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Chapter 5
Potential therapeutic effect of Quercetin,
Zinc and BLE on arsenic induced
Pancreatic Oxidative stress
Part of this chapter has been published
Patel HV and Kalia K. Ameliorating effect of quercetin and zinc on arsenic
induced pancreatic oxidative stress, Asian Journal of Experimental Biological
Science-(Under Preparation)
Patel HV and Kalia K. Effect of bamboo leaves extract (BLE) on arsenic
induced pancreatic oxidative damage and diabetes mellitus in Wistar rats-
(Under Preparation)
Chapter-5
118
5.1 Introduction
The biological mechanism(s) by which arsenic induce diabetes mellitus remains
poorly understood. The common theme that was emerged is the role of reactive oxygen
species (ROS) in the pathogenesis of arsenic-induced diabetes mellitus. It has been shown
in previous experiment that chronic exposure to arsenic induces pancreatic oxidative
damages which might play a role in the development of arsenic induced diabetes mellitus.
Zinc deprivation was also observed in pancreatic tissue on arsenic exposure. Based on the
above observations, it is apparent that use of antioxidants provides a possible and novel
alternative treatment for the arsenic induced diabetes.
A positive relationship has also been established between dietary supplementation
with certain vegetables/plants and the reduction of toxic effects of various toxicants,
environmental agents including heavy metals (Flora et al., 2008). Oxidative stress due to
arsenic toxicity in rats has been ameliorated by therapeutic supplementation of nutritional
antioxidants like ascorbic acid, α-tocopherol or N-acetyl cysteine and some essential
metals such as zinc during chelation therapy (Modi et al., 2005). Deficiency of several
essential elements has been shown to aggravate the toxic effects of metals, and
supplementation of such micronutrients/essential metals ameliorates the toxicity.
Flavonoids are also important for human health. The antioxidant activity of flavonoids
results from the combination of their iron chelating activity and their ability to scavenge
reactive oxygen species. Flavonoids, predominantly quercetin, appear to be key
antioxidants in the treatment of various chronic diseases (Hollman, 1999). Use of herbal
products could be a better alternative to meet the objective of finding a suitable treatment
for arsenic poisoning.
Flavonoids (more than 8000) constitute the largest and most important group of
polyphenolic compounds in plants. Flavonoids are a large family of more than
4,000 secondary plant metabolites, comprising anthocyanins, catechines, flavonols,
flavones, and flavonones. Although the antioxidant activity of the polyhydroxyflavones
seems to be primary a function of their ability to act as free radical acceptors, the metal-
complexing properties may give some contribution to their total antioxidant activity. The
positive health effects associated with the intake of flavonoids have been ascribed to their
Chapter-5
119
well-known antioxidant properties and to inhibiting effects on a wide range of enzymes
(Nijveldt et al., 2001).
Quercetin has received considerable attention because of its overwhelming
presence in foods. Quercetin (3, 3’, 4’, 5-7-pentahydroxyflavone), a chemical cousin of
the glycoside rutin, is a unique flavonoid that has been extensively studied by researchers.
Quercetin, the most frequently studied bioflavonoid in the class of flavonol and is a
strong antioxidant. QCT presents in large amounts in vegetable, fruits, tea, and olive oil,
and because it contains a number of phenolic hydroxyl groups, it exhibits its therapeutic
potential against many diseases (Murota & Terao, 2003). Quercetin itself is an aglycon or
aglucone that does not possess a carbohydrate moiety in its structure. Quercetin is usually
found in plants as glycone or carbohydrate conjugates. Quercetin is of interest because of
its pharmacological function. Quercetin has the ability to boost the endogenous
antioxidant system. Quercetin a common bioflavonoid is present in herbal preparations
consumed by diabetic patients along with routine anti-diabetic agents. It has been
reported that quercetin ameliorated the diabetes-induced changes in oxidative stress.
Quercetin possesses a catalogue of pharmacological actions, including cardio-protection,
anti-ulcer effects, anti-inflammatory, cataract prevention, anti-cancer activity, anti-
allergic, antiviral and antibacterial activities, and so forth (Bentz, 2009).
Zinc is the second most abundant trace element in the body (Zhou et al., 2007). It
is contained in hundreds of enzymes and in many protein domains participating in a
number of cellular processes such as including cellular proliferation, differentiation and
apoptosis (Franco et al., 2009). Zinc plays an important role in the structure and function
of biological membranes. Zinc plays a key role in the regulation of insulin production in
pancreatic tissue. Biologically it is an important enzymatic cofactor, but among its
features, the most interesting is undoubtedly the ability to induce the synthesis of
Chapter-5
120
detoxificant proteins such as metallothioneins or metal binding proteins. (Kondoh et al.,
2003)
Number of studies carried out to assess antioxidant properties of various plants
resulted in the development of herbal medicine and nutritional supplementation in
nutraceuticals. These phytochemicals and natural antioxidants exhibited a wide range of
beneficial biological effect and could neutralize oxidation of biological molecules by
scavenging free radicals and chelating free catalytic metals (Sanchez, 2002). Much
attention has therefore been focused in finding naturally occurring antioxidants form
medicinal plant and food because they are biodegradable to non-toxic products that
replace synthetic antioxidants which are being limited to use because of their adverse side
effects. Bambusa arundinacea, locally known as Bans or bamboo, a perennial fastest-
growing plant on earth is presumed to have origin in Asia. Bamboo is an ancient Chinese
medicine and an Indian folk medicine. Bamboo is considered as a rich source of flavones
glycosides having ability to interact with lipid bilayers by influencing their incorporation
into the cells. The leaves of bamboo tree are stimulant, aromatic and tonic. They are
useful in counteracting spasmodic disorders, and arrest secretion or bleeding. Bamboo
leaves have been used clinically in the treatment of hypertension, arteriosclerosis,
cardiovascular disease, and cancer. Decoction or juice of the fresh bamboo leaves is
applied as a medicine in ulcers. The leaves are useful in killing intestinal worms,
especially threadworms (Muniappan & Sundararaj, 2003). The bamboo leaves, obtained
from the common tall bamboos have recently been utilized as a source of flavonoids (e.g.,
vitexin and orientin), used as antioxidants. Antioxidant of bamboo leaves (AOB), a pale
brown powder extracted from bamboo leaves has been listed in the national standards, i.e.
GB2760, as a kind of food antioxidant in China. The main functional components in AOB
are flavonoids, lactones and phenolic acids (Lu et al., 2006). Bamboo leaf extracts have
typical pleasant fragrance of bamboo leaves and taste slight bitter and sweet. The extracts
can be widely applied in medicine, food, feedstuff, antiaging products and cosmetics.
Therefore the fifth chapter accentuates on the protective role of quercetin (QCT),
quercetin with zinc supplementation and bamboo leaves extract (BLE) individually
against arsenic induced pancreatic oxidative damages in experimental animals, which
may lead to diabetic condition based on oxidative mechanism of arsenic induced diabetes
mellitus.
Chapter-5
121
5.2 Material and methods
5.2.1 Chemical/therapeutic agent
Zinc acetate was procured from Merck chemicals ltd and used as source for the
supplementation as zinc. Quercetin powder was obtained from Hi-media ltd, Bombay.
5.2.2 Bamboo leaves collection and extraction
The mature bamboo leaves were collected from botanical garden of Department
of Biosciences, Vallabh Vidhyanagar, Gujarat in the autumn season. The specimens were
botanically identified as Bambusa arundinacea. The collected leaves were washed 4-5
times by distilled water, shade-dried completely and then powdered with a mixture
grinder and stored in an air-tight container in the refrigerator before use. The dried
bamboo leaf powder was subjected to extraction, three times with 10 volumes of
methanol in electrical shaker for 24 hours at room temperature. Bamboo leave extract
were separately filtered, through Whatman filter paper 1. The residue was re-extracted
with 10 volumes of methanol using same procedure. After procedure was repeated thrice,
all the extracts were pooled and evaporated to dryness at 50°C. The resulting gummy
mass was weighted and suspended in distilled water used for the treatment. The
percentage yield of the bamboo leave extract using methanol as solvent was found to be
13.7%.
Figure 5.1 Bamboo leaves of Bambusa arundinacea (Family:Poaceae; Subfamily:
Bambusoideae; Tribe:Bambuseae)
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122
5.2.3 Experimental protocol
The whole experiment was performed on albino Wistar rats (n=60) and were
divided into different groups as follows After fifteen days of acclimatization, the rats
were randomly assigned into five groups of twelve rats in each group.
Group I (Control): served as a negative control and received only distilled water
without addition of arsenic orally
Group II (As): Animals were administered with arsenic as sodium arsenite at the
dose 1.5 mg/kg body weight daily for a period of 4.0 wk orally and marked as
arsenic exposed group.
Group III (QCT): Administered with arsenic as in group II + simultaneously
received quercetin (QCT) (15.0mg/kg body weight) prepared in DMSO
intraperitoneally (i.p.) once daily
Group IV (QCT + Zinc): Administered with arsenic as in group II + QCT as in
group III + simultaneously administered with zinc acetate (10mg/kg body weight,
orally) as supplement daily
Group V (BLE): Administered with arsenic as in group II + simultaneously
administered with bamboo leave extract (250mg/kg body weight) orally once
daily. The selected dose of the bamboo leave extract was based upon previously
experiments.
After the experimental period was over (5 wks), the animals were kept in overnight
fasting and then sacrificed by light ether anesthesia. A small portion from the gastro-
splenic part of the pancreas was quickly isolated, washed with saline, blotted dry on filter
paper and placed in ice-cold phosphate buffer (pH 7.4). It was kept on a small ice-slab
and cut into small pieces with scissors and homogenized immediately. Pancreatic tissue
from half of the animals from each group was stored at -20 °C for the wet digestion and
used for the metal (arsenic and zinc) estimation. 10.0% pancreatic tissue homogenates
were prepared in 50mM phosphate buffer (pH 7.4) and centrifuged at 10,000 rpm for 15
min at 4ºC. The resultant supernatant was collected in another tube and used for various
biochemical analysis. The blood was collected by cardiac puncture and serum was
Chapter-5
123
separated by centrifugation at 2500 rpm for 15 min and stored at 4 ºC. Blood glucose
level and glycosylated hemoglobin level was estimated by GOD/POD enzymatic and
chemical method described by Chandalia et al. (1980) respectively. Protein concentration
from pancreatic tissue was estimated according to the method of Lowry et al. (1956).
5.2.4 Biochemical estimation of markers of oxidative stress
The biochemical parameters analyzed from pancreatic tissue homogenate were
according to previously described method presented in following table.
No. Biochemical parameters References
1. Thiobarbituric Acid Reactive Substances (TBARS) Ohkawa et al., 1979
2. Protein Carbonyl Content (PCO) Reznick and Packer, 1994
3. Advanced Oxidation Protein Product (AOPP) Kayali et al., 2006
4. Nitric Oxide (NOx) Titheradge et al., 1889
5. Thioredoxin Reductase (TrxR) Smith et al., 2002
6. Glutathione Peroxidase (GPx) Rotruck et al., 1984
7. Reduced GSH Jollow et al., 1974
8. Arsenic and Zinc metal As described previously
5.2.5 Statistical analysis
The mean values ± SD were calculated for each parameter. Percentage restoration
against arsenic induced pancreatic oxidative stress was calculated by considering the
difference between arsenic exposed group and control group as 100% restoration. For
determining the significant difference, One-Way analysis of variance was carried out and
the individual comparison of the group mean value was done using Dennett’s test
followed by least significant difference (LSD) test. P<0.05 was considered as significant.
Chpater-5
124
5.3 Results
5.3.1 Effect of QCT alone or in combination with zinc and BLE individually on
the body weight, blood glucose and glycosylated hemoglobin level
Five week of arsenic exposure did not produce visible clinical signs of toxicity in
the exposed animals. A significant (P<0.05) change on the steady gain in body mass of
rats in arsenic exposed group was recorded when compared to control during the entire
period of the experiment. Co-administration of quercetin alone or in combination with
zinc could able to significantly enhance the reduced gain in body mass in arsenic exposed
rats. Rats from the bamboo leaves extract (BLE) treated groups revealed significantly
(P<0.05) higher gain in mean body mass compared to arsenic exposed animals (Fig 5.1).
It was observed that treatment with BLE show better improvement in the gain in body
mass in arsenic exposed rats compared to QCT alone or QCT in combination with zinc.
Figure 5.1 Effect of quercetin alone or in combination with zinc as well as BLE
individually on body weight in arsenic exposed rats. Values are mean ± SD. of six rats;
*P<0.05 arsenic exposed (control) compared to normal animals;
†P<0.05 compared to
arsenic control. # P<0.05 compared to QCT alone treated group
Results showed that, compared to control, blood glucose and HbA1c level were
significantly increased by 44.89% and 56.81% (P<0.001) in arsenic exposed rats
respectively. Co-administration of QCT alone or in combination with zinc normalized the
arsenic induced elevated level of blood glucose. The blood glucose levels of the BLE-
Control As QCT QCT+Zn BLE
Body weight 210.8 190.6 204.2 209 219
0
50
100
150
200
250
Fin
al
Bod
y w
eigh
t (g
)
† † #
*†
Chpater-5
125
treated rats (83.07 ± 5.98 mg/dl) were significantly (P<0.05) lower than that of the rats of
arsenic exposed group (124.84 ± 8.05).
Figure 5.2 Preventive effects of QCT alone or in combination with zinc as well as BLE
individually on arsenic induced elevated blood glucose level. * Significant difference from
the normal control at P<0.05; †P < 0.05 compared to arsenic control.
Figure 5.3 Preventive effects of QCT alone or in combination with zinc as well as BLE
individually on arsenic induced elevated blood HbA1c level. * Significant difference from
the normal control at P<0.05; †P < 0.05 compared to arsenic control.
Fig 5.2 & 5.3 presented the effect of treatments on blood glucose and glycosylated
hemoglobin level on arsenic exposure respectively. Arsenic induced increased
glycosylated hemoglobin level was declined significantly in groups of rats co-
Control As QCTQCT+Z
nBLE
Blood glucose 86.164 124.84 91.1 90.92 83.07
0
20
40
60
80
100
120
140
Blo
od
glu
cose
lev
el (
mg/d
l)
*
† ††
Control As QCT QCT+Zn BLE
HbA1c 3.696 5.796 3.866 3.808 4.594
0
1
2
3
4
5
6
7
Hb
A1c
lev
el (
g%
)
HbA1c*
†
† †
Chpater-5
126
administered with QCT alone or in combination with zinc. Co-treatment with BLE could
also able to protect (P<0.05) the arsenic induce elevated level of glycosylated hemoglobin
as observed in combined administration of QCT and zinc. BLE treated arsenic exposed
rats exhibits more beneficial effect to reduced blood glucose and HbA1c but non-
significantly over the QCT and QCT + zinc treated group. All the three treatment had the
similar effect on the biomarker for diabetes mellitus.
5.3.2 Effect on pancreatic lipid peroxidation, protein oxidation and nitric oxide
level
Figure 5.4 Effect of simultaneous supplementation of QCT alone or in combination with
zinc or BLE individually on arsenic induced change in TBARS and AOPP level. *P<0.05
arsenic exposed compared to normal animals; †P<0.05 compared to arsenic control;
#
P<0.05 compared to QCT alone treated group
The protective effects of quercetin alone or in combination with zinc and BLE
separately on arsenic induced lipid peroxidation (TBARS) and protein oxidation (AOPP)
are presented in Fig 5.4. Fig 5.5 demonstrated the effect of treatments on PCO level in
arsenic exposed rats. Arsenic exposure led to a significant increase in lipid peroxidation
as evident from elevated (by 95.27%) level of TBARS in arsenic exposed animals
compared to control. Quercetin treatment in these animals could significantly reduce the
production of TBARS (P<0.05), but could not reach to the normal level. Quercetin along
with zinc supplementation could produce significantly much better recovery in lipid
peroxidation (P<0.05) then QTC alone and were found to be similar to that of control
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
TBARS AOPP
nm
ol/
mg o
f p
rote
in
Control As QCT QCT+Zn BLE
*
*
* † #
* †
† #
† # † #* †
Chpater-5
127
rats. Treatment with bamboo leaves extract (BLE) individually normalized the value of
TBARS as compared to control rats. It was noted that treatment with BLE reduced the
TBARS level significantly even compared to control.
Increased production of AOPP and PCO level was observed in pancreatic tissue of
arsenic exposed rats compared to control. The simultaneous administration of QCT alone
was effective in protecting arsenic induced elevated level of protein oxidation in this
tissue to some extent but, could not reach to normal level. Treatment with QCT in
combination with zinc was most effective in reducing arsenic induced protein oxidation.
Oral administration of BLE to arsenic exposed rats was effective in reducing the protein
oxidation. BLE was found to be more beneficial in reducing the protein carbonyl (PCO)
level than combined administration of QCT and zinc, but in case of AOPP level BLE and
QCT + zinc had similar effect.
Figure 5.5 Level of protein carbonyl (PCO) in pancreatic tissue of normal control,
arsenic control and experimental treated group. *P<0.05 arsenic exposed compared to
normal animals; †P<0.05 compared to arsenic control;
# P<0.05 compared to QCT alone
treated group; @
Significantly different from QCT + zinc (P<0.05)
Pancreatic nitric oxide (NOx) level was increased significantly (P<0.05) in arsenic
exposed animals by 72.25%. QCT treatment in these animals could significantly (P<0.05)
diminish production of nitrite by 60.59%, while QCT and zinc combined could produce
much better recovery by 104.85% than QCT alone. Treatment with BLE individually in
Control As QCT QCT+Zn BLE
PCO 1.7028 2.6594 2.0974 1.826 1.6152
0
0.5
1
1.5
2
2.5
3
PC
O (
nm
ol/
mg o
f p
rote
in)
PCO*
* †
† # @† #
Chpater-5
128
these animals had similar effect as observed in combined administration QCT + zinc and
protects the elevated nitric oxide level by 109.44% (Fig 5.6).
Figure 5.6 Effect of QCT alone or in combination with zinc or BLE individually on nitric
oxide level in arsenic exposed rats. Values are given as mean ± SD (n=6) Values are
statistically significant at *P<0.05 arsenic exposed compared to normal animals;
†P<0.05
compared to arsenic control; # P<0.05 compared to QCT alone treated group
5.3.3 Effect on thiol related antioxidative enzymatic system and reduced GSH level
Figure 5.7 Effect of co-administration of QCT alone or combined with zinc or BLE
individually on pancreatic GPx activity on arsenic exposure. Data are mean ± SD (n=6).
Values are statistically significant at *P<0.05 compared to normal animals;
†P<0.05
compared to arsenic control; # P<0.05 compared to QCT alone treated group
Control As QCT QCT+Zn BLE
Nox 3.02 5.202 3.88 2.914 2.814
0
1
2
3
4
5
6
NO
x (μ
mol/
mg o
f p
rote
in)
NOx*
* †
† # † #
Control As QCT QCT+Zn BLE
GPx 8.142 5.96 7.252 7.98 8.262
0
1
2
3
4
5
6
7
8
9
GP
x a
citi
vty
(U
/mg o
f p
rote
in)
*
* †† #
† #
Chpater-5
129
Figure 5.8 Effect of co-administration of QCT alone or combined with zinc or BLE
individually on pancreatic TrxR activity on arsenic exposure. Data are mean ± SD (n=6).
Values are statistically significant at *P<0.05 compared to normal animals;
†P<0.05
compared to arsenic control; #P<0.05 compared to QCT alone treated group;
@significantly different from QCT + zinc (P<0.05)
The effects of quercetin alone or in combination with zinc or BLE separately on
thiol relating enzymes GPx and TrxR activities are shown in Fig 5.7 & 5.8 after chronic
arsenic-exposure. Results indicated that, as compared to control, GPx activity was
decreased significantly (P<0.05) in arsenic exposed animals by 26.79%, while QCT
supplementation in these animals significantly (P<0.05) recovered the activity of GPx by
59.21%. However, QCT and zinc combined could produce further recovery by 92.58% in
GPx activity. Oral administration of BLE could also able to restore (by 105.5%) the
arsenic induced declined activity of GPx to near control levels and exhibits similar effect
as observed in QCT + zinc combined administration. Significantly decreased activities
(37.26%) of TrxR enzyme was observed in arsenic exposed rats. Treatment with QCT
alone could elevate the declined activity of TrxR on arsenic exposure without any
beneficial effect of zinc supplementation with QCT. Oral administration of BLE to
arsenic exposed rats restored TrxR activity by 108.98% to near control levels.
Fig 5.9 showed the effects of either quercetin alone or with zinc supplementation
and BLE separately on GSH level after chronic arsenic-exposure. Arsenic exposure
consistently reduced pancreatic GSH content by approximately 31.17% as compared to
Control As QCT QCT+Zn BLE
TrxR 0.3586 0.225 0.3152 0.3374 0.3706
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
0.45
Trx
R a
ctiv
ity
(U
/mg o
f p
rote
in)
*
†
†† #
Chpater-5
130
control animals. Treatment with QCT alone was significantly (P<0.05; by 67.5%)
restored the pancreatic GSH level and similar effect was observed after QCT with zinc
supplementation. Declined pancreatic GSH content was restored (by 117.14%)
completely and reaches to normal value in BLE treated arsenic exposed rats. BLE showed
the most beneficial effect to improve the GSH content than QCT alone or in combination
with zinc administration. The results indicated that BLE administration proved to be the
most effective to restored GSH and its related enzyme activity.
Figure 5.9 Effect of co-administration of QCT alone or combined with zinc or BLE
individually on pancreatic GSH content. Data are mean ± SD (n=6). Values are
statistically significant at *P<0.05 compared to normal animals;
†P<0.05 compared to
arsenic control; # P<0.05 compared to QCT alone treated group;
@ Significantly different
from QCT + zinc (P<0.05)
5.3.4 Effects on arsenic concentration in pancreatic tissue
Fig 5.10 shows the effects of QCT alone or with zinc supplementation and
administration of BLE individually on arsenic accumulation in pancreatic tissue in arsenic
exposed rats. Co-administration of QCT with arsenic significantly (P<0.05; by 35.18%)
prevented the accumulation of arsenic in studied tissue which was further reduced after
supplementation of zinc with QCT. BLE significantly prevented the accumulation of
arsenic in pancreatic tissue and had similar effect on arsenic accumulation as observed in
QCT + zinc combined administration.
Control As QCT QCT+Zn BLE
GSH 26.618 18.322 23.922 25.1 28.04
0
5
10
15
20
25
30
35
Red
uce
d G
SH
lev
el (
mg/1
00
g)
*
* ††
† # @
Chpater-5
131
Figure 5.10 Effect of simultaneous supplementation of QCT alone or in combination
with zinc and BLE individually on arsenic concentration in pancreatic tissue. Values are
mean ± SD of six rats; *P<0.05 arsenic exposed compared to normal animals;
†P<0.05
compared to arsenic control; #P<0.05 compared to QCT alone treated group
Table 5.1 Percentage changes in the body wt, glucose, HbA1c, TBARS, AOPP, PCO,
NOx, GSH level and activities of GPx & TrxR of experimental groups compared to
control
Groups/Parameters Arsenic (As) QCT QTC + Zinc BLE
Body weight -9.582* -3.131 -0.854 3.889
Plasma glucose 44.887* 5.729 5.519 -3.591
GHbA1c 56.812* 4.599 3.030 24.297
TBARS 95.267* 24.122* -10.941 -19.745
AOPP 76.561* 29.403* -2.737 0.491
PCO 56.251* 23.231* 7.286 -5.099
NOx 72.252* 28.477* -3.509 -6.821
GPx -26.799* -10.931* -1.989 1.474
TrxR -37.256* -12.103 -5.912 3.347
Reduced GSH -31.167* -10.129* -5.703 5.342
Values are statistically significant at *P<0.05 compared to control
Control As QCT QCT+Zn BLE
Arsenic 0.212 13.52 8.838 7.104 7.798
0
2
4
6
8
10
12
14
16
Ars
enic
(μ
g/g
of
tiss
ue)
*
* †
* † # * † #
Chpater-5
132
Table 5.2 Percentage change in the body weight, blood glucose, HbA1c, TBARS, AOPP,
PCO, NOx, GSH level and activities of GPx & TrxR of experimental groups compared to
arsenic exposed group
Groups/ Parameters QCT QTC + Zinc BLE
Body weight 7.135† 9.654
† 14.90
†
Plasma glucose -27.026† -27.171
† -33.458
†
GHbA1c -33.298† -34.299
† -20.738
†
TBARS -36.401† -54.368
† -58.879
†
AOPP -26.679† -44.891
† -43.062
†
PCO -21.121† -31.328
† -39.255
†
NOx -25.413† -43.983
† -45.905
†
GPx 21.678† 33.892
† 38.624
†
TrxR 40.088† 49.955
† 64.711
†
Reduced GSH 30.564† 36.993
† 53.040
†
Arsenic content -31.61† -47.48
† 42.382
†
†P<0.05 compared to arsenic control
Chpater-5
133
Table 5.3 Percentage restorations in the body weight, blood glucose, HbA1c, TBARS,
AOPP, PCO, NOx, GSH level and activities of GPx and TrxR and arsenic content on
different treatment groups
Groups/ Parameters QCT QTC + Zinc BLE
Body weight 67.32 91.08 140.59
Plasma glucose 87.24 87.70 107.99
GHbA1c 91.90 94.66 57.24
TBARS 74.68 111.45 120.72
AOPP 61.71 103.76 99.54
PCO 58.75 87.12 109.16
NOx 60.59 104.85 109.44
GPx 59.21 92.58 105.49
TrxR 67.51 84.13 108.98
Reduced GSH 67.50 81.70 117.14
Arsenic content 35.18 48.21 42.99
Discussion
134
5.4 Discussion
The finding of the present study has indicated that quercetin exerted protective
effect against arsenic induced diabetes mellitus and pancreatic oxidative stress. The
antioxidative effects were more pronounced when quercetin was administered along with
zinc supplement. This study also suggests that methanolic extract of bamboo leaves
(BLE) is effective in ameliorating pancreatic oxidative damage and also prevent the
hyperglycemic effect in arsenic induced experimental diabetes mellitus. In the present
case, we observed not only a moderate effect of quercetin and BLE in reducing pancreatic
oxidative stress but also showed a significant reduction in tissue arsenic burden. To our
knowledge, this is the first in vivo study exhibiting the potential of quercetin and bamboo
leaves extract in reducing arsenic induced pancreatic oxidative damage and also shows
antidiabetic properties.
Results from the present study have suggested that diabetogenic effect of arsenic
could be attributed to arsenic induced pancreatic oxidative stress. Previously, it was
reported that arsenic induced pancreatic oxidative stress play a very important role in the
development of arsenic induced diabetic mellitus (Izquierdo-Vega et al., 2006). In recent
years, studies have shown that the production of reactive oxygen and nitrogen species by
arsenicals are directly involved in oxidative damage to proteins, lipids, DNA and ability
to interact with thiol group of enzymes as well as proteins which can lead to cellular
toxicity and death (Flora et al., 2008). Arsenic exposure altered intracellular redox status
and inhibited glutathione related enzymes like GSH reductase, TrxR, GPx that eventually
has lead to cytotoxicity. Oxidative stress may, therefore, be one of the reasons for arsenic-
induced diabetes mellitus (Patel & Kalia, 2010b). Oxidative stress has recently been
shown to be responsible, at least in part, for pancreatic β-cell dysfunction on arsenic
exposure. Arsenic induced oxidative stress in pancreatic tissue which could lead to
progression of pancreatic β-cell dysfunction because of the relatively low expression of
antioxidant enzymes such as catalase and superoxide dismutase, as pancreatic β-cells may
be vulnerable to ROS attack when the system is under oxidative stress situation
(Kajimoto & Kaneto, 2004). Furthermore, evidence has suggested that hyperglycemia
aggravates the oxidative stress status by autoxidation of glucose and its primary and
secondary adducts (Dave & Kalia, 2007). Based on the above observations, it has
Discussion
135
apparent that uses of antioxidants have provided a possible and novel alternative
treatment for the arsenic toxicity.
Quercetin is reported to have many beneficial effects on human health, including
cardiovascular protection, anticancer activity, antiulcer effects, anti-allergic activity,
cataract prevention, antiviral activity and anti-inflammatory effects (Mi et al., 2007). Due
to the presence of aromatic hydroxyl groups, flavonoids have strong antioxidant
properties. Quercetin, the most abundant of the flavonoids consists of 3 rings and 5
hydroxyl groups. QCT is biologically available and accumulated in the pancreas (Zhang
et al., 2010b). In vivo, the antioxidant effect of quercetin is probably facilitated by its
ability to insert into lipid membranes, due to its planar structure (Van Dijk et al., 2000;
Ionescu et al., 2007). Quercetin is known to act as an antioxidant substance, protecting
the living cells against the damage induced by free radicals (Ortega et al., 2009). QCT has
been known to prevent oxidant injury and cell death by several mechanisms, such as
scavenging oxygen radicals, protecting against lipid peroxidation, and chelating metal
ions (Lakhanpal & Rai, 2007; Laughton et al., 1991).
Diabetogenic effect was observed in arsenic exposed rats as evident from elevated
blood glucose and HbA1c level in present study and is in agreement with previous finding
where increased blood glucose and insulin resistance was observed on arsenic exposure
(Patel & Kalia, 2010b). QCT alone or in combination with zinc administration was shown
to be capable of preventing hyperglycemia induced by arsenic and normalizing blood
glucose and HbA1c level in arsenic exposed rats showed significant anti-hyperglycaemic
activity of QCT in arsenic induced diabetic rats. It has been established that QCT exerting
its beneficial anti-diabetic effects (Vessal et al., 2003). QCT, a flavonoid was shown to
be capable of preventing STZ induced hyperglycemia and normalizing blood glucose
level and oxidative stress in diabetic rats (Adewole et al., 2007; Mahesh & Menon, 2004).
Hii and Howell reported that exposure of isolated rat islets to certain flavonoids such
epicatechin or QCT enhances insulin release by 44–70% (Hii & Howell, 1984). It was
demonstrated that quercetin treatment protected and preserved pancreatic β-cell
architecture and integrity (Adewole et al., 2007). Here, we use zinc as supplementary
agent along with QCT because our previous experiment showed declined zinc content in
pancreatic tissue on arsenic exposure, and supplementation of zinc might be beneficial
and fulfill the requirement for proper pancreatic function. In the present study, zinc
Discussion
136
supplementation in combination with quercetin did not shows any beneficial effect in
reducing blood glucose and HbA1c level over the quercetin alone might be due to
competitive effect on anti-hyperglycemic effect. It was know that zinc plays a key role in
the regulation of insulin production in pancreatic tissue. On the contrary, some authors
reported that zinc supplementation has potential beneficial effects on glucose homeostasis
in chronic diabetes (Chen et al., 1998; Brandao-Neto et al., 2003). The results of the
present study also revealed that oral administration of bamboo leaves extract (BLE)
decreased the blood glucose and HbA1c level in arsenic induced diabetic rats. BLE may
bring about its anti-hyperglycemic effect might be through boosting insulin secretion
from the remnant β-cells and from regenerating β-cells. Hypoglycemic plants act through
a variety of mechanisms such as improving insulin sensitivity, augmenting glucose-
dependent insulin secretion and stimulate the regeneration of islets of langerhans in
pancreas of STZ-induced diabetic rats (Sezik et al., 2005). It has been suggested that
flavonoids may provide effective treatments for type 2 diabetes mellitus and its associated
complication (Peluso, 2006). In the present study, we observed arsenic induced oxidative
stress accompanied by the accumulation of arsenic with impaired activity and depletion of
antioxidant status in pancreatic tissue of arsenic exposed rats. Interestingly, quercetin
alone or in combination with zinc or bamboo leaves extract individually restored the
oxidant status of pancreatic tissue and prevented the hyperglycemia induced by arsenic.
Oxidative stress was reflected in the pancreatic tissue as the depletion of
glutathione content accompanied by marked reduction in activities of GPx and TrxR and
significant elevation of lipid peroxides, protein oxidation and NOx level on arsenic
exposure may be associated with overproduction of ROS and RNS (Mukherjee et al.,
2006). The increased lipid and protein oxidation have an important role in pancreatic
damage associated with arsenic induced diabetes mellitus. However, recent findings have
suggested that induction of nitric oxide formation might play a role in the destruction of
β-cells during the development of diabetes mellitus (Corbbet et al; 1993). These results
were in agreement with previous findings whereby arsenic-treated rats showed marked
increase in pancreatic cells lipid peroxidation (Mukherjee et al., 2006). Concomitant
administration of QCT alone to arsenic exposed rats prevented the increased lipid
peroxidation and protein oxidation but could not restored to normal, whereas
administration of QCT in combination with zinc has brought the lipid peroxidation and
protein oxidation in pancreas to near control levels, which could be a result of improved
Discussion
137
antioxidant status. Quercetin prevented the arsenic induced pancreatic oxidative stress
and also improved the antioxidative status might be due to to its ability to scavenge free
radicals, as it was considered to be a strong antioxidant (Kostyuk & Potapovich, 1998).
The hepatoprotective effect of galactosylated liposome-encapsulated QCT against liver
fibrogenesis was reported on arsenic exposure (Lee et al., 2003; Mandal et al., 2007).
QCT has been also reported to prevent arsenic induced hepatic and kidney oxidative
stress (Mishra & Flora, 2008). Quercetin has the ability to stop the propagation of lipid
peroxidation, and increased glutathione (GSH) levels (Ansari et al., 2008). QCT
treatment decreased the elevated NOx level and restored the reduced antioxidant enzyme
activities (TrxR, GPx) in arsenic exposed animals. In the present study, QCT alone or in
combination with zinc tended to normalize TrxR activity and glutathione level. QCT
causes scavenging of free radicals longer react with nitric oxide, resulting in less damage.
The data revealed a marked protective effect of quercetin against arsenic-induced
elevation of total nitrate/nitrite level in pancreatic tissue. We observed not only a
moderate protective effect of quercetin in reducing oxidative stress but also significantly
reduced tissue arsenic burden.
Quercetin is considered to be a strong antioxidant due to its ability to scavenge
free radicals and bind transition metal ions. Quercetin also has the ability to boost the
endogenous antioxidant system. These properties of quercetin allow it to inhibit lipid
peroxidation (Sakanashi et al. 2008). Quercetin has antioxidant activities, inhibit protein
kinases, inhibit DNA topoisomerases and regulate gene expression (Moskaug et al.,
2004). It was observed that quercetin was found to be effective in preventing arsenic
poisoning by reducing hepatic and blood oxidative stress (Dwivedi & Flora, 2011). It is
documented form the earlier report that QCT could prevent hyperglycemia and prevent
the decrease activity of antioxidant enzymes in pancreas in diabetic animals (Abdelmoaty
et al., 2010). In the present study, the protective potential of quercetin against pancreatic
oxidative damages during arsenic exposure could be due to both of its metal chelating and
antioxidant properties (Moskaug et al., 2004). Reduction of oxidative stress was noticed
by the improvement of antioxidant status and a marked reduction of arsenic content in
pancreas by QCT. The antioxidant efficacy of QCT could be attributed to its higher
diffusion rate into the membranes (Moridani et al., 2003) allowing it to scavenge free
radicals at various sites; (ii) its pentahydroxyflavone structure, allowing it to chelate
metal ions; (iii) regeneration of endogenous and exogenous antioxidants like vitamin C
Discussion
138
and E and glutathione and (iv) presence of sulfhydryl group in the structure justified its
selection in this study against arsenic toxicity. The phenolic groups are also responsible of
the metal-chelating activity. Quercetin may sequester these metal ions by chelation
(Lakhanpal & Rai, 2007).
Interestingly, the results of the present study have shown the more beneficial
effect of zinc over pancreatic oxidative stress and removal of arsenic from tissue. These
studies suggest that zinc supplementation promotes arsenic elimination. Biologically, zinc
is an important enzymatic cofactor and has ability to induce the synthesis of detoxificant
proteins (Kondoh et al., 2003), such as metallothioneins or metal binding proteins which
is an effective scavenger of hydroxyl radicals (Sahin & Kucuk, 2003; Sreedhar et al.,
2004). Zinc has also been shown to have an important mechanism for the antioxidant
function. In addition, zinc protects sulphydryl group against oxidation thereby preventing
protein from oxidation, hence stabilizing the cellular thiol pools (Kraus et al., 1997) may
have been partly responsible for the better improvement in protein oxidation in group
supplement with zinc along with QCT compared to QCT alone. Therefore, the combined
antioxidant activities may have been responsible for the decrease in lipoperoxidative
changes and protective effect in arsenic induced pancreatic oxidative stress observed in
this study. Beneficial role of zinc supplementation during chelation therapy has been
reported on arsenic induced oxidative stress (Modi et al., 2005). In conclusion, the
present study has shown for the first time the ability of zinc to ameliorate the arsenic
induced pancreatic oxidative stress partly due to its antioxidant properties. Zinc may
therefore be useful as a protective agent against arsenic induced toxic damage. Another
interesting and significant observation in the present study was the QCT along with zinc
could significantly reduce the arsenic content from pancreatic tissue. This suggests that
antioxidative and metal chelating activity of QCT was more pronounced when it was
administered along with zinc metal.
The present study has also demonstrated anti-hyperglycemic effect of the
methanolic extract of bamboo leaves in arsenic induced diabetic rats and exhibited
preventive role against arsenic induced pancreatic oxidative damages. The administration
of BLE could significantly reduce the pancreatic lipid peroxidation product and protein
oxidation levels in arsenic exposed rats indicating BLE as potent inhibitor of oxidative
damage to pancreatic tissue. The bamboo leaves extract could restore the reduced GSH
Discussion
139
level and the GPx and TrxR activities in the pancreatic tissue of arsenic exposed rats.
Treatment with BLE also showed the significant protection from elevated nitric oxide
(NOx) level in pancreatic tissue on arsenic exposure indicating its nitric oxide radical
scavenging activity.
Preliminary studies conducted by us revealed the non-toxic nature of the bamboo
leaf extract and the presence of biologically active ingredients such as flavonoid,
phenolics, etc., which may be responsible for its biological activity (Machwan et al.,
2010). Flavonoids can exert their antioxidant activity by various mechanisms, e.g., by
scavenging or quenching free radicals, by chelating metal ions, or by inhibiting enzymatic
systems responsible for free radical generation (Dias et al. 2005). Phenolic compounds
and flavonoids from medicinal plant possess a high anti-oxidant potential due to their
hydroxyl groups and protect more efficiently against free radical-related diseases.
Bamboo leaves have recently been utilized as a source of flavonoids (e.g., vitexin and
orientin) used as antioxidants. It was demonstrated that bamboo oil increased the
superoxide radical, DPPH radical and nitrite scavenging activity in vitro. The glutathione
production and the activities of GPx and CAT were improved in liver on bamboo oil
administration shows antioxidative activity in vivo (Choi et al. 2008). The antioxidant of
bamboo leaves (AOB) a kind of poly-phenols-rich extract from bamboo leaves. It has
been certificated as a natural antioxidant by the Ministry of Health of the People’s
Republic of China in 2003, which has a warrant for use in edible oil, meat product,
aquatic product and puffed food as a novel food additive (Lu et al., 2006) Moreover,
AOB was testified as stronger antioxidant having inhibitory efficacy on transition metal
ion and free radical-induced deterioration of macromolecules in vitro (Hu et al. 2000).
There were no fetotoxic, embryotoxic, teratogenic effect observed for BLE and was safe
to use (Lu et al., 2006). BLE exhibited a concentration-dependent scavenging activity of
DPPH radical. Recently, it has been shown that bamboo extract exhibits antioxidant
activity against the DPPH radical and cytoprotective effects against oxidative damage in
HepG2 cells (Park et al., 2007). S. borealis, one type of bamboo species, could have
novel alternative medicinal uses as an antidiabetic agent. It was demonstrated
that SBwE
has an anti-apoptotic activity protecting endothelial dysfunction possibly caused by high
glucose-induced oxidative stress (Choi et al., 2008), might be associated with directly
scavenging hydroxyl radical, and with increasing the activities of antioxidative enzymes
in mice tissues (Hu et al., 2000). BLE greatly ameliorated antioxidant enzymes mainly,
Discussion
140
GPx and TrxR and prevented the rise in lipid peroxides, protein oxidation in the
pancreatic tissue. These findings might indicate an improvement in oxidant status and
suggested a possible antioxidant activity for BLE. Heijnen et al. (2001) have shown that
particular hydroxyl groups seem to be positively related to abilities of flavonoids to
scavenge peroxynitrite. Recently, protective effect of bamboo leaves flavonoids has been
reported on myocardial injury (Yuan et al., 2009). Fu et al. (2010) have found that
antioxidant of bamboo leaves was capable of blocking chain reactions of lipid auto
oxidation, chelating metal ions of transient state, scavenging nitrite compounds and
blocking the synthetic reaction of nitrosamine. Recently, Sood et al. (2011) have reported
the protective effect of bamboo leaves extract against lead induced oxidative stress in the
kidney and brain tissue.
Our data has suggested that methanolic extract of bamboo leaves acted as an
effective chelating agent in reducing the arsenic load, suppressing arsenic-induced
pancreatic oxidative stress and biochemical alterations and also exhibited anti-
hyperglycemic effect protecting animals from arsenic-induced diabetes mellitus and
pancreatic oxidative stress and in the depletion of arsenic concentration. In conclusion,
protective role of BLE against arsenic induced diabetes and pancreatic oxidative damage
is probably due to its chelating activity and its antioxidative property. The improvement
in pancreatic oxidative damage by BLE might be due to the presence of some biological
active ingredients and phenolic compounds.
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