Orcinol derivative compound with antioxidant properties protects Langerhans islets against streptozotocin damage

Article abstract of DOI:10.1111/jphp.12696

Objective: To design and synthesize an orcinol derivative compound, 3-formyl-2,4-dihydroxy-5,6-dimethyl sodium benzoate (A4), as an antioxidant molecule and to test its effects on oxidative stress in an in-vitro model of apoptosis of pancreatic rat beta cells induced by streptozotocin (STZ). Methods: Scavenger properties of A4 were assessed using its capacity to capture the DPPH radical in vitro. Antiapoptotic properties of A4 were analysed by electron microscopy and TUNEL assay in rat pancreatic islets in a streptozotocin model. Key findings: The results show that A4 displays antioxidant activity in in-vitro assays and induced a significant reduction in STZ-induced beta cell apoptosis and low ultrastructural damage to cellular organelles in the rat pancreatic islets as evidenced by electronic microscopy, this effect could be attributed to its antioxidant activity in a similar manner than resveratrol. Conclusion: The overall results indicate that the new orcinol derivative molecule displays both antioxidant and antiapoptotic effects and protect pancreatic beta cells against STZ damage.

Full text of DOI:10.1111/jphp.12696

Research Paper
Orcinol derivative compound with antioxidant properties
protects Langerhans islets against streptozotocin damage
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Nancy M.V. Sanchez-Lira , Angelica Morales-Miranda , Gustavo Garcıa de la Mora ,
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Juan Carlos Leon Contreras , Ignacio Gonzalez-Sanchez , Norma Valencia , Marco Cerbon and
Sumiko Morimotoa
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Departamento de Biologıa de la Reproduccion, Instituto Nacional de Ciencias Medicas y Nutricion Salvador Zubiran, Tlalpan, Ciudad de Mexico,
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Mexico, Division de Posgrado, Departamento de Quımica Organica, Facultad de Quımica, Universidad Nacional Autonoma de Mexico, Coyoacan,
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Ciudad de Mexico, Mexico, Departamento de Patologıa, Instituto Nacional de Ciencias Medicas y Nutricion Salvador Zubiran, Tlalpan, Ciudad de
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Mexico, Mexico, Departamento de Biologıa, Facultad de Quımica, Universidad Nacional Autonoma de Mexico, Coyoacan, Ciudad de Mexico,
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Mexico, Departamento de Farmacia, Facultad de Ciencias, Universidad Nacional de Colombia, Bogota, Colombia and Unidad de Investigacion en
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Reproduccion Humana, Facultad de Quımica, Instituto Nacional de Perinatologıa, Ciudad de Mexico, Mexico
Keywords
Abstract
cellular morphology; oxidative stress;
pancreatic beta cells; rat; TUNEL
Objective To design and synthesize an orcinol derivative compound, 3-formyl-
2,4-dihydroxy-5,6-dimethyl sodium benzoate (A4), as an antioxidant molecule
and to test its effects on oxidative stress in an in-vitro model of apoptosis of
pancreatic rat beta cells induced by streptozotocin (STZ).
Correspondence
Sumiko Morimoto, Departamento de
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Biologıa de la Reproduccion, Instituto
Methods Scavenger properties of A4 were assessed using its capacity to capture
the DPPH radical in vitro. Antiapoptotic properties of A4 were analysed by elec-
tron microscopy and TUNEL assay in rat pancreatic islets in a streptozotocin
model.
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Nacional de Ciencias Medicas y Nutricion
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Salvador Zubiran, Av. Vasco de Quiroga 15,
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Col. Belisario Domınguez Seccion XVI,
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Tlalpan C.P. 14080, Ciudad de Mexico,
Mexico.
Key findings The results show that A4 displays antioxidant activity in in-vitro
assays and induced a significant reduction in STZ-induced beta cell apoptosis
and low ultrastructural damage to cellular organelles in the rat pancreatic islets as
evidenced by electronic microscopy, this effect could be attributed to its antioxi-
dant activity in a similar manner than resveratrol.
E-mail: sumiko.morimotom@incmnsz.mx
Received September 6, 2016
Accepted December 29, 2016
Conclusion The overall results indicate that the new orcinol derivative molecule
displays both antioxidant and antiapoptotic effects and protect pancreatic beta
cells against STZ damage.
doi: 10.1111/jphp.12696
(SOD), catalase, glutathione peroxidase (GPx) and antioxi-
dants prevent or delay the damage produced by free
Introduction
Diabetes mellitus is a multifactorial metabolic disease char-
acterized by pancreatic beta cells dysfunction and insulin
secretion deficiency resulting in hyperglycaemia.[1,2] This
hyperglycaemia provokes oxidative stress in beta cells,
resulting in an elevated level of free radicals and cell death.
Different types of diabetes have been described; however,
the main representative feature is a progressive deteriora-
tion of pancreatic beta cells mass, leading to insufficient
insulin secretion.[3]
radicals.[6]
Streptozotocin (STZ) is a N-nitrosourea derivative of D-
glucosamine, described as an alkylating agent, inducing
beta cell toxicity and diabetogenic properties, which are
mediated through diverse mechanisms including targeted
uptake of STZ in beta cells by Glut2 transporter,[7]
increased oxidative stress due to nitric oxide (NO) release
and reactive oxygen species (ROS) production.[8,9]
In contrast, resveratrol (RSV) (3,5,4-trihydroxystilbene) is
a compound stilbene related to the family of the phytoalexins
produced by plants in response to disease, injury and stress.
These phenolic compounds are present in fruits and vegeta-
bles, cocoa, chocolate, red wine, green tea and other dietary
sources and exhibit potent free radical scavenging properties
Molecular studies have demonstrated that apoptosis of
pancreatic cells is due to the production of free radicals,
including the superoxide anion (O2), hydroxyl radicals
(OHÀ), hydrogen peroxide (H2O2) and singlet oxygen
(1O2).[4,5] In contrast, enzymes like superoxide dismutase
© 2017 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, ** (2017), pp. **–**
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New antiapoptotic compound in Langerhans islets
Nancy M.V. Sanchez-Lira et al.
in-vitro. The main source of stilbenes is RSV, which is mainly
found in grapes and red wine.[10] This compound improves
antioxidant defence in pancreatic tissue, increases activity of
antioxidant enzymes (SOD, catalase, GPx and glutathione-S-
transferase) and protects cells from free radical damage.[11]
Antioxidant activity of resveratrol in pancreatic islets is of
particular importance given that antioxidant defence of
b-cells is very weak, rendering them susceptible to oxidative
stress.[12] RSV was also found to reverse degenerative changes
in b-cells of STZ-induced diabetic rats[13] and to prevent
STZ-induced b-cell apoptosis.[14]
Compounds with antioxidant effects such as resveratrol
have been studied in DM1 and DM2 and demonstrated
that are useful to partially recover pancreatic beta cell func-
tion by improving glucose levels, insulin secretion and
insulin resistance.[11] Therefore, the search for new
compounds that present antioxidant activity continues to
be of great interest to prevent beta cell damage in diabetes.
In this study, we designed and synthesized a new orcinol
derivative, 3-formyl-2,4-dihydroxy-5,6-dimethyl sodium
benzoate (A4). The design of A4 was based in natural com-
pounds derived from lichens that have shown antioxidant
effects.[15,16] Some compounds like lobariellin and methyl
haematommate derived from lichens have shown protective
effects against oxidative damage and DNA protection in
primary murine neuronal cultured cells and ker-
atinocytes.[15] The structure of the designed molecule was
intended to act as a scavenger for free radicals to prevent
pancreatic damage induced by diabetes and to contribute
in some way to the search for new treatments. Thus, the
aim of this study was to design and synthesize an orcinol
derivative compound 3-formyl-2,4-dihydroxy-5,6-dimethyl
sodium benzoate (A4), as an antioxidant molecule and to
test its effects on oxidative stress in an in-vitro model of
apoptosis of rat pancreatic beta cells induced by STZ.
The compound was in its acid form, and the physical
and spectroscopic proprieties were the following: light yel-
low solid; mp. 176–178 °C; IR (cmÀ1) 3200–2600, 1626;
EMIE (m/z, %) (166, 100), M+: (210, 20), M + 1: (211, 2).
NMR-1H (ppm) (CDCl3): d = 2.13 (s, 3H, Ph-CH3), 2.58
(s, 3H, Ph-CH3), 10.33 (s, 1H, CH=O), 12.89 (bs, 3H, 2Ph-
OH, COOH). NMR-13C (ppm) (CDCl3/TMS): d = 10.41
(–CH3), 19.63 (–CH3), 106.45 (C-1), 114.7 (C-3), 149.17
(C-5), 151.97 (C-4), 164.12 (OH–C=O), 194-2 (CH=O).
To perform the biological tests, the compound was made
water soluble by the preparation of its sodium salt. In brief,
the acid form was suspended in water and treated with one
equivalent of sodium hydroxide. The resulting salt was
precipitated with ethanol, filtered and dried.
Animal care and management
Male albino Wistar rats (180–200 g) were maintained in
the animal facility of the Instituto Nacional de Ciencias
Mꢀedicas y Nutricioꢀn Salvador Zubirꢀan (INCMNSZ), in
Mexico City. All animal handling and procedures were
approved by the Animal Experimental Ethics Committee of
INCMNSZ, in accordance with the Official Mexican Guide-
line for the Care and Use of Laboratory Animals (NOM-
062-ZOO-1999). Rats were under controlled conditions of
temperature (22–24 °C) and 12-h light; 12-h darkness
cycles, with free access to water and fed normal laboratory
chow (Zeigler Rodent RQ 22-5, Gardner, PA, USA). Rats
were euthanatized using a guillotine (Thomas Scientific,
Swedesboro, NJ, USA).
Antioxidant capacity of A4
The 1,1-diphenyl-2-picrylhydrazyl free radical (DPPH)
model was used to assess the free radical scavenging activity
of RSV (50 lM) and A4 (25, 50 and 100 lM). The absorbance
of the preparation samples were measured at 517 nm using
UV–vis spectrophotometer (DU-6 Beckman Atlanta, GA,
USA), every 15 min until a steady state was reached.[19] Anti-
radical activity was defined as the effective concentration of
antioxidant necessary to decrease the initial DPPHÁ concen-
tration by 50% expressed as EC50 = ((sample)/(DPPHÁ)).
Materials and Methods
Synthesis of 3-formyl-2,4-dihydroxy-5,6-
dimethyl sodium benzoate (A4)
Methyl 2,2-dimethyl-4,6-dioxocyclohexan carboxylate was
obtained in four steps using the methodology developed
by Rapson & Robinson.[17] Further transformation to
methyl 4,6-dihydroxy-2,3-dimethylbenzoate was performed
according to the procedure described by Nelson.[18] The
resulting yield was increased by microwave radiation at
90 °C for 40 min in one step. The formulation reaction
was done by treatment of the dihydroxy compound with
dichloride (methoxy) methane in the presence of TiCl4 in
41% yield. Finally, the ester hydrolysis was achieved with
concentrated sulfuric acid at 0 °C during 16 h giving 91%
of the free acid.
Isolation of islets
Intact islets of Langerhans were obtained by digestion with
collagenase.[20] Briefly, a bile duct catheter was introduced
and pancreatic islets isolated and collected individually
using a stereoscopic microscope following collagenase
digestion (5 mg/pancreas) and cultured overnight with
RPMI 1640 medium supplemented with 10% fetal bovine
serum, 100 U/ml penicillin, 100 mg/ml streptomycin
(Invitrogen, Carlsbad, CA, USA) at 37 °C in humidified
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Nancy M.V. Sanchez-Lira et al.
New antiapoptotic compound in Langerhans islets
5% CO2 95% atmospheric air. Islets were washed twice
with a buffer solution (pH 7.4) containing 20 mM HEPES,
115 mM NaCl, 5 mM NaHCO3, 5 mM KCl, 2.6 mM CaCl2,
1.2 mM KH2PO4, 1.2 mM MgSO4, 3 mM D-glucose and
0.1% bovine serum albumin (Sigma, St. Louis, MO, USA).
To standardize the exposure time to STZ (0.5 mM),
which is the dose previously reported to produce apoptosis
in-vitro,[21–23] the islets were incubated for 30, 60 and
120 min. Afterwards, they were stained with toluidine blue
and sectioned into 60 nm cut to evaluate the degree of con-
densation of chromatin by light microscopy.
using the statistical program Sigma Stat 3.5 (Systat Soft-
ware, Point Richmond, CA, USA).
Results
Compound A4 has antioxidant activity
To evaluate the antioxidant activity of A4, we measure its
capacity to capture the DPPH radical as described by
Brand-Williams et al.[19] We tested A4 (Figure 1), prepara-
tions at 25, 50 and 100 lM and the results were compared
to those produced by RSV at the standard dose of 50 lM.
As shown in Figure 2, the DPPH capture increased with
Treatments
To assess the protective effect of A4 as antioxidant, groups
of 50 islets were pretreated during 60 min with different
concentrations of either compound A4 (25, 50 and
100 lM) or with RSV (50 lM) as the positive control of
antioxidant protection. Then, the islets were treated with
0.5 mM of STZ during 60 min. Ethanol was used as vehicle
(final concentration 0.01%). All studies were conducted in
duplicate.
Electron microscopy
After the incubation, islets from each treatment group were
embedded in Epon resin (EMbed-812, Electron Microscopy
Sciences) and 1-lm-thick cuts were made. Sections were
observed by transmission electron microscopy (EM 10CR;
Carl Zeiss, Thornwood, NY, USA), and the morphology
and integrity of the pancreatic beta cells were analysed.
Figure 1 Chemical structure of the compound 3-formyl-2,4-dihy-
droxy-5,6-dimethyl sodium benzoate (A4).
TUNEL assays
The proportion of apoptotic beta cells in complete islets
was assessed by the TUNEL technique[24] (Roche Diagnos-
tic, Mannheim, Germany), following the manufacturer’s
instructions. Briefly, the islets were fixed in 2%
paraformaldehyde for 1 h and make them permeable in
0.1% triton X-100 and 0.1% sodium citrate. Then, 50 ll of
the TUNEL reaction mixture was added to each sample.
Islets were counterstained with DAPI, and the cells were
visualized in the wave length emitted by rhodamine and
DAPI (600 and 461 nm, respectively). Representative
microphotographs were taken using confocal microscopy
(Model FV1000; Olympus,Tokyo, Japan).
Statistical analysis
Figure 2 Percentage of DPPH captured by resveratrol and by differ-
ent concentrations of A4. Each bar represents the mean value of the
percentage of DPPH captured Æ SE. Data were analyzed by Kruskal–
Wallis test followed by Tukey post hoc test (n = 3 independent experi-
ments). Different letters indicate significant differences at each time
point (P < 0.001).
Data of DPPH capture were obtained from three indepen-
dent experiments (n = 3) and the percentage of apoptotic
beta cells (n = 50 islets per treatment) were analysed using
the Kruskal–Wallis test followed by a post hoc Tukey’s test,
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New antiapoptotic compound in Langerhans islets
Nancy M.V. Sanchez-Lira et al.
time, but in all cases the effect was greater (P < 0.001) in
the treatment with RSV than A4. At 30 and 60 min, there
were no statistical significant differences in the percentage
of radical capture by effect of A4 at 100 and 50 lM; the only
significant difference between A4 concentrations was at
25 lM. At this concentration, it was observed almost half of
the effect of capture of DPPH radical produced by RSV. At
90 and 120 min, A4 at concentrations of 25 and 50 lM
showed a similar effect, while 100 lM produced a greatest
effect in DPPH radical capture.
Further observations by electron microscopy were made
in 1-lm cuts of the same islets. Condensation of chromatin
was found after 30 min of STZ treatment, the presence of
apoptotic bodies at 60 min and condensation of chromatin
and destruction of cytoplasm at 120 min, all changes typi-
cal of apoptosis (Figure 3). The observation of these mor-
phological changes allowed us to select the median time of
60 min of treatment with STZ, as the time at which the cel-
lular damage produced to be reversed by the effect of A4
and RSV.
The ultrastructural characteristics of pancreatic beta cells
in different conditions are shown in Figure 4. Top panels
show untreated control islets. It was observed a conserved
cellular structure with nuclear integrity and no chromatin
condensation. The characteristic secretory granules of beta
cells exhibited a central small dense core surrounded by a
clear lucent halo with an external single-layered membrane.
After STZ treatment (0.5 mM), we observed chromatin
condensation and the formation of apoptotic bodies (Fig-
ure 4c and 4d). Cells undergoing apoptosis exhibited cir-
cumscribed chromatin adjacent to the nuclear membrane
in the presence of intact cytoplasmic organelles and mem-
branes besides a reduced number of insulin secretory gran-
ules. These changes were prevented by RSV (50 lM)
pretreatment followed by STZ (0.5 mM). The integrity of
the endoplasmic reticulum was preserved, less chromatin
condensation was evident in the nucleus (Figure 4e and
Treatment with A4 and RSV prevents beta
cell damage
To determine the optimal time of treatment with STZ for
the induction of apoptosis of pancreatic beta cells, and
whether this damage was prevented by A4, we used STZ as
a model of oxidant stress damage of these cells.
Microscopy studies were conducted on islets of Langer-
hans that had been treated during 30, 60 and 120 min with
0.5 mM STZ. Under light microscopy, control islet sections
showed well-defined shape and undisrupted capsule with
cells of intact nuclei and cytoplasm. Islets treated with STZ
showed chromatin aggregation and a central area of lysis
and debris between peripheral cords of intact cells. These
observations indicate that the time of treatment with STZ
was directly proportional to cellular damage.
(a)
(b)
(c)
(d)
Figure 3 Electron microscopy microphotographs of islet cells treated with streptozotocin 0.5 mM (30, 60 and 120 min). Intracellular changes are
shown (arrows) in some organelles, including secretory granules, chromatin condensation, apoptotic bodies and chromatin degradation: (a) con-
trol, (b) streptozotocin treatment for 30 min, (c) streptozotocin treatment for 60 min, (d) streptozotocin treatment for 120 min. N = nucleus,
SG = secretory granules, AB = apoptotic bodies, CC = chromatin condensation, and CD = chromatin degradation.
4
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Nancy M.V. Sanchez-Lira et al.
New antiapoptotic compound in Langerhans islets
(a)
(c)
(e)
(b)
(d)
(f)
Figure 4 Effect of resveratrol pretreatment on ultrastructural characteristics of beta cells after streptozotocin treatment. Control group (a, b);
and treated with streptozotocin alone or in combined treatments: 0.5 mM streptozotocin (c, d); 50 lM resveratrol + 0.5 mM streptozotocin (e, f).
Electronic microscopy was performed according to materials and methods, intracellular structure were analyzed to determine changes in organelles
and secretory vesicles. N = nucleus, SG = secretory granules, ER = endoplasmic reticulum and AB = apoptotic bodies.
4f), and a moderate increase in secretory granules was also
observed.
A protective effect against apoptosis of beta cells was also
observed with A4 at 25 and 50 lM; however, the effect was
significantly lower compared with A4 at 100 lM and RSV
(Figure 6 and Table 1). The percentage of apoptosis of
STZ-treated pancreatic beta cells was reduced in a similar
proportion by effect of a pretreatment with both A4 at
100 lM and RSV.
In the islets pretreated with A4 (100 lM), the greatest
protection was found to be reducing the deleterious effect
produced by STZ (Figure 5). However, at lower concentra-
tions (25 lM) chromatin condensation and damage to the
endoplasmic reticulum was still observed (Figure 5a and
5b), in contrast to the protective effect observed at doses of
50 and 100 lM of A4 (Figure 5c–5f). The islets pretreated
with A4 (100 lM) and RSV (50 lM) showed a similar pat-
tern of beta cell protection (Figure 4e, 4f and Figure 5e, 5f)
and were similar to control islets (Figure 3a).
Discussion
Pancreatic beta cells are highly vulnerable to oxidative
stress due to their restricted capacity for DNA repair
induced by oxidative stress.[6,12] Hyperglycaemia generates
free radicals that in turn induce oxidative stress, a deleteri-
ous condition for the pancreatic beta cells, which leads to
cellular damage and apoptosis. In experimental diabetes
model in rats, antioxidant compounds such as RSV with a
scavenger activity diminish insulin resistance and reduce
the glucose levels.[13,25]
A4 treatment protects against STZ induction
of apoptosis of beta cells
As shown in Figure 6, STZ produces an extensive damage
through apoptosis in beta cells (≥78%). When islets were
pretreated with antioxidant compounds (RSV or A4), the
percentage of STZ-induced apoptosis decreased (Table 1).
RSV showed the greatest protection against apoptosis in
beta cells; a similar effect was observed with A4 at 100 lM.
In the present study, we induced a deleterious effect on
the pancreatic beta cells using STZ as evidenced by ultra-
structural changes characteristic of apoptosis. Although it is
© 2017 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, ** (2017), pp. **–**
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Nancy M.V. Sanchez-Lira et al.
New antiapoptotic compound in Langerhans islets
(b)
(d)
(f)
(a)
(c)
(e)
Figure 5 Electron microscopy of beta cells. The ultrastructural characteristics of islets are shown in cells treated with A4 at 25 lM (a, b); 50 lM
(c, d) and 100 lM (e, f); respectively for 60 min, afterwards 0.5 mM of streptozotocin were added. N = Nucleus, SG = secretory granules and
ER = endoplasmic reticulum.
known that depending on the dose, STZ induces apoptosis
or necrosis,[21–23,26] however, we have previously reported
effect of A4 in pancreatic islets treated with STZ was
demonstrated by the findings in the ultrastructural studies.
After pretreatment with A4 (100 lM), pancreatic beta cells
showed less chromatin condensation and a moderated
increase in secretory granules, besides the percentage of
apoptotic beta cells that significantly decreased compared
with islets treated with other concentrations of A4 (25 and
50 mM) and the STZ-treated islets. Regarding ultrastruc-
tural characteristics of the cells, the islets pretreated with
A4 at 100 lM showed a similar pattern of beta cell protec-
tion as compared to RSV and were similar to control islets,
which mean a conserved cellular structure with nuclear
integrity and no chromatin condensation. It has been
reported that STZ may induce ROS and reactive nitrogen
species, formation and oxidative stress.[29] The data pre-
sented herein support the hypothesis that the apoptotic
damage produced by oxidative stress in pancreatic beta cells
can be prevented by A4 at least in part. This compound at a
concentration of 100 lM showed scavenging activity
that STZ induced early apoptosis of Langerhans islets[27]
;
accordingly, in the present study we used reported doses to
produce apoptosis in beta cells.[26] Cells undergoing apop-
tosis by effect of STZ treatment were observed with a char-
acteristic pattern of structural changes including extensive
cleavage of their DNA, chromatin condensation, cell
shrinking and formation of apoptotic bodies. Additionally,
it has been reported that this cell damage induced by STZ
was prevented by treatment with RSV.[13,28]
To evaluate the antioxidant properties of compound A4,
we used the method of stability of the radical DPPH. The
results proved that A4 presented antioxidant activity. How-
ever, in this assay A4 was less efficient concerning scaveng-
ing activity than RSV at the same concentration.
Once verified the antioxidant capacity of A4, its potential
protective effect on pancreatic beta cells was tested in the
in-vitro model of STZ-induced apoptosis. The protective
6
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Nancy M.V. Sanchez-Lira et al.
New antiapoptotic compound in Langerhans islets
Figure 6 Representative microphotographs of rat pancreatic Langerhans islets. Beta cells in control islets and those treated with streptozotocin
at 0.5 mM are shown. The streptozotocin treatment induces apoptosis in beta cells, which was detected by the TUNEL assay. Pancreatic islets were
pretreated with resveratrol or A4. Apoptotic cells can be appreciated after resveratrol pretreatment at 50 lM followed by streptozotocin at
0.5 mM and after pretreatment with A4 at different concentrations 25, 50 and 100 lM, followed by streptozotocin treatment at 0.5 mM. The total
cell nuclei were stained with DAPI and the apoptotic cells with rhodamine. In the last panel is shown the co-location of both markers.
producing a decrease in the percentage of apoptosis similar
to that produced by RSV.
A4, an orcinol derivative, is in line with those reported
by Perico-Franco et al.[15,16] that showed an antioxidant
capacity of lobariellin and methyl haematommate, and
prevented skin and neurodegenerative damage by oxida-
tive stress. Compounds isolated from lichens have a
b-orcinol core as A4.
In addition, compounds structurally related to A4 iso-
lated from lichens, lobariellin, stereocaullin and methyl
haematommate show high scavenging activity. All these
compounds, including A4, have in their structure a
b-orcinol core, which has been proposed that confers
antioxidant properties.[15,16] The antioxidant activity of
Additionally, Thadhani et al.[30] evaluated the antioxi-
dant activity of some lichen metabolites, they found that
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Table 1 Percentages of streptozotocin-induced apoptosis of beta
cells in rat pancreatic islets pretreated with A4 (3-formyl-2,4-dihy-
droxy-5,6-dimethyl sodium benzoate) or resveratrol (RSV)
It is likely that compounds like RSV and in our case A4
penetrate the membrane of the beta cell due to its polarity
and interact with free radicals derived from the metabolism
of STZ; however, this deserves to be further investigated.
Treatment
Apoptotic nuclei (%)
Control
<1a
STZ 0.5 mM
RSV 50 lM
A4 25 lM
A4 50 lM
A4 100 lM
78 Æ 1b
19 Æ 0.7c
30 Æ 1d
35 Æ 1d
22 Æ 0.7c
Conclusion
The overall results indicate that the new orcinol derivative
molecule displays both antioxidant and antiapoptotic
effects and protects pancreatic beta cells against STZ dam-
age. Further studies are needed to fully elucidate the molec-
ular mechanism of protection, as well as developing in-vivo
models to ascertain whether this compound could have a
potential as a therapeutic agent to prevent the loss of beta
cells in diabetes.
Data are expressed as percentage (ÆSE) of apoptotic beta cells from
total cell number in pancreatic islets. n = 50 islets per treatment,
experiments were conducted in duplicate. Differences in the percent-
age of beta cells per treatment were analysed by Kruskal–Wallis and
Tukey’s tests for multiple comparisons (P < 0.001). Data with different
letters differ statistically.
Declarations
methyl-b-orcinol carboxylate displays a potent NO scav-
enger. Taking into account the aforementioned result, the
protective effect of A4 observed in pancreatic beta cells is
probably due to its scavenger properties and by capture of
free radicals as suggested for RSV,[25] which has a phenolic
ring related to orcinol core.
Conflict of interest
The Author(s) declare(s) that no conflict of interest exists.
Acknowledgements
Although the mechanism of targeted beta cell toxicity of
STZ is not clear, it is generally assumed that it is dependent
upon the DNA alkylating activity of its methylnitrosourea
moiety.[31] The transfer of the methyl group from strepto-
zotocin to the DNA molecule results in DNA fragmenta-
tion. In the attempt to repair DNA, poly (ADP-ribose)
polymerase (PARP) is overstimulated. This diminishes
intracellular NAD+ and subsequently ATP stores, leading
to cell death.[31]
This study was partially supported by grant (CB177624)
from the Consejo Nacional de Ciencia y Tecnologꢀıa (CON-
ACyT). The authors would like to thank to Dr. Yvonne
Grillasca Rangel and Hector Torres Dominguez for the syn-
thesis of the 3-formyl-2,4-dihydroxy-5,6-dimethyl sodium
benzoate. We also wish to thank to Dr. Rogelio Hernꢀandez
Pando for the use of electronic microscopy facilities.
5. Maritim AC et al. Diabetes, oxidative 10. Crozier
stress, and antioxidants: review.
A et al. Dietary phenolics:
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