Article wjpps 1454479295 (2)

www.wjpps.com Vol 5, Issue 2, 2016. 1165
Gowda et al. World Journal of Pharmacy and Pharmaceutical Sciences
STUDY OF ANTIOXIDANT ACTIVITY OF CEE'RICH - HERBAL
VITAMIN C SUPPLEMENT IN EXPERIMENTAL ANIMALS.
Vijayalaxmi Hiremath1
, Supriya CS1
, Khader Shareef KS2
, Nataraj Loganayaki2
,
Shanaz Tejani-Butt3
, Shivalinge Gowda KP1*
,K. Venkateswarlu2.
1
Department of Pharmacology, PES College of Pharmacy, Bengaluru, Karnataka, India.
2
SUGUNA Herbal Division, Suguna Foods Pvt Ltd, Coimbatore, Tamil Nadu.
3
University of the Sciences in Philadelphia, Philadelphia, PA, USA.
ABSTRACT
The antioxidant activity of Cee’Rich Vitamin C supplement was
investigated using carbon tetrachloride (CCl4, 2ml/kg s.c route)
intoxicated albino Wistar rat liver. The Cee’Rich Vitamin C
supplement was administered orally at a dose of 200 and 400mg/kg,
for 10 consecutive days. The doses were determined from the acute
oral toxicity study in albino mice. The CCl4 was administered to all the
groups except group I (normal control). Silymarin (100 mg/kg) was
used as a standard hepatoprotective drug. After 24h of the last
treatment, blood was withdrawn by retro orbital puncture and serum
SGPT, SGOT and LDH were determined. The SOD was calculated
from the liver homogenate. The results indicated that serum SGPT,
SGOT and LDH levels were significantly decreased where as SOD levels were significantly
increased in Cee’Rich Vitamin C treated rats. This study suggests that the presence of
flavonoids in the Cee’Rich Vitamin C supplement exhibit significant hepato protective effect
and could protect tissues from oxidative stress via a free radical- scavenging and antioxidant
mechanism.
KEYWORDS: Cee’Rich Vitamin C supplement, Carbon tetrachloride, Superoxide
dismutase, Lactate dehydrogenase, Antioxidant enzymes, Silymarin and Flavonoids.
INTRODUCTION
Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are produced through
oxidative processes within the mammalian body. These radicals play an important role in
WORLD JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES
SJIF Impact Factor 5.210
Volume 5, Issue 2, 1165-1176 Research Article ISSN 2278 – 4357
Article Received on
12 Dec 2015,
Revised on 02 Jan 2016,
Accepted on 26 Jan 2016
*Correspondence for
Author
Dr. Shivalinge Gowda KP
Department of
Pharmacology, PES
College of Pharmacy,
Bengaluru, Karnataka,
India.

oxidative stress related pathogenesis of various diseases. The human body possesses many
defense mechanisms against oxidative stress, including antioxidant enzymes and non-
enzymatic compounds. For proper maintenance of tissue structure and function, free radicals
and antioxidants should maintain equilibrium.
Exposure to environmental pollutants such as smoke, pesticides and ultra violet radiations
can cause,-the natural antioxidant mammalian mechanism to become insufficient, resulting in
excessive generation of free radicals. These free radicals in turn produce cellular distress and
damage to the structure and function of cell membranes leading to degenerative disorders like
Alzheimer's diseases, cataracts, liver injury, cardiovascular diseases, neoplasm disorders,
atherosclerosis, nephritis, diabetes, rheumatoid arthritis and inflammation.[1,2]
Antioxidants mediate their protective effects by directly reacting with free radicals,
quenching them and preventing damage to cellular components-consequently hindering
disease.[1,2]
Recently, interest has increased considerably in finding naturally occurring
antioxidants to replace synthetic antioxidants, which are restrictive due to their
carcinogenicity.[3]
The antioxidant phyto chemicals such as polyphenols, flavonoids and
related compounds found in medicinal plants have received increasing attention for their
potential role in the prevention of human diseases.[4]
As a result, ethno medicinal plants have
been investigated and reported to have antioxidant and radical scavenging potential.[5,6]
MATERIALS AND METHODS
Cee’Rich Vitamin C supplement: The herbal sample of Cee’Rich Vitamin C supplement
was provided by the sponsor, Suguna Foods and products, Coimbatore, Tamil Nadu.
Drugs and Chemicals: CCl4 was obtained from Loba laboratory reagents; phenazine
methosulphate was obtained from Loba laboratory reagents for the estimation of SOD; liquid
paraffin and nitro blue tetrazolium chloride was obtained from S D Fine & chemo Limited
and served as a vehicle for treatment with CCl4; Kits for Lactate dehydrogenase (LDH),
serum glutamic oxaloacetic transaminase (SGOT) & serum glutamic pyruvic transaminase
(SGPT) measurements were obtained from Anjan distributors, Bengaluru, authorized dealer
for Erba diagnostics.
CCl4: It has been established that CCl4 is accumulated in hepatic parenchyma cells and
metabolically activated by cytochrome P450-dependent monooxygenases to form a tri-

chloromethyl radical (CCl3). The CCl3 radical alkylates cellular proteins and other
macromolecules with a simultaneous attack on polyunsaturated fatty acids in the presence of
oxygen to produce lipid peroxides and other bio-markers leading to liver damage.
Phytochemical Screening
Cee’Rich Vitamin C supplement was dissolved in water and screened for different classes of
phytoconstituents, according to the methods described by Khandelwal.[7]
Table 1 shows the
results of phytochemical screening of Cee’Rich Vitamin C supplement.
Test Animals
Male Albino Wistar rats and female Albino mice were used in the antioxidant study. The
animals were procured from M/S Raghavendra Enterprises, Bengaluru, The animals were
kept in air conditioned animal house and they were exposed to 12h day and 12h night cycle
with a temperature between 25±o
C. The animals were housed in spacious, hygienic,
polypropylene cages with paddy husk as bedding during the experiment period. The animals
were fed with water and rat pellet feed (M/s Krish Scientific Co, Bengaluru) ad libitum. All
the experimental procedures and protocols used in this study were reviewed by Institutional
Animal Ethics Committee ( PESCP/IAEC/16/2015 dated 16/6/2015) and were in accordance
with the guidelines of the CPCSEA (PES College of Pharmacy, CPCSEA registration No:
600/PO/Ere/S/02/CPCSEA).
Acute Oral toxicity study
Female Albino mice weighing between 20-25 g were used for acute toxicity studies (6) and
divided into four groups consisting of 5 animals in each group. The animals were fasted
overnight prior to acute experimental procedures. Acute oral toxicity studies were performed
for Cee’Rich Vitamin C supplement according to the toxic class method 425 as per OECD
guidelines.[8]
Doses of 300, 1000, 2000 and 3000mg/kg body weight were given to the 4
groups. Individual observations were made once during the first 30 minutes and periodically
during the first 24hours with special attention given during the first 4 hours and daily
thereafter for a total of 14 days Additional observations such as changes in skin and fur, eyes
and mucous membranes, respiration, circulation, autonomic, CNS, somatomotor activity and
behavior patterns were also observed.[9]

Experimental Design for Antioxidant activity of Cee’Rich Vitamin C supplement on
CCl4 induced liver toxicity in rats.
The test animals were randomly divided into five - groups (I-V) of six rats for each treatment
group. Group I served as a control and received water (oral route) for 10 consecutive days
and liquid paraffin ( sc) in a dose of 2 ml/kg on the 2nd
, 3rd
, 5th
and 7th
day. Group II was
given water (oral route) for 10 days and CCl4 in liquid paraffin (1:2 v/v), 2 ml/kg bw (sc
route to induce liver toxicity in animals) on the 2nd
, 3rd
, 5th
and 7th
day. Group III and IV were
administered Cee’Rich supplement at a dose of 200 mg/kg or 400 mg/kg bw (oral route)
respectively for 10 consecutive days, Both Groups (III and IV) also received CCl4 in liquid -
paraffin (1:2 v/v), 2 ml/kg bw (sc route), administered to induce liver toxicity in animals) on
the 2nd
, 3rd
, 5th
and 7th
day. Group V was treated with the standard reference drug Silymarin,
at a dose of 100 mg/kg bw (oral route) for 10 consecutive days, with CCl4 in liquid paraffin
(1:2 v/v), 2 ml/kg bw (sc route) on the 2nd
, 3rd
, 5th
and 7th
day.[10]
After 24 h of the last dose, blood was collected from retro-orbital plexus under ether
anesthesia. The blood samples were allowed to clot for 30 min, and the serum was separated
by centrifugation at 2500 g at 37°C. The supernatant liquid (serum) was separated, and
transferred in-to an Eppendorf tube and used for biochemical estimations.[10]
After blood
collection, the animals of all groups were sacrificed under light ether anesthesia, and
dissected.
Preparation of Liver Homogenate
The liver samples were rinsed in sterile ice cold saline and then homogenized in ice –cold
0.25 M sucrose, 1:10 w/v solution using a micro centrifuge. The homogenate was centrifuged
at 5000 rpm for 15 min, supernatant decanted and used for the estimation of antioxidants and
enzyme levels using standard kits.
Serum parameters
The following serum parameters were measured: Serum glutamic oxaloacetic transaminase
(SGOT), Serum glutamic pyruvic transaminase (SGPT) and Lactate dehydrogenase (LDH)
were assayed spectrophotometrically according to the standard procedures using
commercially available diagnostic kits.(Erba kit).[11]
Tissue parameter: Superoxide dismutase (SOD) was measured from tissue homogenates.

In-vivo antioxidant parameters
a. Estimation of SGOT
SGOT catalyzes the reversible transfer of an α - amino group between aspartate and
glutamate. It is an important enzyme in amino acid metabolism. SGOT is found in the liver,
heart, skeletal muscle, kidneys, brain and RBCs. Serum AST and ALT levels and their ratio
(AST/ALT) are commonly measured as clinical biomarkers for liver health. AST levels are
found to be increase in liver diseases, myocardial infarction, muscular dystrophy and
cholecystitis, whereas AST level are decrease in patients undergoing renal dialysis and those
with vitamin B6 deficiency.[12]
b) Estimation of SGPT
SGPT catalyzes the transfer of an amino group from L-alanine to α-ketoglutarate. The
products of this trans-amination are - pyruvate and L-glutamate. Normally, ALT is found
inside liver cells. But if the liver is inflamed or injured, ALT is released into the bloodstream.
Measuring blood levels of ALT provides useful information about the health of the liver
cells.[13]
ALT present in sample catalyzes the transfer of the amino group from L-alanine to α-
ketoglutarate forming pyruvate and L-glutamate. Pyruvate in the presence of NADH and
lactate dehydrogenase (LDH) is reduced to L-lactate. In this reaction NADH is oxidized to
NAD. The reaction is monitored by measuring the rate of decrease in absorbance at 340 nm
due to the oxidation of NADH to NAD. The reagent incorporates a reaction mechanism of
regenerating the NADH for the extended stability of the working reagent.
c) Determination of Serum LDH
LDH catalyzes the reduction of pyruvate with NADH to form NAD. The rate of oxidation of
NADH to NAD is measured as a decrease in absorbance which is proportional to the LDH
activity in the sample.
LDH is found in many body tissues, including the liver. Elevated levels of LDH may indicate
the liver damage. Serum LDH levels increase in myocardial infarction, pulmonary diseases,
hepatic disease, hemolytic anemia, renal diseases and muscular dystrophy.[14]
Calculations
LDH activity in U/L at 370
C = ΔA/min. X 8095

d. Super oxide dismutase (SOD)
SOD is an endogenously produced intracellular enzyme present in essentially every cell in
the body. Cellular SOD is actually represented by a group of metalloenzymes with various
prosthetic groups. The prevalent enzyme is cupro-zinc (CuZn) SOD, which is a stable
dimeric protein (32,000).[15]
SOD is considered to be fundamental in the process of
eliminating Reactive oxygen species ( ROI) by reducing (adding an electron to) superoxide
to form H2O2. Catalase and the selenium-dependent glutathione peroxidase are responsible
for reducing H2O2 to H2O.
Principle
The principle is based upon the method (NBT-NADH-PMS) of generation of oxygen by
NADH in the presence of 02 and the reduction of NBT (from yellow to blue) by 02 mediated
by PMS. SOD activity is measured as the inhibition of the rate of reduction of NBT by the
superoxide radical, observed at 560nm.
Procedure for SOD measurement
SOD activity was assayed by the method of Kono et. al.[16]. The assay system consisted of
EDTA 0.1mM, sodium carbonate 50 mM and 96 mM of nitro blue tetrazolium (NBT). In a
cuvette, 2 ml of above mixture, 0.05 ml hydroxylamine and 0.05 ml of PMS were mixed and
the auto-oxidation of hydroxylamine was observed by measuring the absorbance at 560 nm.
The results were calculated as units/mg protein, where one unit of enzyme is defined as the
amount of enzyme inhibiting the rate of reaction by 100%.[16]
RESULTS
Phytochemical investigation
Table 1: Phytochemical screening of Cee’Rich Vitamin C supplement.
Sl. No Testing compounds Name of the test Inference
1 Flavonoids
Shinoda test: +ve. +ve interference showed the
presence of flavonoids. The flavonoids
are reported to have an anti-oxidant,
hepato protective anti-ulcer and immuno
modularity activity.
Alkaline test:
2 Saponins Foam test:
+ve. Saponins have been reported to
control human cardiovascular diseases,
bactericidal, anti-oxidant, anti-microbial,
anti-viral, anti-inflammatory and
vasodilatory effects.

Acute oral toxicity study: Acute toxicity studies revealed the non-toxic nature of the
Cee’Rich Vitamin C supplement up to a dose level of 2000 mg/kg body weight in mice.
There was no lethality or toxic reaction found at any of the doses selected during the study.
Therefore as per the CPCSEA guideline 420 it was thought that 2000 mg/kg was the LD50
cut off dose. Therefore 1/10th
and 1/5th
dose (200 mg/kg and 400 mg/kg) were selected for all
further in vivo antioxidant studies.
Table 2: Effect of Cee’Rich Vitamin C supplement on biochemical parameters of liver
In rats
Each value, expressed as Mean ± SEM (n = 6) animals in each group, was compared to the
first group (Normal vehicle treated) by non paired t test followed by One way ANOVA,
Dunnett’s multiple comparison test. The table indicates significant alteration in SGOT <p
0.0002, <p0.0001, LDH <p0.0010 and SOD <p0.003 when compared to CCl4 treated group
Effects on SGOT
The CCl4 in liquid paraffin administered rats (group II) showed significant increase in the
SGOT level when results were compared to the water + liquid paraffin administered group
(group I). This confirms the liver toxicity in group II rats. The rats treated with standard
Silymarin + CCl4 in liquid paraffin (group V) showed significant antioxidant effect when
results were compared to group II rats. The rats administered with CCl4 in liquid paraffin +
Cee’Rich Vitamin C supplement (group III and IV) showed a significant reduction in the
SGOT levels, when results were compared to group II rats, indicating a significant
antioxidant effect of Cee’Rich Vitamin C supplement.
Treatment Groups SGOT IU/dl SGPT IU/dl LDH IU/dl
SOD U/mg of
protein
I. Water + liquid
paraffin
32.29 ± 7.51 20.61±3.63 38.60 ± 4.48 455 ± 5.40
II. Water + CCl4 in
liquid paraffin
(1:1v/v) 2ml/kg bw
86.18 ± 9.71***
80.48 ± 5.48****
105.3 ± 16.99** 304 ±4.5***
III. Cee’Rich Vitamin
C(200 mg/kg) + CCl4
in liquid paraffin.
77.54±11.66* 48.27±4.78*** 115.2±22.83
425.0±5.2**
IV. Cee’Rich Vitamin
C (400 mg/kg) + CCl4
in liquid paraffin
48.77± 1.59* 58.86±2.88* 53.05±7.60*
489.05±21***
V. Silymarin
(100mg/kg) + CCl4 in
liquid paraffin
39.59±6.06** 42.26±6.26**** 53.34±5.99*
463.5±4.5***

Effects on SGPT
SGPT level when results were compared to the water + liquid paraffin administered group
results were compared to group II rats.
The rats administered with CCl4 in liquid paraffin + Cee’Rich Vitamin C supplement (group
III and IV) showed significant reduction in the SGPT level, when results were compared to
group II rats, indicating a significant antioxidant effect of Cee’Rich Vitamin C supplement.
Effects on LDH
LDH level when results were compared to the water + liquid paraffin administered group
results were compared to group II rats.
IV) showed a significant reduction in LDH levels, when results were compared to group II
rats, suggesting a significant antioxidant effect of Cee’Rich Vitamin C supplement at 400
mg/kg.
Effects on SOD
SOD level when results compared to the water + liquid paraffin administered group (group I).
This confirms the liver toxicity in group II rats. The rats treated with standard Silymarin +
CCl4 in liquid paraffin (group V) showed significant antioxidant effect when results
compared to group II rats.
III and IV) showed significant elevation in SOD levels, when results were compared to group
II rats. The results indicate a significant antioxidant effect of Cee’Rich Vitamin C supplement
at 200 mg/kg as well as 400 mg/kg.

Graphical representations
G
ro
u
p
I
G
ro
u
p
II
0
5 0
1 0 0
1 5 0
SGOTin
IU/dl
***
G
ro
u
p
IIG
ro
u
p
IIIG
ro
u
p
IV
G
ro
u
p
V
0
5 0
1 0 0
1 5 0
SGOTin
IU/dl
* * *
* **
Fig 1 Fig-2
Fig1: Rats administered CCl4 (Group II-Positive control) showed a significantly
elevated level of SGOT when compared to normal control rats.
Fig 2: Group IV (treated with Cee’Rich Vitamin C, 400 mg/kg) and group V (treated
with reference compound, Silymarin) showed significantly decreased levels of SGOT
compared to group II (disease control) rats. In contrast, Group III treated with
200mg/kg of Cee’Rich Vitamin C did not show any effect on SGOT levels compared to
Group II.
G
ro
u
p
I
G
ro
u
p
II
0
2 0
4 0
6 0
8 0
1 0 0
SGPTin
IU/dl
* * * *
G
ro
u
p
II
G
ro
u
p
III
G
ro
u
p
IV
G
ro
u
p
V0
2 0
4 0
6 0
8 0
1 0 0
SGPTinIU/dl
* * * *
* * * * * * * *
Fig 3 Fig 4
Fig 3: Rats administered CCl4 (Group II-Positive control) showed a significantly
elevated level of SGPT when compared to normal control rats.
Fig 4: Groups III and IV (treated with Cee’Rich Vitamin C) and group V showed a
significantly decreased level of SGPT compared to group II (disease control) rats.

LDHIU/dl
G
ro
u
p
I
G
ro
u
p
II
0
50
100
150
200
* *
LDHIU/dl
G
ro
u
p
II
G
ro
u
p
III
G
ro
u
p
IV
G
ro
u
p
V
0
50
100
150
200
* *
* *
Fig 5 Fig 6
elevated level of LDG when compared to normal control rats.
Fig 6:- Group IV (treated with Cee’Rich Vitamin C) and group V showed significantly
decreased levels of Serum LDH compared to group II (disease control). In contrast,
Group III treated with 200mg/kg of Cee’Rich Vitamin C did not show any effect on
LDH levels compared to Group II.
U/mgofprotein
G
roup
I
G
roup
II
0
100
200
300
400
500
***
U/mgofprotein
G
roup
II
G
roup
III
G
roup
IV
G
roup
V
0
200
400
600
******
**
***
Fig 7 Fig 8
decreased level of SOD when compared to normal control rats.
Fig 8: Groups III and IV (treated with Cee’Rich Vitamin C) and group V showed a
significantly elevated level of liver SOD compared to group II (disease control).
DISCUSSION
Antioxidants work against molecules that form free radicals, destroying them before they can
begin the domino effect that leads to oxidative damage. For example, certain enzymes in the
body, such as superoxide dismutase, work with other chemical to transfer free radical into
harmless molecules. Vitamin C is an antioxidant that may prevent cataracts and cancers of

the stomach; throat, mouth, and pancreas. It may also prevent the oxidation of LDL
cholesterol, lowering the risk of heart disease and liver disease. In the present study,
Cee’Rich Vitamin C was evaluated for antioxidant activity using hepatotoxicity induced by
CCl4 in rat model. Carbon tetra chloride has been extensively studied as a liver toxicant, and
its metabolites such as trichloromethyl radical and trichloromethyl peroxy radical are
reported to be involved in the pathogenesis of the liver. The results of the present study
indicate that Cee’Rich Vitamin C is non toxic upto a dose of 2g/kg body wt. with no
behavioral changes or death. Furthermore, this study found that the Cee’Rich Vitamin C
supplement, in doses of 200 and 400 mg/kg, p.o., significantly elevated SOD levels and
significantly decreased serum SGOT, SGPT and LDH levels when compared to the toxic
control group.
CONCLUSION
From the results of the present study, it is concluded that Cee’Rich–Herbal Vitamin C
supplement, formulated by M/s Suguna Foods Pvt Ltd, Herbal Division, Suguna Lifeherbs
Coimbatore, TN, possesses superior antioxidant properties that may serve to protect users
against diseases associated with oxidative stress.
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