Antidiabetic effect, antioxidant activity, and toxicity of 3′,4′-Di-O-acetyl-cis-khellactone in Streptozotocin-induced diabetic rats

Article abstract of DOI:10.1016/j.bmcl.2016.06.071

Pyranocoumarins are compounds with an important pharmacological profile, such as anti-inflammatory, antioxidant, cytotoxic, antiviral, antibacterial, and hypoglycemic effects. These molecules have a widespread presence as secondary metabolites in medicinal plants used to treat Diabetes Mellitus (DM). The aim of this work was to evaluate antidiabetic activity in Streptozotocin (STZ)-induced diabetic rats and the antioxidant effects of 3′,4′-Di-O-acetyl-cis-khellactone (DOAcK), as well as its toxic potential. We obtained DOAcK with an enantiomeric excess of 70% by chemical synthesis. Our results showed that this compound exerts an important antidiabetic effect: blood glucose decreased in groups treated with DOAcK by 60.9% at dose of 15?mg/kg (p?<0.05) compared with the diabetic control group, and demonstrated a statistically significant increase in weight gain (45.7?±?9.7 in the group treated with DOAcK vs. ?23.0?±?33.1 in the group with diabetes). In a biochemical profile, DOAcK did not modify lipid metabolism and did not cause damage at the renal level. DOAcK administration increased the activities of Catalase (CAT), Glutathione Peroxidase (GPx), and Super Oxide Dismutase (SOD) to levels near those of the healthy group. Histopathological analysis exhibited morphology similar to that of the healthy group and the group treated with DOAcK. DOAcK is not mutagenic by Ames test for Salmonella typhimurium strains TA98, TA100, or TA102, and is not genotoxic by Micronucleus assay; median lethal dose (LD₅₀) >2000?mg/kg and, at this dose, no signs of toxicity or death were reported after 14?days of observation. These results indicate that DOAcK can improve glucose metabolism, which may be due to the increased antioxidant activity of CAT, GPx and SOD. In addition, DOAcK is not toxic in the studies tested.

Full text of DOI:10.1016/j.bmcl.2016.06.071

Accepted Manuscript
Antidiabetic effect, antioxidant activity, and toxicity of 3’,4’-Di-O-acetyl-cis-
khellactone in Streptozotocin-induced diabetic rats
Elix Alberto Domínguez-Mendoza, Jorge Cornejo-Garrido, Eleuterio
Burgueño-Tapia, Cynthia Ordaz-Pichardo
PII:
S0960-894X(16)30682-5
DOI:
http://dx.doi.org/10.1016/j.bmcl.2016.06.071
BMCL 24024
Reference:
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date:
Accepted Date:
7 June 2016
24 June 2016
Please cite this article as: Domínguez-Mendoza, E.A., Cornejo-Garrido, J., Burgueño-Tapia, E., Ordaz-Pichardo,
C., Antidiabetic effect, antioxidant activity, and toxicity of 3’,4’-Di-O-acetyl-cis-khellactone in Streptozotocin-
induced diabetic rats, Bioorganic & Medicinal Chemistry Letters (2016), doi: http://dx.doi.org/10.1016/j.bmcl.
2
016.06.071
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Antidiabetic effect, antioxidant activity, and toxicity of 3’,4’-Di-O-acetyl-cis-
khellactone in Streptozotocin-induced diabetic rats
1
1
Elix Alberto Domínguez-Mendoza , Jorge Cornejo-Garrido , Eleuterio Burgueño-
2
1*
Tapia and Cynthia Ordaz-Pichardo
1
Laboratorio de Biología Celular y Productos Naturales, Escuela Nacional de
Medicina y Homeopatía (ENMH), Instituto Politécnico Nacional, Guillermo Massieu
Helguera 239, Col. La Escalera, 07320 CDMX, México
2
Departamento de Química Orgánica, Escuela Nacional de Ciencias Biológicas
(
ENCB), Instituto Politécnico Nacional, Prolongación de Carpio y Plan de Ayala
s/n, Col. Santo Tomás, 11340 CDMX, México
*Corresponding author: Cynthia Ordaz-Pichardo. Phone: (+52) (55) 5729 6000,
ext. 55535, Fax: (+52) (55) 5729 6000, ext. 55561; E-mail address:
dra_cynthia@hotmail.com
Laboratorio de Biología Celular y Productos Naturales, Edificio de Posgrado,
Primer Piso, Escuela Nacional de Medicina y Homeopatía, Instituto Politécnico
Nacional, Guillermo Massieu Helguera 239, Col. La Escalera Del. Gustavo A.
Madero, 07320 CDMX, México.
Keywords: Antidiabetic activity; antioxidant activity; 3´,4´-Di-O-acetyl-cis-
khellactone; diabetes.
1

Abstract
Pyranocoumarins are compounds with an important pharmacological profile,
such as anti-inflammatory, antioxidant, cytotoxic, antiviral, antibacterial, and
hypoglycemic effects. These molecules have a widespread presence as secondary
metabolites in medicinal plants used to treat Diabetes Mellitus (DM). The aim of
this work was to evaluate antidiabetic activity in streptozotocin (STZ)-induced
diabetic rats and the antioxidant effects of 3’,4’-Di-O-acetyl-cis-khellactone
(
DOAcK), as well as its toxic potential. We obtained DOAcK with an enantiomeric
excess of 70% by chemical synthesis. Our results showed that this compound
exerts an important antidiabetic effect: blood glucose decreased in groups treated
with DOAcK by 60.9% at dose of 15 mg/kg (p <0.05) compared with the diabetic
control group, and demonstrated a statistically significant increase in weight gain
(
45.7 ± 9.7 in the group treated with DOAcK vs. –23.0 ± 33.1 in the group with
diabetes). In a biochemical profile, DOAcK did not modify lipid metabolism and did
not cause damage at the renal level. DOAcK administration increased the activities
of Catalase (CAT), Glutathione Peroxidase (GPx), and Super Oxide Dismutase
(
SOD) to levels near those of the healthy group. Histopathological analysis
exhibited morphology similar to that of the healthy group and the group treated with
DOAcK. DOAcK is not mutagenic by Ames test for Salmonella typhimurium strains
TA98, TA100, or TA102, and is not genotoxic by Micronucleus assay; median
lethal dose (LD50) >2,000 mg/kg and, at this dose, no signs of toxicity or death
were reported after 14 days of observation. These results indicate that DOAcK can
improve glucose metabolism, which may be due to the increased antioxidant
activity of CAT, GPx and SOD. In addition, DOAcK is not toxic in the studies
tested.
2

Diabetes Mellitus (DM) is a chronic disease of impaired glucose metabolism
characterized by hyperglycemia, which is caused by a deficiency in insulin
1
secretion, insulin resistance, or both. The World Health Organization (WHO)
estimates that in 2012, hyperglycemia was the direct cause of 1.5 million deaths.
th
Last year, 9% of adults worldwide had DM, and DM will be the 7 leading cause of
2
death in 2030. Treatment of type 2 DM (DM2) continues to present challenges
because many patients have problems achieving adequate glucose levels. Despite
the availability of many oral and injected antidiabetic drugs, therapeutic efficacy is
regularly accompanied by side effects, such as hypoglycemia, weight increase,
and cardiovascular complications, in addition to presenting high costs and, in many
cases, drugs are not accessible to the entire population. Therefore, the search for
3
novel drugs with better risk-benefit profiles continues. According to recent reviews,
in Mexico there are at least 383 plant species employed for the treatment of
Diabetes Mellitus (DM), but only a few of these have been investigated for their
4
preclinical or clinical efficacy. In a previous work, we found that the ethyl acetate
extract of Arracacia tolucensis carries out significant hypoglycemic activity in
5
Streptozotocin (STZ)-induced diabetic rats, being responsible for the biological
activity, two of the major compounds: the chromone (S)-(+)-4’-O-
angeloylvisamminol, and the pyranocoumarin (3´R,4´R)-(-)-4’-O-acetyl-3’-O-
6
angeloylkhellactone, also known as Praeruptorin A.
On the other hand, many works report that pyranocoumarin derivatives possess
different biological activities, including cytotoxic, anti-inflammatory, antioxidant,
7
antiviral, antibacterial, and hypoglycemic activity. In patients with diabetes, there is
a state of Oxidative Stress (OS), which it is due to a persistent production of
Reactive Oxygen Species (ROS), Reactive Nitrogen Species (RNS), or a decrease
8
in antioxidant enzyme activity. Some coumarins, pyranocoumarins, and their
related molecules have been evaluated in animal models, finding that they have
hypoglycemic and antioxidant activity by increasing the activity of Catalase (CAT),
9,10
Glutathione Peroxidase (GPx), and Super Oxide Dismutase (SOD).
Compounds
that inhibit the increase in oxidant species may be drugs of interest for the
1
1
treatment of DM. Thus, we obtained the pyranocoumarin derivative 3’,4’-Di-O-
3

acetyl-cis-khellactone (DOAcK) with an enantiomeric excess of 70% by chemical
synthesis, and evaluated its antioxidant and antidiabetic activity, as well as its
possible toxic effects (Ames test, Micronucleus assay, Acute oral toxicity) in the
search for a new drug that provides better quality of life for patients with diabetes.
Synthesis of DOAcK (Fig. 1) was performed in three reactions steps. In the first
step, umbelliferone was reacted with 1,1-diethoxy-3-methyl-2-butene in a medium
of xylene, obtaining seseline as a yellow solid after purification by column
chromatography (80%). Then, seseline was reacted with AD-mix α in water:t-
butanol to obtain cis-khellactone with a yield of 80% and enantiomeric excess of
7
0% ([α] = +23). We compared this characteristic with those in the literature, and
asymmetric dihydroxylation was stereo-selective primarily obtaining the R,R
12
configuration. In the final step, DOAcK was obtained by the reaction of cis-
khellactone with acetyl chloride and DMAP in dichloromethane (CH Cl ) to obtain a
2
2
white solid by column chromatography, for which the yield was 41%.
Figure 1. Synthesis of 3’,4’-Di-O-acetyl-cis-khellactone (DOAcK).
Administration of DOAcK increased Body Weight (BW) in a significant manner in
comparison with untreated groups Table 1. Diabetic and vehicle groups decreased
and increased slightly in BW (–23.0 ± 33.1 Standard Error of the Mean [SEM]) and
4

(
2.2 ± 18.5), respectively, in contrast with the healthy group (64.3 ± 28.9). The
group treated with Glibenclamide and DOAcK increased in 54.8 ± 20.4 and 45.7 ±
.7, respectively. There was significance between the treated and untreated
9
groups: the group treated with DOAcK exhibited weight close to that of the healthy
group.
A survey of the literature illustrated that diabetic rats decreased their BW;
however, treatment with extracts, fractions, or compounds from plants with
5
antidiabetic activity can reverse this loss. It has been found that Rutin exhibited an
increase in BW up to 12.1%, and Madhumega chooranam can increase BW by
13,14
4
2.7%.
Insulin comprises the most potent anabolic hormone known and Beta cells are
responsible for producing; it is known that the death of these cells could be caused
by hyperglycemia, and compounds with pyranocoumarin structure can improve
9,15,16
it.
The increase in ROS can lead to death of Beta cells; therefore, an
improvement in BW may be due to that DOAcK carries out better control of
17
hyperglycemia by decreasing ROS.
Table 1. Weight after 15-doses of DOAcK (15 mg/kg) in streptozotocin (STZ)-induced diabetic rats
Group
Day 0
g)
Day 7
(g)
Day 21
(g)
Weight gain
(g)
(
Healthy
Diabetic
Diabetic +
Vehicle
334.5 ± 15.7
371.6 ± 10.2
321.5 ± 11.1
345.0 ± 15.8
370.0 ± 13.0
323.1 ± 6.4
398.8 ± 24.3
348.6 ± 31.49
323.8 ± 14.8
64.3 ± 28.9*
#
-23.0 ± 33.1
#
2.2 ± 18.5
Diabetic +
Glibenclamide
Diabetic +
DOAcK
330.7 ± 9.2
319.4 ± 6.9
341.4 ± 13.1
335.3 ± 10.9
385.5 ± 18.2
365.1 ± 6.7
54.8 ± 20.4*
45.7 ± 9.7*
*
Statistically significant difference vs. diabetic group by multiple comparison Dunnett test;
#
Statistically significant difference vs. healthy group by multiple comparison Dunnett test. Vehicle:
Water 2:1 DMSO. Glibenclamide was administered at 2.5 mg/kg; Weight gain = Weight on day 21,
less weight on day 0 (n = 10; mean ± SEM; p <0.05). Treatment was started on day 7 after STZ-
induction; on day 21, 15-doses of DOAcK were completed. (n = 10, mean ± SEM; p <0.05).
Coumarins and their derivatives have attracted attention due to their anti-
18,19
inflammatory, antibacterial and antiarthritic activities,
as well as antidiabetic
agents. In some studies it has been reported that different coumarins and
5

9
pyranocoumarins decreasing blood glucose by up to 42%, and in some cases it
has been demonstrated that this improvement is due to changes in the activity of
10
Glucose-6-phosphatase and Fructose-1,6-bisphosphatase. These and many
other studies report that coumarins and their derivatives comprise a source of
novel molecules of biological interest.
4
In Mexico, are at least 383 plant species employed for the treatment of DM. We
6
have evaluated extracts and compounds isolated from A. tolucensis with anti-
5
inflammatory and antidiabetic activities in STZ-induced diabetic rats. We now
propose the evaluation of the bioisostere of one of the major metabolites of A.
tolucensis, DOAcK, which is an analogue of Praeruptorin A; we expect that, due to
a structural activity relationship, this compound will exhibit the same or better
20
antidiabetic activity. Table 2 shows the values of fasting blood glucose of the
experimental groups. DOAcK treatment for 15 days at 15 mg/kg decreased blood
glucose values. Blood glucose decreased in the groups treated with Glibenclamide
and DOAcK by 56.6 and 60.9% (p <0.05), respectively, when compared with the
diabetic control group. These results indicate that DOAcK can improve glucose
metabolism. Many works in the literature have evaluated the extracts of plants with
antidiabetic activity. Toddalia asiatica (L.) Lam. Ethyl Acetate (EtOAc) extract
decreasing the blood glucose level from 290.38 ± 1.89 to 108.27 ± 4.38 mg/dL
compared with the diabetic group. In addition, this extract increased SOD, CAT,
21
and GPx activity, and the CH
2
Cl fraction of Kalanchoe pinnata can decrease
2
22
blood glucose in STZ-induced diabetic rats from 359 ± 11 to 109 ± 10 mg/dL.
Other studies has been demonstrated that administration of Skimmin, a
coumarin, decreased blood glucose from 494.4 ± 5.0 to 390.0 ± 10.1 mg/dL; in
addition, Skimmin regulated Transforming Growth Factor beta-1 (TGF-β1) protein
23
levels, which improved nephropathy in rats . Clorichromene and XLF-III-43, other
coumarins, have been evaluated against diabetic nephropathy; both compounds
24
improved diabetic complications by preserving the blood-retinal barrier and
25
inhibiting advanced glycation end products. The glucose values obtained in this
study are similar to the studies discussed previously. DOAcK performed an
antidiabetic effect, with a decrease of blood glucose of 199.2 ± 24.7 mg/dL
6

compared with the untreated diabetic group. This result demonstrated that DOAcK
possesses interesting antidiabetic activity.
Table 2. Fasting blood glucose after 15-doses of DOAcK (15 mg/kg) in streptozotocin (STZ)-
induced diabetic rats
Group
Day 0
mg/dL)
Day 7
(mg/dL)
Day 14
(mg/dL)
Day 21
(mg/dL)
Day 21
(increased
glucose)
Decrease
of blood
glucose
(mg/dL)
-
(
(
mg/dL)
Healthy
Control
61.25 ±
3.0
106.80 ±
4.8
98.30 ± 3.1 90.50 ± 8.5
29.25 ± 9.1*
Diabetic
Control
54.00 ±
2.6
371.80 ±
81.4
326.50 ±
72.3
327.50 ±
23.9
273.50 ±
21.8
-
-
Diabetic +
Vehicle
69.50 ±
8.5
272.00 ±
78.2
368.00 ±
65.3
406.30 ±
38.4
336.80 ±
24.8
Diabetic +
Glibenclamide 7.3
63.75 ±
211.70 ±
45.2
282.80 ±
75.4
142.00 ±
5.5
78.25 ±
24.8*
185.5 ±
24.5
Diabetic +
DOAcK
72.90 ±
5.2
363.20 ±
70.3
280.70 ±
76.4
128.30 ±
6.4
55.35 ±
19.2*
199.2 ±
24.7
*Statistically significant difference vs. diabetic group by multiple comparison Dunnett test. Vehicle:
Water 2:1 DMSO. Glibenclamide was administered at 2.5 mg/kg. Increased glucose = Glucose on
day 21, less glucose on day 0. Decrease of blood glucose = Glucose of diabetic group less glucose
of treated group. Treatment was started on day 7 after STZ-induction; on day 21, 15-doses of
DOAcK were completed. (n = 10, mean ± SEM; p <0.05).
Table 3 depicts the biochemical profile in the experimental groups with and without
treatment. No statistically significant differences were observed between all groups
compared with healthy and diabetic controls; these results suggest that
administration of DOAcK does not modify lipid metabolism and does not cause
damage at the renal level. This is consistent with the absence of macroscopic
pathologic lesions in the liver and kidney of animals treated with DOAcK.
7

Table 3. Biochemical profile in fasting blood after 15-doses of DOAcK (15 mg/kg) in streptozotocin
STZ)-induced diabetic rats
(
Group
Creatinine
mg/dL)
Urea
(mg/dL)
Cholesterol
(mg/dL)
HDL
(mg/dL)
LDL
(mg/dL)
(
Healthy
Control
0.53 ± 0.02
0.58 ± 0.03
0.56 ± 0.03
0.62 ± 0.05
0.51 ± 0.06
49.41 ± 3.26 70.54 ± 2.01
60.85 ± 6.89 68.30 ± 1.28
62.98 ± 6.21 80.09 ± 9.35
58.43 ± 3.89 70.64 ± 2.79
42.04 ± 6.99 70.84 ± 1.51
30.42 ± 1.57 21.66 ± 2.32
28.26 ± 1.54 12.67 ± 1.53
36.31 ± 0.51 23.11 ± 9.82
26.60 ± 1.87 21.71 ± 4.68
29.77 ± 1.09 22.38 ± 3.06
Diabetic
Control
Diabetic +
Vehicle
Diabetic +
Glibenclamide
Diabetic +
DOAcK
DOAck = 3’,4’-Di-O-acetyl-cis-khellactone. (N = 10; mean ± SEM; p <0.05). Vehicle: Water 2:1
DMSO. (n = 10, mean ± SEM; p <0.05).
Oxidative Stress (OS) produced by cellular respiration and environmental factors
is the main cause of DM complications (protein oxidation, retinopathies,
26
nephropathies, and neuropathies). Superoxide anion, hydrogen peroxide and
radical hydroxyl are the most dangerous radicals known. The cell possesses CAT,
GPx, and SOD, enzymes that remove ROS, therefore, the increase of these
27
enzymes provides protection against the harmful effects evoked by ROS.
Extracts of Stevia rebaudiana and Eucalyptus globulus administered in animal
28,29
models of diabetic rats increased CAT and GPx activities.
We performed an
evaluation of DOAcK on CAT, GPx and SOD activities. Table 4 illustrates the
effect on the antioxidant level in the liver of STZ-induced diabetic rats.
Administration of DOAcK and Glibenclamide increased CAT activity to levels near
those of the healthy group, but interestingly, DOAcK increased GPx activity more
than Glibenclamide treatment. On the other hand, administration of DOAcK
increased SOD activity of statistically significant manner. CAT, GPx, and SOD are
three of the major antioxidative enzymes. It is known that there are compounds
that are able to increase the expression of these enzymes. The thiazolidinediones
comprise a family of compounds that possess this activity; however, their toxic
30
effects have removed some of these compounds. Our results provide evidence
that DOAcK can increase the activity of CAT, GPx and SOD, thus increasing
8

antioxidant activity in order to attenuate damage generated by ROS. These results
are in agreement with the results obtained in the Ames assay: DOAcK are not
mutagenic on the TA102 strain, which is able to detect chemical compounds with
the ability to generate ROS.
Table 4. Catalase (CAT), Glutathione Peroxidase (GPx), and Super Oxide Dismutase (SOD)
activity after 15-doses of DOAcK (15 mg/kg) in streptozotocin (STZ)-induced diabetic rats
Group
CAT
GPx
SOD
+
2
H O
2
consumed
NADPH consumed
(µM/min/mg protein)
54.6 ± 5.5
Unit of SOD
(U/min/mg protein)
0.83 ± 0.06
(µM/min/mg protein)
I
Healthy
Control
132.2 ± 5.9
47.5 ± 2.6
68.9 ± 11.1
93.2 ± 4.9*
94.8 ± 7.8*
II
Diabetic
Control
20.8 ± 3.3
0.44 ± 0.01
0.22 ± 0.06
0.55 ± 0.02
0.73 ± 0.03*
III
IV
V
Diabetic +
Vehicle
10.0 ± 0.6
Diabetic +
Glibenclamide
48.75 ± 5.8*
119.0 ± 20.7*
Diabetic +
DOAcK
*Statistically significant difference vs. the diabetic group by the multiple comparison Dunnett test.
Vehicle: Water 2:1 DMSO. U of SOD: One unit (U) of SOD is defined as the amount in µg of
enzyme that causes a 50% decrease of the NBT reduction. (n = 10, mean ± SEM; p <0.05).
Histopathological analysis of the healthy rats group demonstrated normal
architecture with a central vein and hepatocytes surrounding this vein, while the
diabetic group exhibited more sinusoidal spaces due to hepatocyte inflammation
and even death. Groups treated with Glibenclamide and DOAcK showed a
morphology similar to that of the healthy group (Fig. 2); this could be due to
improved glucose control. In other studies it has been reported that Cyclocarya
paliurus and mangiferin in STZ-induced diabetic rats, can decrease the damage
31,32
observed in different tissues, in contrast with the untreated groups.
9

Figure 2. Histopathological analyses of liver in Streptozotocin (STZ)-induced diabetic rats. A)
Healthy control, B) Diabetic control, C) Diabetic + Glibenclamide, D) Diabetic + DOAcK. 1. Central
vein, 2. Hepatocyte, 3. Sinusoids. Images are representative of each experimental group (200X).
Fig. 3 depicts the Islets of Langerhans of the experimental groups. The diabetic
untreated group exhibits a constriction of these Islets, as well as a decrease of β-
cells versus the healthy group. In contrast, the DOAcK-treated group demonstrated
morphology close to that of the healthy group. Patients with diabetes exhibited a
decrease of β-cells after having been diagnosed with DM, due to progressive
33
damage by OS evoked by ROS and hyperglycemia. The morphology observed in
the group treated with DOAcK is probably due to attenuation in ROS levels.
10

Figure 3. Fluorescence microphotographs of the Islets of Langerhans of Streptozotocin (STZ)-
induced diabetic rats. Insulin is displayed in green. A) Healthy control, B) Diabetic control, C)
Diabetic + Glibenclamide, D) Diabetic + DOAcK. Images are representative of each experimental
group (200X).
None of the five DOAcK concentrations tested were mutagenic for S.
typhimurium strains TA98, TA100, or TA102 in the absence or presence of the S9
fraction. There are natural products that possess potential mutagenic activity, such
34
as flavonoids and alkenylbenzenes; therefore, in order to avoid potential health
risks, we evaluated the mutagenic potential of DOAcK by means of the Ames test.
Only positive controls increased the revertants number under all conditions. Table
5
presents the mutagenic index of the doses used, which were determined as
follows: the average number of revertants per plate test divided by the average
number of revertants per baseline control plate. Since 1983, Ohta found that
35
coumarin and 7-hydroxy-coumarin have antimutagenic effects; furthermore, in
004, Marques and Lin found that 4-hydroxy-coumarin can be an antimutagen
agent. This compound might form hydrogen-bonds between its carbonyl group and
2
36
amino group of mutagenic compounds to produce stable complexes. Although it
is necessary to conduct other studies, we think that DOAcK and other coumarins
would possess a mechanism similar to that demonstrated by 4-hydroxy-coumarin.
11

Table 5. Mutagenicity index of DOAcK in Salmonella typhimurium strains TA98, TA100, and TA102
Substance
Concentration
µM/plate
TA98
TA100
TA102
With
S9
Without
S9
With Without
With
S9
Without
S9
S9
S9
DOAcK
DOAcK
DOAcK
DOAcK
8.65
1.1
1.2
1.2
1.1
1.4
1.1
0.7
1.0
0.8
1.1
0.4
0.4
0.9
0.9
0.8
0.9
1.0
0.9
1.3
1.0
1.0
0.9
1.4
1.0
0.8
1.2
1.4
1.2
0.9
1.1
0.7
0.7
1.0
1.1
0.8
1.0
17.30
34.60
50.00
100.00
141.00
0.19
DOAcK
DMSO 0.1%
Picrolonic acid
N-Methyl-N'-nitro-
N-nitrosoguanidine
27.3
0.007
22.9
4
-Nitroquinoline-1-
oxide
-Aminoanthracene 0.005
0.003
3.1
2
54.1
19.1
3.5
DMSO = Dimethyl Sulfoxide.
In vivo Micronucleus assay can detect genotoxic agents that cause damage to
37
the chromosomes or to the mitotic spindle. We evaluated the potential of DOAcK
to produce micronucleus 48 h after treatment was administered. Fig. 4 depicts the
percentage of micronuclei of treated groups. The positive control
(
(
Cyclophosphamide) was significantly increased compared with the vehicle control
water 2:1 DMSO), whereas administration of DOAcK did not affect the percentage
of micronuclei. When a new drug is proposed for use in humans, it is necessary to
analyze its toxic activity in order to avoid partial or mortal complications.
Glibenclamide has been reported as not evocating any significant increase in the
38
frequencies of micronucleated cells. However, recently it was reported that
39
Metformin and Glimepiride increased the frequency of micronucleated cells. The
presence of genotoxicity in drugs currently employed to treat DM is evident; in
contrast, our results show that DOAcK are not aneuploid or clastogenic agent.
12

Figure 4. Percentage of erythrocytes with micronucleus in the different groups. Data are expressed
as mean ± Standard Error of the Mean (SEM). *Significant statistical differences (p <0.05) against
the negative control. CP: Cyclophosphamide; vehicle (water 2:1 DMSO).
The OECD-423 test was conducted to evaluate the acute toxicity of DOAcK at
4
0
the initial dose of 2,000 mg/kg. At this dose, no signs of toxicity or death were
reported after 14 days of observation. Animals treated with DOAcK increased BW
in the same manner as the untreated control group (Table 6). Hence, according to
the Globally Harmnonized System (GSH) system, the LD50 of DOAcK is >2,000
mg/kg. Although DOAcK LD50 has not been previously determined, its precursor,
4
1
the coumarin, has an LD50 >700 mg/kg, both toxicity values suggesting that the
acute toxicity of this family of compounds is relatively elevated. The biochemical
profile in diabetic rats showed similar values to healthy group, indicating no toxic
activity at the dose tested (Table 3).
Table 6. Body Weight (BW) of animals after 14 days of treatment with 2,000 mg/kg of DOAcK
Day 1
Day 14
Weight gain
14
Control
22.3 ± 1.8
DOAcK
Con-trol
35.7 ± 1.5
DOAcK
Control
DOAcK
Weight
21.0 ± 1.0
34.0 ± 0.0 13.3 ± 0.3
13.0 ± 1.0
The search for new drugs to treat DM is essential, despite that there are many
medicaments available to the public and many others under investigation; these
13

drugs have different degrees of effectiveness and side effects. The
pyranocoumarins are compounds that possess an important pharmacological
profile; we have provided evidence regarding how DOAcK can improve glucose
metabolism, which may be due to the increased antioxidant activity of CAT, GPx,
and SOD, in addition to not presenting high toxicological potential. Therefore,
DOAcK could be an alternative in treating DM. However, more studies are
necessary to warrant this: it is important to evaluate DOAcK in a different animal
model of diabetes and to determine its sub-chronic and chronic oral toxicity. In this
moment, we are evaluating DOAcK in an animal model of diet-induced obese mice
to evaluate its potential for treating metabolic syndrome.
Acknowledgments
We thank Ricardo Gaxiola-Centeno, MVZ, for his excellent technical support in
the model of Streptozotocin-induced diabetic rats. The authors are very grateful for
the financial support from SIP-IPN (20140974 and 20150719). Our thanks also go
to CONACYT for the fellowship granted to EAD-M (240114), he studied in the
program of “Doctorate of Science in Biotechnology on Network of the National
Polytechnic Institute”.
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Graphical abstract
17

Highlights


DOAcK exerts an important antidiabetic effect.
DOAcK administration increased the activities of Superoxide Dismutase (SOD),
Catalase (CAT) and Glutathione Peroxidase (GPx).

DOAcK is not mutagenic by Ames test and are not genotoxic by Micronucleus
assay.


DOAcK not presenting high toxicological potential.
DOAcK could be an alternative to treat Diabetes Mellitus.
18