PROPOLIS: THERAPEUTIC PERSPECTIVES AGAINST SILICA INDUCED TOXIC MANIFESTATIONS

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Chapter
PROPOLIS: THERAPEUTIC
PERSPECTIVES AGAINST SILICA
INDUCED TOXIC MANIFESTATIONS
Monika Bhadauria,1
∗
Satendra Kumar Nirala,1
Amita Jaswal,1 Suchita Raghuvanshi,1 Renu Bhatt,2
and Sangeeta Shukla1
1School of Studies in Zoology, Jiwaji University, Gwalior 474011, India
2Department of Psychology and neuroscience, Florida State
University, Tallahassee 32306, US6, US
ABSTRACT
Silica is one of the most documented workplace contaminants. Long-term
occupational exposure to silica is associated with an increased risk for respiratory
diseases such as silicosis, tuberculosis, chronic bronchitis, chronic obstructive pulmonary
disease and lung cancer. Protective role of propolis extract (200 mg/kg, po) was
evaluated against silica (SiO2; 40 mg/kg; ip for 6 weeks) induced subchronic toxic
manifestations in liver, kidney and lung and effectiveness was compared with positive
control silymarin. Toxicological consequences were evident by decreased hemoglobin
and enhanced level of serum transaminases, alkaline phosphatase, angiotensin converting
enzyme, protein, cholesterol, creatinine and blood sugar after exposure to silica. Tissue
biochemistry revealed raise in acid phosphatase, whereas fall in alkaline phosphatase,
adenosine triphosphatase, total protein and glycogen content in liver, kidney and lung
after silica toxicity. Hepatic glucose-6-phosphatase was found to be decreased after silica
administration. Significant raise in lipid peroxidation and decreased level of reduced
glutathione, super oxide dismutase and catalase showed oxidative stress consequences in
these organs after silica exposure. Propolis extract showed therapeutic potential and
 Corresponding author: Monika Bhadauria. Email: bhadauria_monika@rediffmail.com;
monikabhadauria@yahoo.com.

Monika Bhadauria, Satendra Kumar Nirala, Amita Jaswal et al.
reverses silica induced oxidative stress and biochemical alterations in liver, kidney and
lungs. Silica intoxication inhibited the activity of aniline hydroxylase of CYP2E1
enzymes, and enhanced peroxidative damage in hepatic microsomal fraction. Conjoint
treatment of propolis protected alteration in aforesaid biochemical variables significantly
and preferred histological features of liver, kidney and lung towards control. In this
chapter, we advocate the therapeutic perspective of propolis against silica induced toxic
manifestations.
Keywords: Propolis, Silica toxicity; Liver, Kidney and Lung
INTRODUCTION
Silica is found in the earth’s crust in percentage of 27.70% with an enormous diversity of
minerals. Chronic inhalation of crystalline or free silica by workers, especially sandblasters,
miners, tunnellers, silica millers, abrasives and flour workers, ceramic workers, glassmakers,
quarry and foundry workers leads to a pulmonary fibrosis called silicosis [1]. It is a
progressive, chronic, nodular, fibrosing disease of the lung caused by prolonged exposure to
silica, which also causes respiratory failure due to fibrotic reaction [2, 3]. Exposure is
associated with many other different disorders besides pulmonary silicosis, such as
progressive systemic sclerosis, emphysema, rheumatoid arthritis, dermatomyositis,
glomerulonephritis and vasculitis [4, 5]. Exposure to silica also induces hepatic and renal
injury because of their vital role in synthesis, metabolism and excretion. It was found that
lactate dehydrogenase, β-glucuronidase, N-acetylglucosaminase and total protein levels are
increased in animals instilled with silica fluid in a dose-related manner [6]. There are about 3
million workers at high potential risk of silica exposure [7]. The crushing of silica generates
the free radicals Si• and SiO• at cleavage zones that may react with water to yield OH [8].
Smokers are at an increased risk as smoking and silica act synergistically causing chronic
obstructive disease in the lungs [9-11]. LD50 of silica through intraperitoneal route is 400
mg/kg of body weight [12] and supposed to be probable prooxidant due to its free radical
property. Free radicals are damaging in nature and use of natural antioxidants is thought to be
the alternative to combat oxidative stress [13]. Involvement of flavonoids in protection
against silica induced cell injury is well reported [14].
Propolis is a natural product derived from plant resins collected by honeybees. It is used
in folk medicine all over the world. Many medicinal properties, including bacteriostatic,
antimycotic, anti-inflammatory, antiprotozoan, antiviral, spasmolytic, astringent, immuno-
stimulatory and free radical scavenging [15-21] have been ascribed to propolis. It contains a
number of chemical constituents, such as polyphenols (flavonoids, phenolic acids and their
esters, phenolic aldehydes, alcohols and ketones), coumarins, steroids, amino acids and
inorganic compounds but the composition differs greatly due to variation in its geographical
and botanical origin. The biological activities of propolis may be due to the presence of a
large number of flavonoids [22]. Propolis is relatively safer with no observed effect level
(NOEL) of 1400 mg/kg body wt/ day in mouse [23]. No untoward effects or mortality was
noted in mice at doses up to 2 g/kg, po [24]. Thus, looking on to a variety of biological
properties of propolis, we made en extensive literature survey and scientific experimentation
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Propolis: Therapeutic Perspectives …
to advocate the protective value of propolis against silica induced toxicity in liver, kidney and
lung.
SCIENTIFIC APPROACH TO ASSESS PROTECTIVE
POTENTIAL OF PROPOLIS
Female albino rats of body weight 150±10 g were selected from the departmental animal
house where standard husbandry conditions (25°±2°C temp, 60-70% relative humidity and 12
h photoperiod) were made available to maintain them and allowed to food and water ad
libitum. Experiments were conducted in accordance with the guidelines set by the Committee
for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), India.
Suspension of silicon dioxide (SiO2) was prepared in triple distilled water at doses of 40
mg/kg body weight and was administered intraperitoneally. A series of extraction was
performed to yield ethanolic extract of propolis (62.8% w/w) and kept at 4°C until use.
Aqueous suspension of propolis (200 mg/kg, p.o.) and silymarin (50 mg/kg, p.o.) were
prepared in gum acacia [25] and silymarin was used as positive control. Equal amount of 1%
gum acacia suspension (GAS, 5 ml/kg) was administered as vehicles to control animals
Twenty four animals were assigned into four groups of six animals each. Group 1 was
normal control and received normal saline and GAS; groups 2-4 were administered SiO2 at
the dose of 40 mg/kg, ip for continue six weeks. Group 2 was treated as experimental control
and received GAS conjointly after three weeks of SiO2 administration. Group 3 received
propolis extract (200 mg/kg, po) and group 4 received silymarin (50 mg/kg, po) conjointly for
last three weeks. On day 1 of 7th week blood was collected from the animals by puncturing
retro-orbital venous sinus and animals of the entire group were euthanized. Experimental
design is given in table 1.
Blood was used for determination of blood sugar [26] and hemoglobin [27]. Blood
samples were centrifuged and obtained serum was used to determine transaminases (AST and
ALT) [28], serum alkaline phosphatase (SALP) [29], serum and tissue protein [30], serum
cholesterol [31] and angiotensine converting enzyme (ACE) [Kit method]. Immediately after
necropsy, liver, kidney and lung samples were excised, blotted free of adhering fluid and kept
at –20°C for tissue biochemistry. Homogenates of liver, kidney and lung tissues were
prepared in chilled hypotonic solution to determine alkaline phosphatase (ALPase) and acid
phosphatase (ACPase) [29]; adenosine triphosphatase (ATPase) [32] and glucose-6-
phosphatase [33]. Fresh tissues were used to determine glycogen [34]. Homogenates were
prepared in 0.15% KCl and 1% sucrose solution for determination of lipid peroxidation
(LPO) [35] and reduced glutathione (GSH) [36] respectively. Superoxide dismutase (SOD)
[37] and catalase (CAT) [38] were determined in liver, kidney and lung tissues. Hepatic
microsomes were prepared [39] to determine peroxidative damage, total proteins and activity
of drug metabolizing enzyme, aniline hydroxylase [40]. For histopathological observations,
small pieces of the tissues of liver, kidney and lung were fixed in Bouin’s fixative and
hematoxylin-eosin stained slides were observed [41]. All the results are expressed as mean ±
SEM. Comparison between two independent groups was made by the analysis of variance
(ANOVA) followed by students ‘t’ test and P<0.05 was considered to be statistically
significant. The data were analyzed manually with the help of personal computer.
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Monika Bhadauria, Satendra Kumar Nirala, Amita Jaswal et al.
Table 1. Experimental schedule
Treatments 1-3 weeks 4-6 weeks 1st day of 7th week
Group 1: Control Vehicle (ip) Vehicle (ip, po) Euthanasia
Group2:Exp Control SiO2
(40 mg/kg, ip, daily)
SiO2 (40 mg/kg, ip,
daily)
+ Vehicle (po)
Euthanasia
Group 3: Therapy SiO2
(40 mg/kg, ip, daily)
SiO2 (40 mg/kg, ip,
daily)
+ Propolis extract
(200 mg/kg, p.o.)
Euthanasia
Group 4: Therapy SiO2
(40 mg/kg, ip, daily)
SiO2 (40 mg/kg, ip,
daily)
+ Silymarin
(50 mg/kg, p.o.)
Euthanasia
ANALYSES OF BLOOD AND TISSUE BIOCHEMICAL VARIABLES
Alterations in different blood and tissue biochemical variables were observed after
intraperitoneal administration of silicon dioxide (Table 2). Significant enhance (P≤0.05) was
noticed in the release of serum transaminases and serum alkaline phosphatase. Similarly,
significantly increased level of blood sugar, serum cholesterol, serum creatinine, serum
proteins and serum ACE were observed after six weeks of silica intoxication (P≤0.05).
Hemoglobin content was found to be reduced significantly after silica toxicity (P≤0.05).
Treatment with propolis extract protected these diagnostic biochemical variables and rendered
them more towards control in a significant manner. Effectiveness of propolis could be well
compared with positive control silymarin showing its excellent therapeutic value against
silica toxicity.
Significant fall in the activity of ATPase in liver, kidney and lung as well as in hepatic G-
6-Pase was observed after silica induced toxicity (P≤0.05). Propolis extract recovered the
activity of ATPase in liver, kidney and lung and revived hepatic G-6-Pase significantly
towards control (P≤0.05; figure 2). Glycogen and protein contents in liver, kidney and lung
tissues were found to be decreased significantly after six weeks of silica exposure (P≤0.05;
figure 3). Treatment with propolis as well as silymarin was found almost equally effective in
bringing the status of glycogen and protein towards control in all the three organs.
4

Table 2. Effect of propolis extract on blood biochemistry against silica induced alterations
Treatments AST ALT ALP ACE Blood
sugar
Cholesterol Creatinine S Protein Hemoglobin
IU/l IU/l mg
Pi/100ml/h
IU/l mg/100 ml mg/dl mg/dl mg/100ml g%
Control 69.2±3.82 46.9±2.59 213±11.7 49.1±2.71 108±5.97 54.3 ±
3.00
0.34±0.018A42.6±2.35 15.8±0.87
SiO2116±6.41*101±5.58*1702±94.1*184±10.2*138±7.62*94.0±5.19*0.59±0.032*54.8±3.02*11.0±0.61A
SiO2 +
Propolis
89.3±4.9486.1±4.75898±49.6116±6.41121±6.68 57.0±3.15

0.45±0.02449.0±2.71 15.4±0.85B
SiO2 +
Silymarin
87.1±4.8156.6±3.12970±53.6124±6.85118±6.52 62.0±3.42

0.43±0.02352.5±2.90 14.8±0.82B
F Variance 17.3¤43.3¤124¤73.7¤4.12¤28.2¤19.7¤4.45¤9.23*
Significant analysis of variance at P≤0.05¤; *Control vs Silica; Silica vs propolis/ silymarin for Students‘t’ test (P≤0.05).

Monika Bhadauria, Satendra Kumar Nirala, Amita Jaswal et al.
ACPase activity was increased and ALPase was decreased significantly in liver, kidney
and lung after silica intoxication (P≤0.05; figure 1). Propolis extract decreased ACPase and
increased ALPase significantly in these organs (P≤0.05).
a) b)
c) d)
e) f)
*Control vs Silica; Silica vs propolis/ silymarin for Students‘t’ test (P≤0.05).
Significant analysis of variance at P≤0.05¤; Abbreviations: Pro (Propolis), Sily (Silymarin)ACPase:
27.1(Hepatic)¤, 7.94(Kidney)¤, 29.3(Lung)¤ ALPase: 22.6(Hepatic)¤, 20.1(Kidney)¤, 82.2(Lung)¤.
Figure 1. (A-F): Effect of silica and propolis on hepatic, renal and lung ACPase and ALPase activity.
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Propolis: Therapeutic Perspectives …
a) b)
c) d)
*Control vs Silica; Silica vs propolis/ silymarin for Students‘t’ test (P≤0.05).
Significant analysis of variance at P≤0.05¤; Abbreviations: Pro (Propolis), Sily (Silymarin).
ATPase: 23.1 (Hepatic)¤, 13.7 (Kidney)¤, 16.9 (Lung)¤ G-6-Pase: 2.95 (Hepatic).
Figure 2. (A-D): Effect of silica and propolis on ATPase and G-6-Pase activity.
OXIDATIVE STRESS AND CELLULAR ANTIOXIDANTS POOL
Figure (4) shows oxidative stress consequences in liver, kidney and lung after silica
intoxication. Significant rise in LPO and decrease in GSH level indicated marked oxidative
stress in these organs (P≤0.05). Propolis extract suppressed the oxidative stress consequences
by decreasing LPO and increasing GSH level in a significant manner as evident by ANOVA
(P≤0.05). The efficacy of propolis was found to be almost equal to positive control
silymarine. Exposure to silica significantly reduced the activity of enzymatic antioxidants, the
SOD and CAT in liver, kidney and lung (P≤0.05; figure 5). Treatment of propolis extract and
silymarin were equally effective in reviving the cellular enzymatic antioxidant pool. Thus,
SOD and CAT were found towards control in these organs in a significant manner by both the
treatments.
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Monika Bhadauria, Satendra Kumar Nirala, Amita Jaswal et al.
a) b)
c) d)
e) f)
*Control vs Silica; Silica vs propolis/ silymarin for Students‘t’ test (P≤0.05).
Significant analysis of variance at P≤0.05¤; Abbreviations: Pro (Propolis), Sily (Silymarin).
Glycogen: 66.1(Hepatic)¤, 7.82 (Kidney)¤, 66.3 (Lung)¤ Protein: 16.4(Hepatic)¤, 9.97(Kidney)¤,
42.3(Lung)¤.
Figure 3. (A-F): Effect of silica and propolis on glycogen and protein contents.
MICROSOMAL FRACTION AND DRUG
METABOLIZING ENZYME (CYP2E1)
Exposure to silica markedly affected hepatic microsomal fraction. Significant raise in
LPO and decrease in protein was observed after silica toxicity (P≤0.05; figure 6). Silica
instillation significantly reduced the drug metabolizing enzyme, the aniline hydroxylase of
CYP2E1 cycle. Conjoint treatment of propolis for three weeks significantly inhibited
microsomal LPO and revived AH activity and protein content in microsomal fraction
(P≤0.05). Propolis extract was found almost equally effective as silymarin positive control.
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Propolis: Therapeutic Perspectives …
VISUALIZATION OF RECOVERY PATTERN BY MICROSCOPIC
STUDIES
The histopathological observations of liver, kidney and lung also supported biochemical
data confirming the effectiveness of propolis extract. Figure 7A showed hepatic histological
features of control. Silica exposure caused loss of cord arrangement of hepatocytes and
sinusoidal spaces with degenerated plasma membranes and nuclei (Figure 7B).
a) b)
c) d)
e) f)
*Control vs Silica; Silica vs propolis/ silymarin for Students‘t’ test (P≤0.05).
Significant analysis of variance at P≤0.05¤; Abbreviations: Pro (Propolis), Sily (Silymarin).
LPO: 105(Hepatic), 101(Kidney)¤, 153(Lung)¤ GSH: 6.04(Hepatic)¤, 3.06(Kidney), 11.3(Lung)¤.
Figure 4. (A-F): Effect of silica and propolis on hepatic, renal and lung lipid peroxidation and
glutathione.
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Monika Bhadauria, Satendra Kumar Nirala, Amita Jaswal et al.
a) b)
c) d)
e) f)
*Control vs Silica; Silica vs propolis/ silymarin for Students‘t’ test (P≤0.05).
Significant analysis of variance at P≤0.05¤; Abbreviations: Pro (Propolis), Sily (Silymarin).
SOD: 20.9(Hepatic)¤, 29.2(Kidney)¤, 16.4(Lung)¤ CAT: 14.0(Hepatic)¤, 4.39(Kidney)¤, 8.63(Lung)¤.
Figure 5. (A-F): Effect of silica and propolis on hepatic, renal and lung SOD and CAT activity.
Conjoint treatment of propolis depicted better formed central vein with cordially arranged
hepatocytes with appearance of polygonal shape, nuclei and sinusoidal spaces around the CV
(Figure 7C). Figure 7D showed renal histological features of control. Silica exposure caused
swelling and dilatation in glomeruli of kidney (Figure 7E). Treatment of propolis protected
alterations in renal histoarchitecture showing well formed glomeruli in Bowman’s capsule
(Figure 7F). Figure 7G showed histological features of control lung. Section of silica exposed
lung showed degenerative changes in lungs (Figure 7H). Therapy of propolis showed well
preserved lung histoarchitecture (Figure 7I).
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Propolis: Therapeutic Perspectives …
a) b)
c)
*Control vs Silica; Silica vs propolis/ silymarin for Students‘t’ test (P≤0.05).
Significant analysis of variance at P≤0.05¤; Abbreviations: Pro (Propolis), Sily (Silymarine).
CYP2E1 activity: 25.2¤, Microsomal LPO: 35.5¤, Microsomal protein: 12.5¤.
Figure 6. (A-C): Effect of silica and propolis on microsomal fration.
THERAPEUTIC PERSPECTIVE OF PROPOLIS
The deposition of silica particles in the lungs of human and experimental animals leads to
silicosis, an industrial era disease. The relationship between crystalline silica and silicosis, a
non-malignant fibrosis of the lung, has been known for decades [42]. This chapter advocates
protective potential of propolis extract silica induced biochemical and histopathological
alterations in liver, kidney and lung. Considerable increase in transaminases and alkaline
phosphatase in serum indicated hepatobiliary dysfunction due to exposure to silica that might
be due to increased activity of phagocytosis and necrosis of liver. Propolis extract decreased
elevated transaminases probably by protecting cellular membranes from silica induced
oxidative degeneration. It is well known antioxidant and can break oxidative chain reactions
occurring in phospholipids of cell membrane and cytosol [43], which ultimately maintained
the integrity of plasma membrane and checked the leakage of transaminases and SALP.
Significant raise in the level of blood sugar, serum protein, serum cholesterol and serum
creatinine was found after subchronic exposure to silica. These elevations in blood sugar and
serum cholesterol, and decrease in glycogen content might be due to dysfunctional changes in
carbohydrate and lipid metabolism in liver.
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Monika Bhadauria, Satendra Kumar Nirala, Amita Jaswal et al.
Figure 7. Photomicrograph of liver of control (A), Silica exposed liver (B), liver with propolis treatment
(C); kidney of control (D), Silica exposed kidney (E), kidney with propolis treatment (F); lung of
control (G), Silica exposed lung (H), lung with propolis treatment (I).
Similarly, increase in serum protein might be due to increased level of leaked out
enzymes from different organs as well as due to cellular destruction. Propolis might reduced
silica induced oxidative burden [44] and improved the metabolic process, which ultimately
reduced hyperglycemia, serum protein and cholesterol. It helped in checking leakage of
different enzymes into circulation by maintaining membrane integrity [45] thus, reduced
serum protein level. Propolis might protect RBCs degradation or modulate hemopoetic
system so that hemoglobin tends towards control [46]. It is established that creatinine, a
catabolic product of creatine can also be secreted via the renal tubules in addition to
glomerular filtration [47,48] thus, regarded as a kidney function test. Severe damage in
kidney due to silica toxicity caused less excretion of creatinine, thus, increased its level in
serum, which is in agreement with other findings [49]. Propolis helped to cope with
alterations in cellular structure of kidney and thus, effective in renal protection. ACE is
secreted by pulmonary and renal endothelial cells [50]. It catalyses the conversion of
angiotensin I to angiotensin II, a potent vasoconstrictor in a substrate concentration dependent
manner [51]. ACE degrades bradykinin, a potent vasodilator, and other vasoactive peptides
[52]. These two actions make ACE inhibition a goal in the treatment of conditions such as
high blood pressure, heart failure, diabetic nephropathy, and type 2 diabetes mellitus. Propolis
therapy reduced ACE level in serum indicating improvement in lung tissues.
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Propolis: Therapeutic Perspectives …
Enhanced LPO in different organs after exposure to silica can be suggested as its toxic
consequences, which is in agreement with other reports [17, 53]. Silica induced oxidative
stress could be due to silica free radicals and subsequent generation of reactive oxygen
species (ROS) upon reaction with oxygen [54-57]. Exposure to silica has been demonstrated
to cause enhanced oxidative DNA damage and formation of silica induced ROS in the lungs
[58-59]. The ROS have been implicated in pulmonary diseases caused by crystalline silica
and many other occupational and environmental pollutants [54, 60]. Aggravated oxidative
stress is associated with pathogenesis of silica associated hepatic, renal and pulmonary
disease. Silica is also reported to be nephrotoxic as the exposure manifests toxic effects on
kidneys. Both the therapy groups were able to suppress LPO. Propolis extract reduced LPO
because flavonoids like quercetin, kaempferol and myericetin, naringenine etc. [61] which are
generally present in the extract have been reported to scavange free radicals like OH and
superoxide and inhibit LPO [22-24, 62]. GSH is involved in maintaining intracellular thiol-
disulfide ratio that is vital for the functioning of a variety of enzymes. One of its most
important function is to provide protection against oxidative damage caused by ROS through
enzymatic and non- enzymatic reactions [63-64]. Fall in GSH level during sub chronic
exposure to silica indicated excessive use of GSH in combating silica induced ROS.
Similarly, reduction in enzymatic antioxidants, SOD and CAT after silica exposure indicated
overwhelming production of free radicals due to silica toxicity. Administration of propolis
extract could restore the GSH as well as revive SOD and CAT level in different organs
because propolis might be well absorbed in these tissues [65] and could suppress silica
induced ROS and maintained cellular antioxidants pool.
The use of ATPase determination was considered as an appropriate index of cytotoxicity
[66-67]. Concurrent fall in the ATPase pattern in liver, kidney and lung is also in agreement
with other reports [68-69]. Treatment with propolis extract significantly recovered the activity
of ATPase. Liver is rich in ATPase and supplies energy for the differential permeability of the
cell and mediates membrane transport. Decreased activity of ATPase clearly indicated
hepatic, alveolar and nephrotoxic property of silica, which caused dysfunction in these
organs. Propolis extract maintained ATPase activity in liver, kidney and lung towards control.
Modulation of hepatic G-6-Pase by propolis treatment also indicated its protective effect on
carbohydrate metabolism and cellular integrity. The cytoplasmic organelle, lysosomes,
contain acid and responsible for the several metabolic processes. Significantly increased
activity of ACPase was observed in liver, kidney and lungs after silica exposure, which might
be due to damage in the lysosomes. During silicosis, liberated ACPase enzyme also caused
injury to the surrounding tissues and thus, increased activity of ACPase was found. Propolis
extract decreased the activity of ACPase considerably in liver, kidney and lung probably due
to its antioxidant effects. It is widely accepted that silica-induced cytotoxicity is due, in part,
to the disruption of phagolysosomal membrane integrity. After phagocytosis of silica, reactive
particle surfaces may interact with phagolysosomal membranes leading to the release of
lysosomal enzymes into the cytosol and cell death [70-71]. Lysosomes contribute to caspase
activation and apoptosis in an in vitro model of silica-induced apoptosis. Silica particles were
shown to induce lysosomal permeability and apoptosis in a response that required an acidic
lysosomal environment, lysosomal cathepsin D activity and lysosomal acidic
sphingomyelinase activity [72]. This might be the possible cause of reduction of ALPase in
tissues after silica exposure. Treatment with propolis might help in improvement of
membrane integrity and thus, maintained lysosomal enzymes. The histopathological
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Monika Bhadauria, Satendra Kumar Nirala, Amita Jaswal et al.
observations of liver, kidney and lung after silica exposure followed by propolis extract
treatment convinced the biochemical data. Thus, we may draw a conclusion that propolis
extract has an excellent potential in treatment of silica induced damage in liver, kidney and
lung.
ACKNOWLEDGEMENTS
Correspondent author is thankful to the Council of Scientific and Industrial Research
(CSIR), India for financial assistancec provided her through Senior Research Associateship
(13 (8271)/2008-09/Pool).
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