect of Combretum lanceolatum
Carlos Roberto Porto Dechandt et al.
OH
3'
4'
2'
HO
O
7
OH
5'
1'
2
Improvement in body and tissues weights of diabetic rats
treated with ClEtOH
Body weight
6'
3
OH
5
OH O
1
Hippocratic test
In the acute toxicity assay, it was demonstrated
that the treatment with ClEtOH in doses ranging from 100
to 5,000 mg/kg did not promote any behavioral changes
or mortality in animals. The doses of 250 and 500 mg/kg
were selected for the evaluation of the antidiabetic activity
of ClEtOH in the subchronic experiment.
At the beginning of the experiment, rats from
different groups showed similar body weight values. The
treatment of diabetic rats with 500 mg/kg of ClEtOH
promoted a 2-fold increase in the diary body weight gain
when compared to DC group (Table 1), leading to an increase
(21%) in the inal body weight (Table 1, Figure 2A). The
body weight in DT500 group was higher in comparison
to the beginning since the 12th day of treatment until the
end of the experiment. Although the animals treated with
metformin or with 250 mg/kg of ClEtOH presented a daily
body weight gain signiicantly higher than controls, the
inal body weight of animals from these groups was not
statistically different of values from untreated animals
(Table 1, Figure 2A).
Weight of peripheral tissues
It was observed an increase of 20% in the
weight of liver from diabetic rats treated with ClEtOH,
in both doses, as well as in metformin-treated rats, when
compared to DC. The treatment of diabetic rats with 500
mg/kg of ClEtOH also promoted increase in the weight
of epididymal (92%) and retroperitoneal (4-fold) adipose
tissues and of soleus (28%) and EDL (29%) muscles (Table
2), which corroborated with the increased body weight
gain of animals from DT500 group.
Reduction on food intake, liquid intake and urinary volume
of diabetic rats treated with ClEtOH
Figure 1. A. Chromatographic proile of Combretum
lanceolatum lowers ethanolic extract (ClEtOH); B. quercetin
standard; and C. ClEtOH with quercetin standard. Analysis in
HPLC-UV with detection at 368 nm.
There were no changes in the daily values of
food and water intake or urinary volume between DT250
and DC groups (Table 1). The treatment with 500 mg/
kg of ClEtOH promoted decrease of 14, 33 and 39%,
respectively, in daily food intake, water intake and urinary
volume in comparison to DC group (Table 1, Figure 2B,
2C and 2D). As expected, the metformin-treated rats also
presented a decrease in daily values of food and water
ingestion and of urinary volume in comparison to controls.
The food and water ingestion and the urinary volume of
diabetic rats treated with 500 mg/kg of ClEtOH were not
different to values of metformin-treated rats, showing
that plant extract improved these parameters likewise
metformin.
Treatment with ClEtOH
parameters of diabetic rats
Plasma glucose
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Rev. Bras. Farmacogn. Braz. J. Pharmacogn. 23(2): Mar./Apr. 2013
ameliorates
biochemical
Antidiabetic effect of Combretum lanceolatum
Carlos Roberto Porto Dechandt et al.
Prior to treatment beginning, all the groups
presented blood glucose levels at very similar values (DC =
493.0±17.6; DMet = 505.4±56.36; DT250 = 473.3±17.22;
DT500 = 484.9±27.77, mg/dL, n=8-9 animals). At the end
of the treatment, diabetic animals that received ClEtOH
presented a reduction of 16% (250 mg/kg) and 22 %
(500 mg/kg) in the glycemia levels when compared to
non-treated rats. As expected, the glycemia levels were
also reduced in diabetic rats treated with metformin, in
comparison to DC and DT250 groups (DC = 505.6±17.50;
DMet = 338.4±13.53; DT250 = 426.8±6.62; DT500 =
391.6±21.13, mg/dL, n=8-9 animals). It is important to
highlight that the glycemia values from DT500 and DMet
groups were similar at the end of the experiment. In the
same way, the area under the curve (AUC) of glycemia
from DMet, DT250 and DT500 groups were also lower
than controls (Figure 3).
Table 1. Values of body weight, food intake, water intake and urinary volume from diabetic rats treated during 21 days with 500
mg/kg of metformin (DMet) and with 250 (DT250) or 500 mg/kg (DT500) of Combretum lanceolatum lowers ethanolic extract.
DC (n= 8)
DMet (n= 9)
DT250 (n=9)
DT500 (n=8)
Initial body
weight (g)
203.7±3.91
200.0±7.64
213.2±3.77
199.2±14.03
Final body
weight (g)
235.4±6.03
269.9±11.66
264.8±7.08
283.7±12.19**
Daily weight
gain (g)
1.45±0.30
3.33±0.41**
2.60±0.32*
3.44±0.35**
Daily food
intake (g)
35.1±1.12
25.3±0.84**
34.9±1.40##
30.1±1.64*
Daily water
intake (mL)
151.3±10.70
64.1±6.60**
142.3±10.79##
100.7±13.03*
Daily urinary
volume (mL)
109.2±9.61
36.8±8.31**
105.1±8.15##
66.1±12.11*,##
Values represent mean±SEM; n: number of animals; *p< 0.05 and **p<0.01 vs DC; ##p<0.01. vs DMet.
Figure 2. Physiological parameters of diabetic rats non-treated (DC) and treated during 21 days with 500 mg/kg of metformin
(DMet) and with 250 (DT250) or 500 mg/kg (DT500) of Combretum lanceolatum flowers ethanolic extract. A. Body weight,
g; B. food intake, g; C. water intake, mL; D. urinary volume, mL. Each point represents mean±SEM of 8-9 animals. *p<0.05
and **p<0.01 vs DC; ##p<0.01 vs DMet.
Rev. Bras. Farmacogn. Braz. J. Pharmacogn. 23(2): Mar./Apr. 2013
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Antidiabetic effect of Combretum lanceolatum
Carlos Roberto Porto Dechandt et al.
values from DC group. As expected, the glycosuria values
from rats treated with metformin were also lower than
values from DC and DT250 groups, as well as the urinary
urea levels (Figures 4 and 5). There were no differences
in the urinary glucose and urea levels between DT500
and DMet groups. The AUC of glycosuria and urinary
urea from diabetic rats treated with 500 mg/kg of ClEtOH
were, respectively, 32% and 23% lower than values from
DC group (Figures 4 and 5).
Figure 3. Plasma glucose levels (mg/dL) during the experiment
(A) and area under the curve of glycemia (B) from diabetic rats
non-treated (DC) and treated during 21 days with 500 mg/kg of
metformin (DMet) and with 250 (DT250) or 500 mg/kg (DT500)
of Combretum lanceolatum lowers ethanolic extract. Each point
represents mean±SEM of 8-9 animals. *p<0.05 and **p<0.01 vs
DC.
Glycosuria and urinary urea
At the end of the experiment, the urinary glucose
(Figure 4) and urea levels (Figure 5) of diabetic rats treated
with 500 mg/kg of ClEtOH were, respectively, 43% and
38% lower than values from diabetic non-treated rats.
The glycosuria and urinary urea levels from diabetic rats
treated with 250 mg/kg of ClEtOH were not different of
Figure 4. Urinary glucose levels (g/24h) during the experiment
(A) and area under the curve of urinary glucose (B) from diabetic
rats non-treated (DC) and treated during 21 days with 500 mg/
kg of metformin (DMet) and with 250 (DT250) or 500 mg/kg
(DT500) of Combretum lanceolatum lowers ethanolic extract.
Each point represents mean±SEM of 8-9 animals. *p<0.05 vs
DC; #p<0.05 vs DMet.
Table 2. Weight of white adipose tissues (epididymal, retroperitoneal and perirenal), skeletal muscles (EDL and soleus) and
liver from diabetic rats treated during 21 days with 500 mg/kg of metformin (DMet) and with 250 (DT250) or 500 mg/kg
(DT500) of Combretum lanceolatum flowers ethanolic extract.
Epididymal (g)
DC (n= 9)
DMet (n= 8)
DT250 (n=8)
DT500 (n=8)
0.920±0.116
1.871±0.244**
1.334±0.091
1.765±0.228**
Retroperitoneal (g)
0.278±0.077
1.210±0.274*
0.733±0.111
1.116±0.260*
Perirenal (g)
0.146±0.074
0.240±0.054
0.132±0.017
0.254±0.064
EDL (g)
0.209±0.008
0.242±0.021
0.244±0.008
0.270±0.026*
Soleus (g)
0.252±0.012
0.269±0.012
0.279±0.018
0.322±0.019*
Liver (g)
10.78±0.293
12.83±0.782*
12.90±0.406*
12.67±0.687*
Values represent mean±SEM; n: number of animals; *p<0.05 and **p<0.01 vs DC.
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Antidiabetic effect of Combretum lanceolatum
Carlos Roberto Porto Dechandt et al.
Figure 6. Phosphorylation levels of AMPK (% of basal values)
in liver slices incubated in the presence of metformin [10 µg/mL
(Met10) and 50 µg/mL (Met50)], quercetin [10 µg/mL (Querc10)
and 50 µg/mL (Querc50)] and Combretum lanceolatum lowers
ethanolic extract [50 µg/mL (ClEtOH50) and 100 µg/mL
(ClEtOH100)]. Values are means±SEM of 3-5 liver slices. *p<
0.05 basal.
Figure 5. Urinary urea levels (g/24h) during the experiment (A)
and area under the curve of urinary urea (B) from diabetic rats
non-treated (DC) and treated during 21 days with 500 mg/kg of
metformin (DMet) and with 250 (DT250) or 500 mg/kg (DT500)
of Combretum lanceolatum lowers ethanolic extract. Each point
represents mean±SEM of 8-9 animals. *p<0.05 and **p<0.01 vs
DC; #p<0.05 vs DMet.
Liver glycogen content
The liver glycogen content was 90% higher
in diabetic animals treated with 500 mg/kg of ClEtOH
in comparison to DC group (DC = 11.36±1.17; DMet =
19.00±4.42; DT250 = 20.39±4.62; DT500 = 21.58±4.21,
mg/g, n = 8-9 animals).
ClEtOH promotes in vitro AMPK activation in rat liver
slices
As expected, the incubation of liver slices with
50 µg/ml metformin promoted an increase (23%) in
the phosphorylation levels of AMPK. There was also
observed a 29% raise in the AMPK phosphorylation when
liver slices were incubated with 10 µg/mL quercetin.
Incubation of liver slices with 50 µg/mL of ClEtOH
resulted in an increase in the phosphorylation levels of
AMPK (31%) in a similar magnitude of that observed to
metformin or quercetin. No changes were observed in the
AMPK phosphorylation after incubation with metformin,
quercetin and ClEtOH in the doses of 10, 50 and 100 µg/
mL, respectively (Figure 6).
Discussion
Crescent attention has been given to the
investigation of the antidiabetic activity of many plant
species all around the world, using different experimental
models and several methodologies. However, information
about the safety of plant-derived medicines is essential,
considering that medicinal plants are easily acquired by
the population and that many toxicological effects may
be observed after the use of herbal preparations. In this
way, it is common the use of mice in the experimental
investigation of the acute toxic effects of plant extracts
orally administered in high doses (Mu et al., 2011;
Jesus et al., 2012). Data from this study showed that the
Combretum lanceolatum Pohl ex Eichler, Combretaceae,
extract did not have any acute toxicity in animals, at every
tested dose. In front of the lack of toxicity, the doses of
250 and 500 mg/kg were chosen to the evaluation of the
antidiabetic activity of C. lanceolatum lower extract in
STZ-diabetic rats.
In insulin deiciency and/or resistance conditions,
the hyperglycemia is a consequence of the combination
of increased endogenous glucose production, related to
the higher rates of glycogenolysis and gluconeogenesis
processes, and reduced glucose uptake by peripheral
tissues. It has been proposed that the search and/or
development of new compounds that correct these glucose
metabolism disturbances will be important to highlight
new options for diabetes treatment (Wu et al., 2005; Leney
& Tavaré, 2009).
Speciically to gluconeogenesis, it is well known
that insulin deiciency lead to a marked skeletal muscle
Rev. Bras. Farmacogn. Braz. J. Pharmacogn. 23(2): Mar./Apr. 2013
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Antidiabetic effect of Combretum lanceolatum
Carlos Roberto Porto Dechandt et al.
proteolysis, increasing the low of amino acids into the
liver and providing substrates to gluconeogenesis. Since
our results showed a reduction in the urinary urea values
(Figure 5) and an increase in the weight of soleus and EDL
skeletal muscles (Table 2), it is reasonable to speculate that
the improvement in the carbohydrate metabolism of diabetic
rats treated with C. lanceolatum could be explained, at least
in part, by a decrease in the rate of hepatic gluconeogenesis,
modulating the activity and/or gene expression of the
rate-limiting enzymes of gluconeogenesis, as example
phosphoenolpyruvate carboxykinase (PEPCK) and/or
glucose-6-phosphatase (G6Pase). If C. lanceolatum extract
exerts an antidiabetic activity through gluconeogenesis
inhibition, its action can be related to that promoted by
metformin, a classical hypoglycemic drug that lead to
gluconeogenesis inhibition (Hundal & Inzucchi, 2003).
Recent indings have described that the hypoglycemic
effect of metformin occurs through activation of adenosine
monophosphate-activated protein kinase (AMPK), a
serine/threonine protein kinase that exerts a central role in
the regulation of cellular metabolism and energy balance
(Hardie, 2007). Indeed, it was demonstrated that metformin
downregulates PEPCK and G6Pase expression through
liver AMPK activation (Lochhead et al., 2000; Zhou et al.,
2001). Nowadays, AMPK activation has been pointed as
a potential target for the treatment of diabetes and other
metabolic disorders (Towler & Hardie, 2007; Viollet et al.,
2007). In addition, the search of natural compounds that
activate this kinase showed that some polyphenols activate
AMPK during its beneicial effects on metabolic disorders,
as example quercetin (Aguirre et al., 2011), curcumin
(Aggarwal, 2010), epigallocatechin-3-gallate (Collins et
al., 2007) and resveratrol (Shin et al., 2009). Therefore, it
can be suggested that polyphenols are potential candidates
of natural origin to diabetes treatment (Hwang et al.,
2009), because they stimulate a cellular target similar
to that of metformin. Corroborating with this, data from
this study showed that AMPK phosphorylation levels
were increased in liver slices incubated in the presence
of C. lanceolatum extract (Figure 6) in a similar way
of that observed with quercetin or metformin, showing
that the antihyperglycemic effect of the extract could be
attributed, at least in part, to AMPK stimulation followed
by inhibition of gluconeogenesis process. Conirming this
hypothesis, our results also demonstrated that the in vivo
beneicial effects of the extract on several parameters were
very similar to that promoted by metformin, as example
reduction on glycemia (Figure 3), glycosuria (Figure
4) and urinary urea (Figure 5). Current investigations
have been carried out in our laboratory to conirm if the
in vivo AMPK activation is involved in the inhibition
of hepatic gluconeogenesis by C. lanceolatum extract,
through the analysis of the AMPK phosphorylation in
liver from diabetic rats treated with the extract, as well as
the changes in the PEPCK expression and in the hepatic
298
Rev. Bras. Farmacogn. Braz. J. Pharmacogn. 23(2): Mar./Apr. 2013
glucose production (Siqueira et al., 2012). Taken together,
it is believed that quercetin, identiied and isolated from
C. lanceolatum extract (Figure 1), activates AMPK in
vivo, contributing to the treatment of diabetes symptoms.
Corroborating our indings, quercetin has been reported
to be the active compound that explains the antidiabetic
activity of some plant species (Eid et al., 2010; Veerapur et
al., 2010). In fact, both in vivo and in vitro studies performed
with quercetin have also demonstrated the beneicial
effects of this lavonoid on several parameters altered in
diabetes (Aguirre et al., 2011). In this way, the isolation
of a potential antidiabetic compound in C. lanceolatum
extract, together with the beneicial effects observed in
STZ-diabetic rats treated with the extract reinforces our
purpose, focused in the continuity of the studies with C.
lanceolatum, obtaining suficient data to justify its future
indication into phytotherapic formulations to treat diabetes
symptoms.
Because liver glycogen content was increased in
diabetic rats after C. lanceolatum treatment, it is possible
that the extract promotes decrease in liver glycogenolysis
and/or increase in glycogenesis. In addition, since the
extract treatment also promoted increase in the weight
of adipose tissues (Table 2) and reduction in the food
intake (Figure 2B) of diabetic rats, similar to metformin
effects, it can be suggested that the extract promotes a
better glucose disposal by peripheral tissues. Considering
that quercetin, found in the extract, stimulates AMPK, a
probable beneicial effect on glucose uptake promoted
by C. lanceolatum can be referred as a metformin-like
response, since this hypoglycemic drug stimulates glucose
transporter type 4 (GLUT4) translocation to plasma
membranes of peripheral tissues in an AMPK-dependent
manner (Lee et al., 2011). These possibilities of the extract
actions are the focus of current studies.
In summary, the indings of the present study
indicated that the ethanolic extract of C. lanceolatum
lowers has antihyperglycemic activity, as well as that
quercetin is the major compound in the extract. The
antidiabetic effect of C. lanceolatum can be attributed, at
least in part, to the AMPK activation in liver by quercetin,
in a similar manner of metformin, inhibiting hepatic
glucose production. Further investigations are ongoing
in our laboratory to detail the mechanisms of action that
explains the antidiabetic effect of this plant extract.
Acknowledgments
The authors are grateful to Air Francisco Costa
for the technical assistance and to Prof. Evandro Luiz
Dall’Oglio for the support on the chromatographic
analysis. This work was supported by grants from the
Programa Institutos Nacionais de Ciência e Tecnologia
em Áreas Úmidas of CNPq/MCT and Centro de Pesquisas
do Pantanal. During this study CRPD received fellowship
Antidiabetic effect of Combretum lanceolatum
Carlos Roberto Porto Dechandt et al.
from Fundação de Amparo à Pesquisa do Estado de Mato
Grosso (FAPEMAT 697415/2010).
Authors contributions
CRPD and JTS contributed in collecting plant
sample, running the laboratory work and analysis of the
data. DLPS running the laboratory work. LCA contributed
to chromatographic analysis. CS, PTSJ, CMBA and
NHK, co-designed experiments, discussed analyses and
contributed to critical reading of the manuscript. AMB
designed the study, supervised the laboratory work, analysis
and interpretation of the data and drafted the paper.
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*Correspondence
Amanda Martins Baviera
Universidade Federal de Mato Grosso
Av. Fernando Correa Costa, 2367, 78060 900 Cuiabá-MT,
Brazil
baviera@ufmt.br
Tel.: +55 65 3615 8765
Fax: +55 65 3615 8798/99