Catechins as antidiabetic compounds of Bridelia ferruginea Benth root bark extract

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Vol. 10(10), pp. 182-186, October 2018
DOI: 10.5897/JPP2018.0528
Article Number: 66FBD6159007
ISSN: 2141-2502
Copyright ©2018
Author(s) retain the copyright of this article
http://www.academicjournals.org/JPP
Pharmacognosy and Phytotherapy
Full Length Research Paper
Catechins as antidiabetic compounds of Bridelia
ferruginea Benth root bark extract
Batomayena Bakoma1,2*, Bénédicte Berké2, Aboudoulatif Diallo1, Kwashie Eklu-Gadegbeku1,
Kodjo Aklikokou1, Messanvi Gbeassor1, and Nicholas Moore2
1Department of Pharmacy, Faculty of Health Sciences, University of Lome, Togo.
2Department of Pharmacology, University of Bordeaux, 33076 Bordeaux, France.
Received 8 August, 2018; Accepted 18 September, 2018
The present study was carried out to evaluate the antidiabetic activity of catechins isolated from
Bridelia ferruginea in previous studies. Epigallocatechin (EGC) and Epigallocatechin gallate (EGCG)
isolated from B. ferruginea were administrated to streptozotocin-induced diabetic mice to evaluate their
anti-hyperglycemic and anti-hyperlipidemic effects. Then, biochemical parameters were assayed in
different groups of streptozotocin-induced diabetic mice. The level of fasting blood glucose levels,
triglycerides (TG) and total cholesterol (TC) in streptozotocin-induced diabetic mice were significantly
decreased after daily oral administration of EGC and EGCG at doses of 10 mg/kg/day, for 21 days.
Glucose intolerance was significantly reduced in streptozotocin induced diabetic mice treated with
catechins. These results suggest that catechins constituents from B. ferruginea, revealed significant
anti-hyperglycemic and antihyperlipidemic activity in type 2 diabetes.
Key words: Bridelia ferruginea, epigallocatechin, streptozotocin, diabetes, medicinal plant.
INTRODUCTION
Diabetes mellitus (DM) is one of the most severe
metabolic disorders characterized by hyperglycemia as a
result of a relative or an absolute lack of insulin secretion,
or/and insulin action on its target tissue (Leila al., 2007).
There are other symptoms, including hyperlipidemia,
which can lead to the development of microvascular
complication of diabetes Sunth (Taskinen, 2003).
There are mainly two types of diabetes, type 1 and type
2. Type 1 diabetes is known as insulin-dependent-
diabetes-mellitus (IDDM), and results from a cellular
mediated autoimmune destruction of the β cells of the
pancreas leading to absolute insulin deficiency (Gavin et
al., 1997). Type I diabetes commonly occurs in child-
hood and adolescence, but can occur at any age. This
form of the disease may account for 5 to 10% of all cases
of diabetes (Stumvoll et al., 2005). Type 2 diabetes,
which is responsible for more than 90% of all diabetes
patients and previously referred to as non-insulin
dependent diabetes mellitus (NIDDM), or adult-onset
diabetes, is a term used for individuals who have insulin
resistance and usually have relative insulin deficiency
(Gavin et al., 1997). The risk of developing this form of
diabetes increases with age, obesity and lack of physical
activity. Obesity and type 2 diabetes are closely
correlated.
*Corresponding author. E-mail: bbakoma@univ-lome.tg. Tel: +228 91000199.
Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution
License 4.0 International License

Most of the conventional synthetic chemical antidiabetic
drugs have low rates of response; they have also severe
adverse effects. Accordingly, it is necessary to introduce
more effective hypoglycemic agents with lower adverse
effects (Sun et al., 2008).
In our previous studies, the effects of B. ferruginea
hydroethanolic extract were proven on some parameters
of metabolic syndrome in type 2 diabetes (Bakoma et al.,
2011); there was lack of apparent toxicity, acute or sub-
chronic, at doses greater than those that induce an effect
in animal disease models (Bakoma et al., 2013). The
ethyl acetate (EtOAc) soluble fraction of the
hydroethanolic extract from the roots of B. ferruginea
were found to be the most active fraction on diabetes
and catechins were isolated (Bakoma et al., 2014 ; 2015).
To the best of our knowledge, the active ingredients with
antidiabetic activity and their probable mode of action
have not been investigated so far. The present study was
designed to identify the active compounds of B.
ferruginea using streptozotocin induced diabetic mice.
MATERIALS AND METHODS
Plant material
The roots of B. ferruginea were collected in August 2012 from
Tsévié area, 35 km North East of Lomé (Togo). Botanical
authentication was confirmed at the Department of Botany,
University of Lomé, where a voucher specimen of B. ferruginea was
deposited at the herbarium (No. 83, 2010).
Animals
Male Swiss mice (BW 30 to 35 g) purchased from Elevage Janvier
(France) were maintained under standard conditions with a 12 h
light/dark cycle and had free access to standard laboratory diet and
water. Prior to initiation of dosing, all rats and mice were acclimated
for 7 days. After, mice were randomized to different groups on the
basis of their body weights. Principles of laboratory animal care as
described in the European Community guidelines were followed
(Official Journal of European Union L197 vol. 50, July 2007). This
study was approved by the ethical committee on animal
experimentation of the University of Bordeaux.
Extraction and fractionation
The air-dried and powdered root bark of B. ferruginea (1230 g)
were sliced and macerated in 9000 ml ethanol-water (80:20) three
times for 72 h at room temperature. The extract was then
evaporated under vacuum (40°C). The residue (172 g) was
dissolved in distilled water and partitioned three times with hexane
(3X400 ML), dichloromethane, DCM (3X400 ML), ethyl acetate,
EtOAc (3X400 ML).
Ethyl acetate fractions were used to isolate epigallocatechin and
epigallocatechin gallate in previous studies (Bakoma et al., 2015).
Diabetes induction and treatment
Forty Swiss male mice (30 to 35 g) were randomly divided into 6
groups of 8 animals. Diabetes was induced in animals of group 2, 3,
Bakoma et al. 183
4, 5 and 6 with a single streptozotocin (STZ) intraperitoneal
injection, at 135 mg/kg weight, in 0.1 M citrate buffer, pH 4.5. Group
1 received the same volume of STZ vehicle (citrate buffer). A week
after STZ delivery, mice with blood glucose above 200 mg/dl were
included in the study, and 24 h later the animals were treated as
follows: group 1 (normal control, NC) and group 2 (diabetic control,
DC) received isotonic saline solution; group 3 (EA) received the
ethyl acetate fraction (50 mg/kg wt); group 4 (EGC) and group 5
(EGCG) received respectively epigallocatechin (10 mg/kg wt) and
epigallocatechin 3-O gallate (10 mg/kg wt); group 6 (MET) received
metformine (50 mg/kg).
Drugs and vehicle were administered daily by gavage for 21
days, and water and food intake were recorded. During the
experimentation, blood glucose level was measured on the first, 7
th, 14th and 21st day after 12 h fasting using Free style papillon
Glucometer.
Oral glucose tolerance test (OGTT) in STZ induced diabetic
mice
OGTT was performed after 21 day treatment, during which
mice were fed with normal diets. Mice were fasted over night;
glucose (2 g/kg) was fed 30 min after administration of drugs.
Blood was withdrawn from tail-vein at 0, 30, 60 and 120 min after
glucose loading. Blood glucose level was measured immediately
using Free style papillon Glucometer.
Estimation of biochemical parameters
Mice were anesthetized with pentobarbital (50 mg/kg i.p) and blood
was collected by heart puncture and centrifuged at 3000 g for 15
min and the plasma was aliquoted and frozen for blood glucose,
plasma total cholesterol (Ch), triglycerides (TG), aspartate
aminotransferase (AST), alanine aminotransferase (ALT) level
determination using commercial kit (Biomerieux,Marcy l’Etoile,
France).
Insulin concentrations were determined from frozen plasma
samples using the Rat and mice Insulin Enzyme Immunoassay Kit
(SPI-BIO, Montigny Le Bretonneux, France).
The index of insulin resistance was estimated by homeostasis
model assessment (HOMA) according to the following formula:
HOMA-IR= Insulin (mUI/L) x Plasma glucose (mmol/ L)/22.4
(Matthews et al., 1985).
Statistical analysis
GraphPad Prism 5.00 (USA) software was used to process the
results. They are expressed as mean value with the standard error
of the mean (M ± E.S.M). These results are analyzed using the
variance analysis (ANOVA) followed by the Tukey posttest, to
compare the batches. The materiality threshold is set at P <0.05.
RESULTS
Effect of substances on STZ-induced diabetic mice
blood glycaemia during the experiment mice
The anti-hyperglycemic effect of EGC and EGCG was
evaluated in STZ-induced diabetic rats. Blood glucose
level was measured in normal and experimental rats on
days 0, 7, 14, 21 of drug treatment. Streptozotocin
administration (100 mg/kg) led to over 2.8 fold elevation
of glycemia in a time-dependent manner (p<0.001)
compared to normal mice. STZ-induced diabetic mice

184 J. Pharmacognosy Phytother.
Table 1. Effect of substances on blood sugar level during the experiment.
Groups
Blood glucose (mg/dl)
Day 0
Day 7
Day 14
Day 21
Normal Control
813.1
862.8
8836
8531
Diabetic control
226±20
241±17###
282±0.8###
317±25###
EA
213±12
222±24
210±13***
225±26**
EGC
2478.6
23935
21515**
22113**
EGCG
26418
271 16
24810*
20510 ***
MET
25025
21813
2089***
18224***
Data were expressed as value with the standard error of the mean (M ± E.S.M, n = 8)
and evaluated by ANOVA followed by Tukey’s test at 5% *P < 0,05 ; **p < 0,01;
***P<0,001 (vs DC); ## P < 0,01 ; ### P < 0,001 (vs C).
Table 2. Effect of substances on oral glucose tolerance test in STZ-induced
diabetic mice.
Groups
Blood glucose (mg/dl)
0 min
30 min
60 min
120 min
Normal Control
853.1
1462.8
1803.6
1623.6
Diabetic control
317±25
441±17###
382±8
412±8
EA
225±26
321±34 **
291±26 **
278±9
EGC
22113
39925*
34515**
285±18
EGCG
20510
341 16***
31810***
220±15***
MET
18224
31813**
2499**
190±11**
Data were expressed as value with the standard error of the mean (M ± E.S.M, n = 8)
and evaluated by ANOVA followed by Tukey’s test at 5% *P < 0,05 ; **p < 0,01;
***P<0,001 (vs DC); ## P < 0,01 ; ### P < 0,001 (vs C).
treated respectively with ethyl acetate fraction (50 mg/kg
wt), EGC (10 mg/kg wt) and EGCG (10 mg/kg wt) for 3
weeks showed a significant (p<0.001) decrease in
glycemia compared to diabetic control group. Normal
control mice did not show any alteration in their glycemia
through the duration of the experiment significantly (Table
1).
Oral glucose tolerance test
Administration of glucose (2 g/kg,) produced significant
increase in blood sugar level of normal control mice.
Treatment with EGC, EGCG, and metformin significantly
reduced blood glucose level at 30 min, 60 min and
120 min compared to diabetic control mice (Table
2).
Effect of substances on plasma biochemical
parameters
At the end of the study, fasting blood sugar level of STZ-
diabetic control (27116 mg/dl) was high compared to
normal control group (12934 mg/dl) significantly (p <
0.001).
Ethyl acetate fraction, catechins and metformin treated
groups showed significant (p < 0.001) decrease of
glycaemia over 21 days of treatment compared to STZ-
diabetic control group. Plasma triglycerides and total
cholesterol levels at the end of the study were
significantly (p < 0.001) higher in the STZ-diabetic control
group (167±13; 1314.9 mg/dl) than in normal control
group (1215.6; 1173.9). Treated groups showed a
significant (p < 0.01) reduction of plasma cholesterol and
triglycerides level, neither EGCG treated group. AST
level was significantly (p <0.001) higher in diabetic control
group (28642 UI/L) compared to normal control group
(1147.3 UI/L), only metformin treated group showed a
significant (p <0.01) reduction of plasma AST.
Plasma insulin concentrations were significantly lower
in diabetic control group (0.47±0.5 ng/ml) compared to
normal control group (0.85±0.2 ng/ml) but only EGCG
and metformin treated groups showed significant
increase of plasma insulin concentrations compared to
diabetic control group (p < 0.01) (Table 3).
DISCUSSION
EGC and EGCG were tested in diabetic mice. To induce

Bakoma et al. 185
Table 3. Effect of catechins on plasma biochemical parameters, insulin index in control, diabetic and treated
mice.
Parameters
NC
DC
EA
EGC
EGCG
MET
Plasma glucose (mg/dl)
12934
27116###
22836***
21821
19716
144±28***
Insulin (ng/ml)
0.85±0.2
0.47±0.05##
0.42±0.8
0.37±0.5
0.590.3
0.54±0.3
AST (UI/L)
1147.3
28642###
15515**
23841
1271.6**
16621**
ALT (UI/L)
646.8
71 6.1
4810*
6510
676.2
637.7
TG (mg/dl)
1215.6
15713##
1189**
11224**
1158.2
935**
Ch (mg/dl)
1173.9
1314.9##
1308.7**
11010*
12611
10410**
Data were expressed as value with the standard error of the mean (M ± E.S.M, n = 8) and evaluated by ANOVA
followed by Tukey’s test at 5% *P < 0,05 ; **p < 0,01; ***P<0,001 (vs DC); ## P < 0,01 ; ### P < 0,001 (vs C).
diabetes in vivo, Streptozotocin were used, a molecule
produced by Streptomyces achromogenes; it is a
substance with antineoplastic, oncogenic and
diabetogenic activities (Like and Rossini, 1976). It
destroys selectively pancreatic-cells by oxidative stress
(Szkudelski, 2001; Long-Ze, 2008). Streptozotocin
induces type 1 or Type 2 diabetes depending on
administered dose (Islam and Loots, 2009). Multiple low-
dose of STZ leads to diabetic rats resembling type 1
diabetes in humans characterized by insulitis with
accumulation of inflammatory cells and degranulation of
cells. A single high-dose administration of STZ causes
toxicity to cells, with inflammation free islet lesions and
degranulation, which is like type 2 diabetes (Islam and
Loots, 2009).
In this study, a single high-dose administration of STZ
significantly induced hyperglycemia accompanied by
hypoinsulinemia. Oral administration of catechins and
metformin for 21 days induced a marked anti-
hyperglycemic activity in STZ-induced-diabetic mice by
reducing glycemia and showing a significant
improvement in glucose tolerance.
This effect can be the result of intestinal glucose
absorption with extra pancreatic action including the
stimulation of peripheral glucose utilization or enhancing
glycolytic and glycogenic process.
Hypercholesteremia and hypertriglyceridemia are
factors seen in the development of atherosclerosis and
coronary heart disease which are some complications of
diabetes (Ananthan et al., 2003). Catechins and
metformin significantly reduced serum triglycerides and
total cholesterol in STZ-diabetic mice. Thus, it is
reasonable to conclude that catechins of B. ferruginea
could modulate blood lipid abnormalities.
Liver is the vital organ of metabolism, detoxification,
storage and excretion of toxic agents and their
metabolites. ALT and AST are markers of liver function
(Ohaeri, 2001). An increase in the activities of ALT and
AST in plasma might be due to the leakage of these
enzymes from the liver cytosol into the blood stream
which gives an indication of hepatotoxic effect of STZ
Ramesh et al. (2010). Treated diabetic mice showed a
reduction of these enzymes activities in plasma
compared to the diabetic untreated mice and
consequently alleviated liver damage caused by STZ-
induced diabetes. Significant reductions in the activities
of these enzymes in treated diabetic mice indicated the
hepato protective role in preventing diabetic
complications.
Some authors indicate also that catechins are
hypoglycaemic properties and act to control diabetes
(Kao et al., 2000; Mai and Chuyen, 2007). Catechins
are powerful antioxidants; increase the sensitivity of cells
to insulin, inhibit the lipogenic enzymes and fat
absorption, (Thielecke and Boschmann, 2009; Roghani
and Tourandokht, 2010; Cherniack, 2011; Rains et al.,
2011; Sae-tan et al., 2011). These data confirms our
hypothesis that catechins are responsible for the activity
of the ethyl acetate fraction and suggest the mechanism
by which this fraction is useful in the treatement of type 2
diabetes.
Conclusion
This study suggests that catechins can be some of B.
ferruginea active molecules. EGC and EGCG tested
improved blood sugar level and glucose tolerance in STZ
induced diabetes. This confirm that catechins of B.
ferruginea root bark are responsible for the antidiabetic
activity.
CONFLICT OF INTERESTS
The authors have not declared any conflict of interests.
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