potential therapeutic effect of quercetin, zinc and ble on...

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)

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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

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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

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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.

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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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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

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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

)

† † #

*†

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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)

*

† ††


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*

†

† †

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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


*

*

* † #

* †

† #

† # † #* †

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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


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*

* †

† # @† #

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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


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*

* †

† # † #


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)

*

* †† #

† #

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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


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)

*

†

†† #

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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.


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.


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)

*

* ††

† # @

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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


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)

*

* †

* † # * † #

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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

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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.

potential therapeutic effect of quercetin, zinc and ble on...

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