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Original Article
a r t i c l e i n f o a b s t r a c t
Article history: Stevia rebaudiana (Bertoni) Bertoni, Asteraceae, is a plant with hypoglycemic and antihyperlipidemic
Received 25 July 2014 properties. S. rebaudiana (SrB) has become a lead candidate for the treatment of the diabetes melli-
Accepted 4 March 2015 tus. However, chronic administrations of S. rebaudiana are required to cause the normoglycemic effect.
Available online 30 March 2015
Importantly, nanomaterials in general and titanium dioxide (TiO2 ) in particular have become effective
tools for drug delivery. In this work, we obtained TiO2 nanomaterials with SrB at different concentra-
Keywords: tions (10, 20 and 30 M) by solgel method. After this nanomaterials were characterized by Fourier
Diabetes
transform infrared spectroscopy and transmission electron microscopy. Where it was demonstrated, the
Stevia rebaudiana
Nanomaterials
presence of the S. rebaudiana in TiO2 nanomaterials, which were observed as hemispherical agglomerated
Hypoglycemic particles of different sizes. The nanomaterials were evaluated in male rats whose diabetes mellitus-
Antihyperlipidemic phenotype was induced by alloxan (200 mg/kg, i.p.). The co-administration of TiO2 -SrB (20 and 30 M)
induced a signicant and permanent decrease in the glucose concentration since 4 h, until 30 days
post-administration. Likewise, the concentrations of insulin, glycosylated hemoglobin, cholesterol, and
triacylglycerides showed a signicant recovery to basal levels. The major nding of the study was that
the TiO2 -SrB (20 and 30 M) has a potent and prolonged activity antidiabetic. TiO2 can be considered like
an appropriated vehicle in the continuous freeing of active substances to treat of diabetes mellitus.
2015 Sociedade Brasileira de Farmacognosia. Published by Elsevier Editora Ltda. All rights reserved.
http://dx.doi.org/10.1016/j.bjp.2015.03.004
0102-695X/ 2015 Sociedade Brasileira de Farmacognosia. Published by Elsevier Editora Ltda. All rights reserved.
146 A. Langle et al. / Revista Brasileira de Farmacognosia 25 (2015) 145151
for a period approximately of 500 h (h) (Lpez et al., 2010a,b). Here, Committee uses of laboratory animals of the BUAP (VIEP-3450-
we performed the synthesis of TiO2 nanomaterials from solutions 2013).
of titanium n-butoxide and the aqueous extract of SrB concentrate
obtained from the dried leaves of the plant. This with the purpose of Induction of diabetes in rats
using it as a controlled release system SrB extract. For the above, this
study aimed to evaluate the antidiabetic activity of intraperitoneal The animals were injected with alloxan dissolved in 0.1 M
injection of TiO2 -SrB nanomaterials in a diabetic rat model. citrate buffer, pH 4.5 at a dose 200 mg/kg body weight (i.p.). The
alloxan dose was selected based on various reports (Raskovic et al.,
Materials and methods 2004; Szkudelski, 2001). Three day after of the administration of
alloxan, blood samples were taken and blood glucose levels were
Preparation of SrB-extract determined. Animals that had higher concentrations of glucose
(150 mg/dl) were considered diabetic.
Stevia rebaudiana (Bertoni) Bertoni, Asteraceae, was grown on
campus grounds of the Benemrita Universidad Autnoma de SrB-TiO2 administration protocols
Puebla (BUAP), Mxico. Dr. Albino Moreno-Rodrguez conrmed
the identication, additionally; a sample was deposited in the In the experiment a total of forty rats were used, which were
botanical garden of the BUAP (le 21-09/14). Leaves SrB (10 g) divided in ve groups (n = 8 per group).
washed and dried in an Elisa-550 oven for 24 h. Then it pulverized
and macerated with a solution of ethanol and water (with a ratio of Group 1: normal rats treated with TiO2 nanomaterials (vehicle
80:20). The solution obtained from the mashing process is ltered group)
and the SrB aqueous extract is placed on a rotary evaporator of the Group 2: diabetic group treated with TiO2 nanomaterials
ESEVE-402-2 brand to eliminate to 80% of solvent (water and alco- Group 3: diabetic rats with TiO2 -SrB nanomaterials to 10 M
hol) under reduced pressure. Thus we obtain the SrB concentrated Group 4: diabetic rats with TiO2 -SrB nanomaterials to 20 M
extract to 80 wt. Group 5: diabetic rats with TiO2 -SrB nanomaterials to 30 M
Preparation of TiO2 -SrB nanomaterials by solgel method The nanomaterials were injected by i.p. The route in all animals
depending of treatment.
The homogeneous solutions were prepared separately with Subsequently, both the empty TiO2 nanomaterials (1 g/kg in
different concentrations of the SrB concentrated extract (10, 20 sterile water at 37 C) and SrB (10, 20 and 30 M) (1 g/kg in sterile
and 30 M). Each homogeneous solution containing 150 ml of water at 37 C) were administered, respectively.
anhydrous 1-butanol (99.8%, SigmaAldrich), 10 ml of deionized
water and 0.5 g of polyvinylpyrrolidone (with an average molecular Method for determination of plasma blood glucose level
weight of 40,000, SigmaAldrich) and the required concentration
of the SrB concentrated extract (10, 20 and 30 M). To evaluate the plasmatic concentration of glucose with respect
For each homogeneous solution was added 21.5 ml of titanium at course of time in the animals injected with SrB nanomaterials, the
n-butoxide (97%, SigmaAldrich) in a reux system at 70 C with animals were deprived of the food for 5 h before the determination.
constant agitation. The nal solution with properties of gel was The blood glucose levels were determined at 0, 4, 8 and 24 as well
immersed in a container with ice for 15 min at 3 C. The solvent was as at 5, 10, 15 and 30 days after administration of each treatments.
removed on a rotary evaporator at 50 C under vacuum conditions In the experiment the blood glucose level of the animals were esti-
to nally obtain the TiO2 -SrB nanomaterials (Lpez et al., 2010a,b). mated by Glucose OxidasePeroxidase Enzymatic Method using a
digital glucometer (ACCU-CHEK brand active). Blood from the tail
Characterized of TiO2 -SrB nanomaterials vein was collected.
OH
OH
Stevioside
OH
Steviol
OH
OH CH 3
HO
HO
HO
O
HO
HO
O
HO
C
O
H3C
H3
CH 3
HO
CH
O O
3
O
O
HO
OH OH
CH 3
Ti+ O Ti+ O Ti+ O Ti+ O Ti+ O Ti+ O Ti+
Stevioside
H O H
O
CH2 H O
HO H CH2
HO CH2 H OH
+ + TiO2 + +
In treated rats with alloxan the blood glucose levels at 4, 8 TiO2 -SrB at a concentration of 2030 M (one-way ANOVA, Bon-
or 24 h, increased (160180 mg/dl), respect to the vehicle group ferroni post-test, p < 0.05).
(80.43 mg/dl) (Fig. 4B). Treatment with TiO2 -SrB nanomaterials in Laboratory studies, developed 31 days after administration of
alloxan-treated animals showed a progressive decrease in glucose treatments, show a signicant increase in the concentration of glu-
levels at 4, 8 and 24 h after administration, which, when it was cose (86%) of the hyperglycemic group + TiO2 , compared to the
compared with the alloxan + TiO2 treated group, was observed a normal rats treated with TiO2 (Fig. 5A and B). Consequently, the
signicant difference in the animals treated with TiO2 -SrB 20 and concentration of glycosylated hemoglobin in the alloxan + TiO2
30 M (Fig. 4B). (One way ANOVA, Bonferroni post-test, p < 0.05). group, was higher (38%) compared to the normal rats + TiO2 (Fig. 5C)
Moreover, comparing the glucose concentration at 5, 10, 15 and (one-way ANOVA, post-Bonferroni test, p < 0.05).
30 days in the hyperglycemic rats after administration of TiO2 - Moreover, the animals of groups of alloxan + TiO2 -SrB 20 and
SrB, was observed, a signicant decrease in the treated animals 30 M, had a lower concentration of glucose (20% and 56%) and
with TiO2 -SrB nanomaterials at 20 and 30 M with respect to the glycated hemoglobin (25% and 41%) compared to the alloxan + TiO2
alloxan + TiO2 group (Fig. 4 C). In this regard, the decrease of glu- group (Fig. 5A and C). Also, groups with alloxan + TiO2 -SrB 20
cose levels was observed during 30 days after administration of and 30 M showed a higher insulin concentration compared to
Fig. 3. (A) Micrograph of TiO2 . (B) SrB/TiO2 -10. (C) SrB/TiO2 -20, and (D) SrB/TiO2 -30 nanomaterials.
A. Langle et al. / Revista Brasileira de Farmacognosia 25 (2015) 145151 149
[glucose] mg/cl
###
250 ### ##
Weight (g)
100 ###
225
50
200
175 *** 0
Vehicle Alloxan Alloxan Alloxan Alloxan
150 + TiO2 + TiO2 + TiO2 + TiO2
SrB (10M) SrB (20M) SrB (30M)
1 2 3 4
Week B 40 ##
##
B 200
30
[Insuline] mg/cl
***
150
20 ***
[Glucose] mg/gl
100 ## 10
###
0
50
Alloxan + TiO2-SrB (10 M) Vehicle Alloxan Alloxan Alloxan Alloxan
Vehicle + Vehicle
+ TiO2 + TiO2 + TiO2 + TiO2
Alloxan + TiO2 Alloxan + TiO2-SrB (20 M)
SrB (10M) SrB (20M) SrB (30M)
Alloxan + TiO2-SrB (30 M)
0 C % Glycated Hb (HbA1c) 10
0 4 8 24 8
Hours ***
C 200
6
#
##
***
4
150
[Glucose] mg/gl
2
###
100 ###
0
Vehicle Alloxan Alloxan Alloxan Alloxan
+ TiO2 + TiO2 + TiO2 + TiO2
50 SrB (10M) SrB (20M) SrB (30M)
Vehicle + Vehicle Alloxan + TiO2-SrB (10 M)
Alloxan + TiO2 Alloxan + TiO2-SrB (20 M) Fig. 5. Effect of administration of TiO2 -SrB nanomaterials on glucose, insulin and
Alloxan + TiO2-SrB (30 M) glycosylated hemoglobin in administered rats with alloxan. Quantication was per-
0
formed in plasma at 31 days after administration of TiO2 -SRB (10, 20 and 30 M) or
0 4 10 15 20 25 30 TiO2 in treated rats with alloxan (200 mg/kg) or vehicle. The graph (A) glucose con-
Days centration (mg/ml) in plasma and shown in graphs (B) and (C) insulin concentration
(mU/ml), and the percentage of glycosylated hemoglobin, respectively are shown.
Fig. 4. Effect of administration of TiO2 -SrB nanomaterials on the weight and the The data show the mean SE of each group respectively. (One-way ANOVA, Bon-
time course of the glycemic levels in treated rats with alloxan. The animals were ferroni post-test, ***p < 0.001: Vehicle vs alloxan + TiO2 and ## p < 0.01, ### p < 0.001:
weighed every week for 4 weeks (A). The blood glucose levels were determined at alloxan + TiO2 -SrB vs alloxan + TiO2 ).
0, 4, 8 and 24 h (B), as well as 5, 10, 15 and 30 days (C) after administration of the
nano-TiO2 SrB (10, 20 and 30 M) in diabetic rats (alloxan, 200 mg/kg). The vehicle
group were administered with the vehicle, and with empty nanomaterials (Vehicle normal rats treated with TiO2 (227% and 185%, respectively) (Fig. 6).
and alloxan + TiO2 ) under the same experimental conditions (n = 8 per group). The
Moreover the treatment with TiO2 -SrB nanomaterials to 20 or
data show the average weight (g) and glucose concentration (mg/dl) SE of each
group respectively. (One-way ANOVA, Bonferroni post-test, ***p < 0.001: Vehicle vs 30 M in diabetic rats decreased the cholesterol (62 and 64%,
alloxan + TiO2 ; # # p < 0.01 and ### p < 0.001: alloxan + TiO2 -SrB vs alloxan + TiO2 ). respectively) and triacylglycerides levels (59 and 63%, respectively)
respect to diabetic rats with TiO2 (Fig. 6A and B). (One way ANOVA,
the group with alloxan + TiO2 (80% and 100%) (Fig. 5B). (One Bonferroni post-test, p < 0.05). Treated animals with TiO2 -SrB nano-
way ANOVA, Bonferroni post-test, p <0.05). This showed for the materials to 10 M did not modify the biochemical parameters
rst time, that nanomaterials TiO2 -SrB (20 and 30 M) reversed evaluated.
hyperglycemic effect induced by alloxan, due to the biochemi-
cal parameters measured (glucose, glycosylated hemoglobin and
insulin) are statistically similar to the vehicle group + TiO2 . Discussion
TiO2 -SrB nanomaterials treatment effect on hyperlipidemic effects In the present study, we synthesize the TiO2 -SrB nanomateri-
induced by alloxan als by the solgel method, which were characterized by FTIR and
SEM, showing the interaction of Stevia rebaudiana (Bertoni) Bertoni,
The results show that the diabetic group treated with TiO2 Asteraceae, nanomaterials with TiO2 , as agglomerates of differ-
increased the cholesterol and triacylglycerides levels respect to ent sizes. Subsequently, we investigate the antidiabetic activity of
150 A. Langle et al. / Revista Brasileira de Farmacognosia 25 (2015) 145151
A 300
-pancreatic cells and induce the insulin secretion constantly. This
insulin promotes the decrease of glucose and glycated hemoglobin
*** levels in the plasma of the diabetic rats (Park and Cha, 2010).
[Triglycerides] mg/cl
200
It is noteworthy that interaction, SrB, with nanomaterials TiO2
decreases progressively, once they are in the aqueous medium,
which provides the blood stream, therefore, the SrB is released and
##
100
distributed peripherally to reaching the sites of action and exercise
##
the respective pharmacological effects. According as suggested by
some work with nanomaterials of TiO2 (Lpez et al., 2010a,b, 2015).
0 However, it is necessary to demonstrate this process in the future.
Vehicle Alloxan Alloxan Alloxan Alloxan In DM, high lipid levels are present and play an important
+ TiO2 + TiO2 + TiO2 + TiO2
SrB (10M) SrB (20M) SrB (30M) toxic role. The present study show high lipid levels in diabetic rats
induced with alloxan but the treatment with nanomaterials of TiO2 -
SrB normalize the prole lipids demonstrating the hypolipidemic
B 300 effect of SrB. However, the molecular mechanism to regulating
*** glucose or lipids of the SrB are unknown. At the moment, stud-
250
ies indicate that SrB has antioxidant and antiapoptotic effects
[Cholesterol] mg/cl
200 (Shivanna et al., 2013), which probably helps to enhance the SrB
therapeutic effect. According to Misra et al. (2011) suggest that Ste-
150
via extract produced good antidiabetic effects together with lesser
### ### loss in body weight and propose that can be used in the prepa-
100
ration of cough syrups and cold beverages for diabetes patients
50
(Himanshu et al., 2011).
0 For above expose, this study describes for the rst times that is
Vehicle Alloxan Alloxan Alloxan Alloxan necessary the delivery system like TiO2 nanomaterials to release
+ TiO2 + TiO2 + TiO2 + TiO2
SrB (10M) SrB (20M) SrB (30M) SrB and generate regulation on hyperglycemia and hyperlipi-
demia in diabetic rats induced with alloxan. Our results open a
Fig. 6. Effect of administration of TiO2 -SrB nanomaterials on concentration of new opportunity for the treatment of the DM; however more
cholesterol and triacylglycerides in administered rats with alloxan. Quantication
studies are needed to better understand the potential use of TiO2 -
was performed in plasma at 31 days after administration of TiO2 -SrB (10, 20 and
30 M) or TiO2 in treated rats with alloxan (200 mg/kg) or vehicle. The graph nanomaterials and SrB in metabolic diseases.
(A) is shown cholesterol concentration (mg/dl) and in the graph (B) triacylglyc-
erides (mg/dl) are shown respectively. Data show the mean concentration SE of
Author contributions
each group respectively. (One-way ANOVA, Bonferroni post-test, ***p < 0. Vehicle vs
alloxan + TiO2 and ## p < 0.01, ### p < 0.001: alloxan + TiO2 -SrB vs alloxan + TiO2 ).
AL, MGC, ST, AD, BV, GI, GC and AMR design the study and wrote
the protocol. AL, AD and ST performed the experiments. AL, AD,
TiO2 -SrB nanomaterials in diabetic rat model induced with alloxan, AMR, ST, BV and GC managed the literature searches and analy-
which is a toxic hepato-pancreatic agent, to generate a similar sis; MGC and AD undertook the statistical analysis. All contributing
metabolic changes to the DM. The alloxan is employed as an exper- authors have approved the nal manuscript.
imental diabetogenic to evaluate the effectiveness of new drugs
with hypoglycemic properties (Szkudelski, 2001).
Glycemia and lipemia evaluations, determines the degree of Conicts of interest
metabolic damage that exists in experimental animals. This study
shows that the treatment with TiO2 alone have no effect on glucose The authors declare no conicts of interest.
and lipids values, while TiO2 -SrB at 20 or 30 M caused a hypo-
glycemic and antihyperlipidemic effect in diabetic rats induced Acknowledgments
with alloxan, which is a toxic glucose analog, which selectively
destroys insulin-producing beta cells in the pancreas when is The authors thank Dr. Carlos Escamilla for his help with the ani-
administered to rodents (Jeppesen et al., 2000), and causes that mal care and thanks to Thomas Edwards, PhD for editing the English
secreted insulin are insufcient to regulate blood glucose, there- language text.
fore in this study the rats exhibited continuous hyperglycemia. This
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