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Cardiovascular & Haematological Disorders-Drug Targets, 2017, 17, 142-152
Ajebli M, Eddouks M*
Faculty of Sciences and Techniques Errachidia, Moulay Ismail University, BP 509, Boutalamine, Errachidia,
52000, Morocco.
Keywords: diabetes; Buxus sempervirens L.; streptozotocin; antihypergycemic. Oral glucose test tolerance; phytochemical, histology.
A R T I C L E H I S T O R Y
Received: September 05, 2017
Revised: May 16, 2018
Accepted: May 30, 2017
DOI:
10.2174/1871529X17666170918140817
142
RESEARCH ARTICLE
Buxus sempervirens L improves streptozotocin-induced diabetes
mellitus in rats
Cardiovascular & HematologicalDisorders - Drug
Targets
Abstract: Buxus sempervirens L. (Boxwood) is a medicinal plant used in Moroccan traditional
medicine for diabetes treatment in Morocco. However; there is no experimental support of this
ethnopharmacological use. The present investigation aimed to study the antidiabetic and the
preliminary phytochemical of the aqueous extract of the leaves of Buxus sempervirens (AELBS).
AELBS (5 mg/kg) was administered by gavage to normal and streptozotocin-induced diabetic rats and
blood glucose levels were measured for 6 hours (acute study) and 15 days (chronic study). Oral glucose
test tolerance (OGTT) and histopathological examination of liver and pancreas were carried out using
standard methods. Furthermore, preliminary phytochemical screening and quantification of phenolic
and flavonoid contents were also realized.
AELBS reduced the blood glucose of both healthy and diabetic rats. This extract was also able to
improve oral glucose tolerance in diabetic rats and it ameliorated hepatic histology, however, any
enhancement was noticed in pancreatic histology. Preliminary phytochemical study revealed that
AELBS possesses many chemical compounds such as alkaloids, phenolic compounds (flavonoids,
tannins, and cyanidins), carbohydrates, reducing sugars and mucilage. Additionally, this extract is rich
on phenolic compounds including flavonoids. According to all these findings, we conclude that
AELBS at a low dose demonstrated a potent antihyperglycemic effect in streptozotocin rats and further
investigations should be necessary of supporting the use of this plant in the management of diabetes
by the Moroccan population.
1. INTRODUCTION
The incidence of diabetes mellitus is high all over the
world. Different types of oral hypoglycaemic agents such
as insulin, biguanides and sulphonylureas are available for
the treatment of diabetes mellitus, but their side effects
constitute a limiting factor, without forgetting all the same
certain novel strategies as for example beta cell
replacement approaches include human whole pancreas or
islet transplantation, bio-artificial pancreas, automated
insulin delivery devices and genetically engineered insulin
secreting cells [1]. Consequently, interest in herbal
remedies is growing due to the minimal side effects and
relatively low costs of these natural products [2, 3].
*Address correspondence to this author at the Faculty of Sciences
and Techniques Errachidia, Moulay Ismail University, BP 509,
Boutalamine, Errachidia, 52000, Morocco; Tel: +212 35 57 44 97;
Fax: +212 35 57 44 85; E-mail: mohamed.eddouks@laposte.net
Several herbs or herbal products were used and continue to
be used as effective treatment against diabetes mellitus in
several folklores. In Morocco, exactly in the region of
Tafilalet, many medicinal plants have showed their
potential antihyperglycemic agents in Moroccan health-
care system [4]. Buxus sempervirens L. (Boxwood) named
locally as “Azazer” is one among a large range of
hypoglycemic herbs that are used in traditional medicine of
Tafilalet to manage diabetes mellitus. This species
belonging to the Boxaceae is found mainly in southern and
central Europe with a clear division into east and west
regions, as well as in North Africa. In traditional medicine
preparations of Buxus sempervirens (B. sempervirens)
were used internally for rheumatism and constipation, for
malaria and pneumonia, and externally for rashes, hair loss,
gout and rheumatic complaints. Homeopathically, this herb
is known to be used for greasy scalp with dandruff and for
hair loss [5]. Additionally, SPV30, a preparation from
sempervirens has shown beneficial effects in
1002-8801/17 $58.00+.00 © 2017 Bentham Science Publishers
B. sempervirens has shown beneficial effects in
HIV asymptomatic patients [6]. In addition, some chemical
compounds were isolated from this plant including
triterpenoidal alkaloids, triterpenes and sterols [7, 8].
Because no pharmacological study was performed
until now to support the antidiabetic use of B. sempervirens
by Moroccan population, we aimed through this
investigation to evaluate the antihyperglycemic effect of
the aqueous extract from the leaves of Buxus sempervirens
L. in normal and diabetic rats after a single and repeated
oral administration (5 mg/kg). In addition,
histopathological examinations and oral glucose test
tolerance were carried out in this investigation. Finally, a
preliminary phytochemical screening and estimation of
total phenolic and flavonoids contents in this aqueous
extract were realized.
1. Material and methods
1.1. Plant material
Specimens of Buxus sempervirens L. were
collected from the Tafilalet region (semi-arid area) of
Morocco in March–April 2016, and air-dried at 40 °C. The
plant was taxonomically identified and authenticated and
voucher specimen was deposited at the herbarium of the
Faculty of Sciences and Techniques, Errachidia.
1.2. Preparation of the aqueous extract
Plant material was prepared according to the
traditional method used in Morocco (decoction): leaves of
Buxus sempervirens L. after air-dried were ground in a
coffee mill, then, 1 g of powdered leaves, mixed with 100
ml distilled water, was boiled for 10 min and then cooled
for 15 min in a Soxhlet apparatus. Thereafter, the aqueous
extract was filtered using a Millipore filter (Millipore 0.2
mm, St Quentin en Yvelines, France) to remove particulate
matter. Finally, the filtration of the extract was lyophilized
in a lyophilizator (LABCONCO, G.BOYER, materiel de
laboratoire, Casablanca). The dose administered was 5 mg
of lyophilized aqueous extract per kg of body weight, we
consider that this dose is the minimal dose which must to
be administered to rats; it should be pointed out that a
preliminary screening was performed in order to determine
the adequate dose.
1.3. Preliminary phytochemical screening and
quantification of phenolic and flavonoid
contents
For a preliminary phytochemical screening of the
aqueous extract of leaves of Buxus sempervirens (AELBS),
tests of the presence of alkaloids, polyphenols, flavonoids,
cyanidins, tannins, glucosides, saponins, quinones,
anthraquinones, mucilage, sterols, sesquiterpenes,
reducing sugars, carbohydrates, terpenoids were carried
out according to standard methods [9-14]. The total
phenolic and flavonoids contents were performed as it has
been described previously [15, 16]. Total phenolic and
flavonoid contents were represented as mean of three
measures ± standard deviation (SD), where values were
estimated by calculation using linear equation illustrated in
the calibration curve.
1.4. Experimental animals
Healthy albino adult male rats (Wistar strain)
weighing 180-280 g were kept in individual polyethylene
cages and maintained in standard condition (23±1 ◦C, with
55±5% humidity and a 12 h/12 h light/dark cycle), and the
animals were fed ad libitum with normal laboratory chow
standard pellet diet.
1.5. Induction of diabetes and
1.9. Induction of diabetes and determination of
parameters
Diabetes was induced in the overnight fasted male
albino wistar rats by a single intraperitoneal injection (i.p.)
of streptozotocin (65 mg/ kg body weight) dissolved in 0.1
M citrate buffer (pH = 4.5), Normal control rat received
distilled water as vehicle. After 3 days, induction of
diabetes injection of STZ blood sample was collected from
the retro-orbital of the rat eyes and plasma glucose level
were determined. The animals confirmed diabetic by the
elevated plasma glucose levels 300 mg/dl were used for the
study.
Normal and diabetic rats were randomly assigned
to three different groups containing six rats each. One
control group received distilled water, a second treated
group received the aqueous extract of leaves of Buxus
sempervirens L. at a dose of 5 mg/kg (5 mg of lyophilized
aqueous extract of leaves of Buxus sempervirens L. per
kilogram of body weight) and the third group received a
reference drug (glibenclamide at a dose of 5 mg/kg). The
dose of 20 mg/kg was used according to the Moroccan
traditional use. For single oral administration, distilled
water (control), glibenclamide or the aqueous extract were
administered and blood glucose levels were monitored
during 6 h. For repeated oral administration, rats were
treated once daily for 15 days and blood glucose levels
were followed during this period. The rats (n = 6 in each
group) were treated orally. All experiments were
performed in fasted rats. Blood samples from rats were
collected from the retro-orbital sinus under ether anesthesia
in order to determine their plasma lipid profile. Blood
glucose levels were determined by the glucose oxidase
method using a reflective glucometer (Contour™ TS) from
Bayer Diabetes Care.
Body weight of all animals was followed during
the experiments.
1.10. Oral glucose tolerance test (OGTT)
Normal and STZ-diabetic albino wistar rats were
fasted for 16h before being subjected to an oral glucose
tolerance test (OGTT) by intragastric gavage with a
glucose solution to achieve a glucose load of 2 g/kg. The
rats were randomly divided into four groups (n = 6).
Group I served as a normal control and received distilled
water only.
Group II served as diabetic control and received standard
drug (glibenclamide), at the oral dose of 5 mg/kg body
weight.
Group III consisting of normal rats and received aqueous
extract, at the oral dose of 5 mg/kg as a fine aqueous
suspension.
Group IV consisting of STZ-diabetic rats and received
aqueous extract, at the oral dose of 5 mg/kg as a fine
aqueous suspension. After 30 min of extract
administration, the rats of all groups were orally treated
with 2 g/kg of glucose. Blood glucose levels were
measured immediately by glucose oxidase method using a
reflective glucometer (Contour™ TS) just prior to glucose
administration and at 30, 60, 90 and 120 min after glucose
loading.
Total glycemic responses to OGTT were
calculated from respective areas under the curve (AUC) of
glycemia during the 120 min observation period according
At the end of the experiment, the organs (pancreas
Buxus sempervirens L improves streptozotocin-induced diabetes mellitus in
rats
M Ajebli and M Eddouks
2. Results
2.1. Effect of AELBS on body weight
in normal and STZ-induced diabetic rats
Oral administration of AELBS (5 mg/ kg) during
15 days was not associated with any significant change
concerning the body weight, either for normal or STZ-
diabetic groups. The same remark was observed in control
group treated by glibenclamide as compared to control
untreated (Figure 1).
of total flavonoids was estimated to be equal to
45.68±0.014 mg of equivalent of quercetin per one gram of
the extract (45.68 mg±0.14 EQ/ g of AELBS).
2.3. Effect of Buxus sempervirens on
blood glucose levels
2.3.1. Single oral administration
Figure 2 represents the effect of AELBS on blood
glucose levels on both normal (panel a) and STZ-diabetic
rats (panel b) after a single oral administration (6 hours of
treatment) of the extract at a dose of 5 mg/kg body weight.
In normal group, blood glucose levels were significantly
decreased 4 hours after gavage (p<0.05); whereas, a
maximal effect (p<0.0001) was observed at the end of the
experiment (after 6 hours of treatment) in comparison with
baseline values (t0). While for control group, any effect
was observed during this test. In glibenclamide-treated
group a significant reduction was shown from the 2nd hour
(p<0.05) until the 6th hour (p<0.0001) of treatment. In STZ-
diabetic rats, a remarkable reduction on blood glucose
levels (p<0.001) was revealed after 6 hours of oral
administration of AELBS; whereas, glibenclamide
administration showed a more pronounced antidiabetic
activity in diabetic rats after 2, 4 and 6 hours of oral
administration (p<0.0001) wile any significant change not
was noted concerning control STZ group.
Figure 2: Plasma glucose levels over 6 h after single oral
administration of the aqueous extract of the leaves of B.
sempervirens extract (5 mg/kg) in normal (Panel a) and
diabetic rats (Panel b). Data are expressed as means ±
S.E.M., n = 6 rats per group. *p < 0.05; **p < 0.01; ***p <
0.001 and ****p<0.0001 when compared to baseline
values.
1.1.1. Repeated oral administration
The effect of repeated oral administration of AELBS on
blood glucose levels in normal (panel a) and STZ-diabetic
(panel b) rats is presented in Figure 3. Control rats did not
Figure 1: Effect of the aqueous extract of aerial part of Buxus
sempervirens L. on body weight in normal (panel a) and
STZ-diabetic (panel b) rats. (All data are expressed as mean ± SEM
(n=6). (*) p<0.05; (**) p<0.01; (***) p<0.001 vs. control.
1.1. Preliminary phytochemical screening and
quantification of total phenols and flavonoid
contents
The results of preliminary phytochemical screening of
AELBS are summarized in Table 1. The results revealed the
presence in this extract of alkaloids, phenolic compounds
(flavonoids, tannins, and cyanidins), carbohydrates, reducing sugars
and mucilage. While, other phytochemical compounds such as
quinones, anthraquinones, saponins, sterols and sisqueterpens are
not present in the same extract.
Total phenols content was estimated to be equal to
507.785±0.22 mg of equivalent of gallic acid content per one gram
of AELBS (507.785 mg±0.22 EGA/g of AELBS), concentration
was calculated using the linear equation showed on the calibration
curve obtained using gallic acid as standard phenol (y=0.0005x +
0.0014), taking into consideration dilutions realized during this test.
While, content of total flavonoids was determined using the
equation illustrated in the calibration curve obtained using quercetin
as standard flavonoids (y = 0.0225x – 0.0171) taking into
consideration dilutions realized during this test; therefore, content
of total flavonoids was estimated to be equal to 45.68±0.014 mg of
Buxus sempervirens L improves streptozotocin-induced diabetes mellitus in
rats
M Ajebli and M Eddouks
exhibit any significant alterations in blood glucose levels. In
addition, AELBS administration caused a significant reduction of
blood glucose levels since the 4th day of treatment (p<0.05), this
significant effect was maintained at the 7th and 15th days. In
glibenclamide-treated group, a significant reduction of blood
glucose levels was noticed at the 4th day (p<0.05) of treatment and
this effect was more pronounced at the 7th day (p<0.01) and 15th day
of treatment (p<0.0001).
For STZ-diabetic rats, the glucose concentration of the
diabetic control rats remained high at all intervals, administration of
AELBS at dose of 5 mg/kg caused an important decease on blood
glucose levels at the end of treatment (p<0.0001). glibenclamide
caused also an important antihyperglycemic effect which is
comparable to AELBS treatment (Figure 3).
Figure 3: Plasma glucose levels over once daily repeated oral
administration of the aqueous extract of leaves of B. sempervirens
(5 mg/kg) for 15 days in normal (Panel a) and diabetic rats (Panel
b). Data are expressed as means ± S.E.M., n= 6 rats per group. *p <
0.05; **p < 0.01; ***p < 0.001 and ****p < 0.0001 when compared
to baseline values.
1.1.1. Effect of AELBS on OGTT in normal and STZ-
induced diabetic rats during 120 min
The oral glucose tolerance experiment indicates the body’s
ability to utilize glucose. The effect of AELBS at a dose of 5 mg/kg
body weight on glucose tolerance was evaluated by measuring the
blood glucose levels at different time points of 30, 60, 90 and 120
min after glucose challenge for both normal and STZ-diabetic rats.
No changes were noticed in normal rats after oral administration of
AELBS, while, in glibenclamide-tretaed group a significant
reduction was observed in glucose levels (p<0.001). The percentage
of this reduction was 42, 49 and 55 %, (after 60, 90 and 120 min
respectively) compared to control (Figure 4). The previous findings
were supported by calculating Areas Under Curve (AUC)
which demonstrated a significant reduction on blood
glucose levels (p<0.05) for normal rats treated by
glibenclamide compared with vehicle control. While no
significant change in AUC was observed for group treated
by AELBS as compared to normal control group.
The results showed also that AELBS reduced
significantly the glucose blood levels 120 min after glucose
load in STZ-diabetic rats (p<0.05), AUC corresponding to
this group indicated also a significant reduction as
compared to normal control rats (p<0.01). Significant
reduction was revealed 60, 90 and 120 min after glucose
load in the glibenclamide-treated group (p<0.01, p<0.01
and p<0.001 respectively), this reduction was also
confirmed by the AUC values (p<0.001).
Figure 4: Effect of the aqueous Mentha suaveolens extract
(20 mg/kg) on oral glucose tolerance in STZ-diabetic rats
and Area Under Curves (panel a), and normal rats and Area
Under Curves correspondents (panel b). Values are means ±
SEM; n=6. (*) P<0.05; (**) P<0.01; (***) P<0.001 vs.
control.
1.1.1. Histopathological examinations
Histopathology of the liver (Figure 5) in control
rats (Fig. 5A) showed normal hepatic cells with hepatocytes
of normal and abundant clear cytoplasm and well-preserved
nucleus and central vein. Furthermore, fat vacuoles may be
seen in histological tissues of normal rats, but usually only
in small numbers. In diabetic control, liver section showed
marked changes in liver morphology accompanied by
severe fatty change and the production of both
microvesicular and macrovesicular vacuolation was
noticed. Moreover, sinusoidal dilation and marked
periportal inflammation fibrosis were visible in this section
(Fig. 5B); number of hepatocytes and diameter of their core
were significantly decreased when compared with section
of normal control (Table 2). In normal glibenclamide-
treated rats morphological and quantitative features were
normal. However, in diabetic rats treated with
glibenclamide, liver section maintained normal architecture
and no fatty changes were observed, mild sinusoidal
Buxus sempervirens L improves streptozotocin-induced diabetes mellitus in
rats
M Ajebli and M Eddouks
Number of IL
Area of IL (µm2)
Normal control
4
2698.15± 346
Glibenclamide normal
4
5224.25± 921
AELBS normal
4
4007.16 ± 502
STZ control
2
1725.36 ± 700
Glibenclamide STZ
3
3568.45 ± 1981
AELBS STZ
2
1523.30 ± 425
congestion and severe feathery degeneration noticed in STZ-
diabetic control have been degenerated (Fig. 5D), in addition,
number of hepatocytes and size of their cores were increased
when compared to the section of diabetic control. In normal rats
treated with AELBS (5 mg/kg), liver section showed normal
hepatic cells with well-preserved cytoplasm, nucleus, and central
vein. This section (slide E) showed many similar features with
section of normal control group. In STZ-diabetic treated with
AELBS, histopathological sections showed normal hepatic
parenchyma and hepatocyte structure in response to the chronic
treatment with AELBS, injuries and inflammations were
remarkably cicatrized and sinusoids have recovered their normal
form and size and the number and diameter of cores of
hepatocytes became close to those of normal control. Indeed, the
damage to the liver cells was reversed in all the AELBS treated
groups. The results show then that repeated oral administration of
AELBS at a dose of 5 mg/kg leads to the improvement of the liver
in STZ-diabetic rats.
resulting from STZ.Figure 6 illustrates histopathological
tissues of pancreas. Histopathological tissues of pancreas
for normal control rats showed normal pancreatic
parenchyma cells and islet cells (Fig. 6G).
In the untreated diabetic rats, streptozotocin caused severe
necrotic changes of the pancreatic islets, particularly in the
β-cells and some nuclear changes were observed in some
places as well as residues of the destroyed cells (Fig. 6K).
In addition, a relative reduction was observed in the size and
number of the islets especially those around the central
vessel together with severe reduction in the beta cells (Table
3). While the pancreas of the positive control diabetic rats
showed increased size of pancreatic islets (Table 3) and
regeneration of the β-cells (Fig. 6L). Indeed, islets of
Langerhans were partially restored and they have recovered
their natural architecture. In AELBS-treated normal rats, the
existence of a morphological similarity with normal control
was observed and the number and the size of islets were also
relatively comparable to normal control group (Table 3).
Table 3: quantitative data of histopathological section of pancreas illustrated in Figure 6, obtained from analysis
with ImageJ software.
Figure 5: Histopathological examinations of the liver of different
experimental groups at the end of the experiment. A: normal
control, B: diabetic control, C: normal treated with standard drug
(Glibenclamide), D: diabetic treated with standard drug
(Glibenclamide), E: Normal treated AELBS and F: diabetic treated
with AELBS. Green arrow indicates hepatocytes, withe arrow
indicates sinusoids, the Yellow one indicates central vein and the
bleu one indicates injuries provoked by intoxication
3. Discussion
Pharmacological investigation and screening of
antidiabetic agents have been extensively investigated in the
past decades and research of phytochemical compounds as
antidiabetic agents becomes more interesting [17]. In fact,
several bioactive compounds isolated from medicinal plants
were reported to possess hypoglycemic or
antihyperglycemic effects. Likewise, more than 1000 plants
have been recounted as efficacious in the treatment of
diabetes mellitus [18-22]. Buxus sempervirens is commonly
used in traditional medicine in Tafilalet region to treat
diabetes mellitus [23]. The present study was carried out to
confirm experimentally the ethnomedicinal use of the
aqueous extract of leaves of Buxus sempervirens as
antidiabetic agent in normal and STZ-diabetic rats. To
analyze the antihyperglycemic influence of AELBS, acute
and chronic study was designed in STZ rats. Streptozotocin
is commonly used as a drug to induce type 1 diabetes in
animal models, providing a useful tool for studying the
pathophysiology and potential treatment of diabetes and its
complications [24].
Oral administration of AELBS (5 mg/ kg) during
15 days was associated with any significant change on body
weight either for normal or STZ-diabetic groups. Significant
hypoglycemic and antihyperglycemic effects were observed
at a dose of 5 mg/kg of AELBS as compared to positive (rats
treated by glibenclamide) and negative (untreated) control
groups. In fact, this effect was marked in both normal and
diabetic groups for both a single and repeated oral
administrations.
Buxus sempervirens L improves streptozotocin-induced diabetes mellitus in
rats
M Ajebli and M Eddouks
groups. In fact, this effect was marked in both normal and
diabetic groups for both a single and repeated oral
administration. The significant reduction in the blood glucose
levels of normal and STZ-diabetic rats following administration
of AELBS indicated that Buxus sempervirens L. leaves possess
a significant glucose lowering activity. Furthermore, significant
action of this extract from the 4th (p<0.05) and 6th hour
(p<0.0001) after its administration demonstrates its high
efficiency as hypoglycemic agent. This may be attributed to the
phytochemical constituents of the extract such as alkaloids,
polyphenols, flavonoids, cyanidins and sterols. It was reported
that the phytochemical content of the plant has been dominated
by alkaloids [25-27] as well as some triterpenoids and sterols
[8], and anthocyanins [28]. Those secondary metabolites could
be implicated in the potent antidiabetic action of this plant. On
the other hand, the results showed that AELBS contains an
important amount of phenolic and flavonoids compounds.
Many experimental studies have demonstrated the involvement
of these phytochemical compounds in the improvement
oxidative stress and progression of diabetes mellitus due to
pancreatic β-cell dysfunction after damage caused by reactive
oxygen species ROS [29-31]. In this context, a previous study
has reported that some fractions of aerial part of Buxus
sempervirens L. exhibited an interesting antioxidant activity
[32]. Hitopathological examinations revealed that AELBS leads
to the enhancement of hepatic tissue. Distorted central vein and
hepatocytes were reinstated to normal in liver of STZ-diabetic
rats treated with AELBS when compared to normal and diabetic
untreated control, while, AELBS did not produce any
improvement in pancreatic tissue. Several reports implicate
diabetes-related oxidative stress in the liver in the pathogenesis
of diabetes. The role of liver in glucose homeostasis is well
known and the beneficial action of AELBS on liver should be
implicated in the observed antidiabetic action of this aqueous
extract. OGTT studies showed that AELBS significantly
declined the level of the blood glucose when compared to the
glibenclamide (p<0.001) and untreated control, this finding was
supported also at the level of the area under the curve AUC as
compared with untreated control for diabetic group, whereas no
change was produced in normal rats. Recent pharmacological
investigations have revealed that modifications of systemic
glycemia in OGTT reflect the activity of the intestinal glucose
transporter SGLT1 [34, 35]. In this study the results showed
clearly that treatment with AELBS significantly improves
glycemic status. This effect is explained partially by the
effective protection of liver and improvement of OGTT.
However other mechanisms of action should be implicated in
this pharmacological effect.
1. Conclusion
In conclusion oral administration of AELBS when
administered at a dose of 5 mg/kg improved considerably the
glycemia in normal and STZ-diabetic rats. Furthermore, the
extract possesses and hepatoprotective effect and it enhances
oral glucose tolerance. The precise mechanisms of action and
the active compounds should be investigated in further
experiments in addition to toxicological studies in order to
support the safe use of Buxus sempervirens L which is rarely
investigated.
GRANTS
This work was supported by the CNRST under grant N°
PPR/2015/35.
CONFLICTS OF INTEREST
The authors declare no conflict of interest.
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