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Citations Using
KRISHGEN Products
Immunoassays
Leptin ELISA
Anti-obesity
potential of Clerodendron glandulosum. Coleb leaf aqueous
extract
Journal of Ethnopharmacology, Volume 135, Issue 2, 17 May 2011,
Pages 338-343, ISSN 0378-874 10.1016/j.jep.2011.03.020.
(http://www.sciencedirect.com/science/article/pii/S0378874111001565)
Ravirajsinh N. Jadeja, Menaka C. Thounaojam, Umed V. Ramani,
Ranjitsinh V. Devkar, A.V. Ramachandran,
Abstract:
Ethnopharmacological relevance Clerodendron glandulosum.Coleb
leaf aqueous extract (CG) is traditionally used by people of
North-East India to alleviate symptoms of diabetes, obesity and
hypertension. Previous study from our laboratory have documented
anti-diabetic and anti-hypertensive properties of CG extract
but, till date there are no pharmacological studies available on
its anti-obesity potential. This inventory investigates the
underlining molecular mechanism/s of CG induced regulation of in
vivo HFD induced obesity and in vitro adipocyte differentiation.
Aim: To evaluate effects of CG extract on (i) expression of
genes regulating visceral adiposity and (ii) in vitro adipocyte
differentiation and LEP release.
Materials and methods: Body weight, lee index, plasma lipids and
LEP, mRNA expression of PPARγ-2, SREBP1c, FAS, CPT-1 and LEP in
epididymal adipose tissue of control and experimental groups
were evaluated. Also, potential of CG extract on in vitro
adipocyte differentiation and LEP release was assessed.
Results: Supplementation of CG extract to HFD fed mice
significantly prevented HFD induced increment in bodyweight, lee
index, plasma lipids and LEP, visceral adiposity and adipocyte
hypertrophy. Also, CG extract supplementation resulted in down
regulation of PPARγ-2, SREBP1c, FAS and LEP expression along
with up-regulation of CPT-1 in epididymal adipose tissue
compared to HFD fed mice. In vitro study recorded significant
anti-adipogenic effect of CG extract that resulted in decreased
adipogenesis, TG accumulation, LEP release, G3PDH activity along
with higher glycerol release without significantly altering
viability of 3T3L1 pre-adipocytes.
Conclusions: Clerodendron glandulosum.Coleb extract prevents
adipocyte differentiation and visceral adiposity by down
regulation of PPARγ-2 related genes and Lep expression thus
validating its traditional therapeutic use in controlling
obesity.
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Sida rhomboidea.
Roxb Leaf Extract Down-Regulates Expression of PPARγ2 and Leptin
Genes in High Fat Diet Fed C57BL/6J Mice and Retards in Vitro
3T3L1 Pre-Adipocyte Differentiation
International Journal of Molecular Sciences Received: 3 May
2011; in revised form: 30 May 2011 / Accepted: 7 June 2011 /
Published: 19 July 2011
Menaka C. Thounaojam 1 , Ravirajsinh N. Jadeja 1 , Umed V.
Ramani 2 , Ranjitsinh V. Devkar 1,* and A. V. Ramachandran 1
1 Division of Phytothrapeutics and Metabolic Endocrinology,
Department of Zoology, The M. S. University of Baroda, Gujarat
390002, India 2 Department of Animal Biotechnology, College of
Veterinary Science and Animal Husbandry, Anand Agriculture
University, Anand, Gujarat 388001, India
Abstract:
Sida rhomboidea. Roxb leaf extract (SRLE) is being used by the
populace of North-East India to alleviate symptoms of diabetes
and obesity. We have previously reported its hypolipidemic and
anti-diabetic properties. In this study, we report the effect of
SRLE on (i) in vivo modulation of genes controlling high fat
diet (HFD) induced obesity and (ii) in vitro 3T3L1 pre-adipocyte
differentiation and leptin release. Supplementation with SRLE
significantly prevented HFD induced increment in bodyweight,
plasma lipids and leptin, visceral adiposity and adipocyte
hypertrophy. Also, SRLE supplementation reduced food intake,
down regulated PPARγ2, SREBP1c, FAS and LEP expressions and
up-regulated CPT-1 in epididymal adipose tissue compared to
obese mice. In vitro adipogenesis of 3T3L1 pre-adipocytes was
significantly retarded in the presence of SRLE extract. Also
decreased triglyceride accumulation, leptin release and
glyceraldehyde-3-Phosphate dehydrogenase activity along with
higher glycerol release without significant alteration of
viability of 3T3L1 pre-adipocytes, was recorded. Our findings
suggest that prevention of HFD induced visceral adiposity is
primarily by down regulation of PPARγ2 and leptin gene
expression coupled with attenuation of food intake in C57BL/6J
mice. SRLE induced prevention of pre-adipocytes differentiation,
and leptin release further substantiated these findings and
scientifically validates the potential application of SRLE as a
therapeutic agent against obesity.
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Human TNFα ELISA
Bresol inhibits
phosphodiesterase 4 gene expression and modulates the levels of
select mediators of inflammation in human monocytic cells
Journal of Immunotoxicology, October-December 2011, Vol. 8, No.
4 , Pages 315-323
R. Sandeep Varma,
G. Ashok, S. Vidyashankar, K. S. Nandakumar, P.S. Patki
Department of Cell biology and Immunology, Research and
Development, The Himalaya Drug Company, Bangalore
Abstract :
Bresol–a poly-herbal formulation, has been reported to be
effective against bronchial asthma and allergic rhinitis in
children. In vivo studies have supported the anti-histaminic and
anti-anaphylactic action of bresol. However, the mechanism of
action of bresol in modulation of inflammation has not been
studied at the cellular and molecular level. The present study
was aimed to elucidate the mechanism(s) of action of bresol at
the cellular and molecular levels, using human monocyte leukemia
cells. The effects of bresol on phosphodiesterase 4B (PDE4B)
gene expression were analyzed using human monocytic U937
leukemia cells. The ability of bresol to stimulate cAMP
formation in these cells, as well as its effects on mediators of
inflammation like tumor necrosis factor-α (TNFα), nitric oxide
(NO), and cycloxygenase-2 (COX-2) in lipopolysaccharide
(LPS)-stimulated U937 cells, were also studied. The results here
indicated that bresol exhibited potential anti-inflammatory
properties by inhibiting LPS-induced PDE4B gene expression in
the cells. Bresol also dose dependently activated cAMP
formation, and inhibited TNFα, NO, as well as COX-2 formation in
the LPS-stimulated cells. Based upon the results, we concluded
that the reported anti-inflammatory activity of bresol might be
attributed to its abilities to inhibit PDE4B and thus elevate
cAMP levels in human monocytes. The anti-inflammatory effects of
bresol might also be a result of the capacity of bresol to
modulate the formation of TNFα, NO, and COX-2 in monocytes.
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Antibacterial
activity of a synthetic peptide that mimics the LPS binding
domain of Indian mud crab, Scylla serrata Anti-lipopolysaccharide
Factor (SsALF) also involved in the modulation of vaginal immune
functions through NF-kB signaling
Microbial Pathogenesis, Received 18 August 2010; revised 21
December 2010; Accepted 22 December 2010. Available online 30
December 2010.
Sachin Sharmaa, 1, R.D. Yederya, 1, M.S. Patgaonkara, C.
Selvaakumarb, K.V.R. Reddya, ,
a Division of Molecular Immunology, National Institute for
Research in Reproductive Health (NIRRH), Indian Council of
Medical Research (ICMR), Jehangir Merwanji Street, Parel, Mumbai
400 012, India
b Department of Bioinformatics, Padmashree Dr. D.Y. Patil
University’s Department of Biotechnology and Bioinformatics, CBD
Belapur, Navi Mumbai 400 614, India
Abstract:
Recently the cDNA coding for anti-lipopolysaccharide factor
(ALF) has been identified from the Indian mud crab, Scylla
serrata and has been named S. serrata anti-lipopolysaccharide
factor (SsALF). SsALF protein sequence demonstrated the presence
of two highly conserved cystine residues between which the
putative lipopolysaccharide (LPS) binding domain is known to be
located. In this study, we have designed and synthesized a 24
amino acid linear (lSsALF24) and a cyclic (cSsALF24) peptides
based on this putative LPS binding domain and demonstrated the
ability of these peptides to bind to LPS. The peptides were
active against vaginal pathogens demonstrated by MIC, CFU and
phagocytosis assays. cSsALF24 did not show toxicity to human
vaginal epithelial cells (HeLa-S3), macrophages and rabbit
erythrocytes even at high concentration (64.64 μM). Flow
cytometry results demonstrated that cSsALF24 peptide suppressed
LPS induced phagocytosis of FITC labeled E. coli. HeLa cells
were stimulated with LPS (10 μg/ml) alone for 6 h or after two
washings with PBS, treated for 1 h with cSsALF24 (64.64 μM).
After washing, the cells were cultured for 24 h in fresh media.
The spent media as well as cells were collected for the
determination of cytokine/chemokine levels such as interleukin-6
(IL-6) interleukin-8 (IL-8), monocyte chemotactic protein-1
(MCP-1) and interleukin-1α (IL-1α) using ELISA and RT-PCR
respectively. Similar results were obtained with LPS stimulated
cells treated with c/nSsALF24 or unstimulated cells treated with
c/nSsALF24. The expression of cytokine/chemokines and mRNA’s
coding these proteins were unaffected in c/nSsALF24 treated
cells. In contrast, in LPS stimulated cells, the expression
levels of these molecules were up-regulated via the induction of
nuclear factor kappa-B (NF-kB) levels. However, the expression
of these pro-inflammatory markers was decreased in LPS
stimulated cells following the treatment with cSsALF24,
attributing anti-inflammatory potential of the peptide.
Collectively, these findings suggest that cSsALF24 might
regulate the vaginal epithelial cell immune responses indirectly
through modulation of LPS-TLR4 binding in NF-kB pathway.
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KineticBlue Reagent
A Rabbit Vaginal
Cell-Derived Antimicrobial Peptide, RVFHb {alpha} P, Blocks
Lipopolysaccharide-Mediated Inflammation in Human Vaginal Cells
In Vitro
Mandar Patgaonkar, Clara Aranha, Gauri Bhonde, K.V.R. Reddy,
Identification and characterization of anti-microbial peptides
from rabbit vaginal fluid,
Veterinary Immunology and Immunopathology, Volume 139, Issues
2-4, 15 February 2011, Pages 176-186, ISSN 0165-2427,
10.1016/j.vetimm.2010.10.012.
(http://www.sciencedirect.com/science/article/pii/S016524271000348X)
Abstract :
Antimicrobial peptides (AMPs) serve as a first line of host
defense and represent an important, though poorly understood
components of the innate immune system. The present study was an
attempt to identify and characterize the major molecules having
anti-bacterial activities from the vaginal fluid of rabbit,
Oryctologus cuniculus. AMPs from the rabbit vaginal fluid (RVF)
were identified in the acid extracts of pooled RVF samples after
RP-HPLC purification. The protein, RVFAMP was effective against
Gram negative (Escherichia coli and Pseudomonas aeruginosa) and
Gram positive (Staphylococcus aureus and Streptococcus pyogenes)
bacteria. The results of acid urea-PAGE-gel overlay assay
(AU-PAGE-GOA) demonstrated clear zone of growth inhibition of E.
coli corresponding to 6 and 15 kDa protein bands. LC–MS
data of these proteins indicated that 15 kDa protein
consists of lysozyme, lipopolysaccharide binding protein (LBP),
hemoglobin-α and β subunits (Hb-α/β), whereas 9 kDa protein
band consists of transthyretin and calcyclin while uteroglobulin
and neutrophil antibacterial peptide-5 (NAMP-5) are present in
the 6 kDa protein band. Of the eight proteins, Hb-α derived
protein was further characterized, as it showed the highest
Probability Based Mowse Score (PBMS) of 288. A 25 mer
peptide, RVFHbαp was active against several clinical pathogens
as demonstrated by minimum inhibitory concentration (MIC) and
radial diffusion assays (RDA). The interaction of RVFHbαP with
bacterial liposome membrane was assessed by calcein dye leakage
assay. RVFHbαP did not show cytotoxicity against human
endocervical cells (End1/E6E7) or erythrocytes. RT-PCR and
immunofluorescence results revealed the expression of RVFHbαP
mRNA and protein in rabbit vaginal tissue. To the best our
knowledge, this is the first report describing the detection of
AMPs in RVFs. In conclusion, these studies indicated that
vaginal epithelial cells (VEC) derived RVFHbαP may have
therapeutic potential in the management of reproductive well
being of rabbits. The major reason for undertaking this study in
rabbits is that, it forms an excellent in vivo model system for
human's studies.
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A Rabbit Vaginal
Cell-Derived Antimicrobial Peptide, RVFHb {alpha} P, Blocks
Lipopolysaccharide-Mediated Inflammation in Human Vaginal Cells
In Vitro
Clinical and Vaccine Immunology, MS Patgaonkar, A Sathe… -
Clinical and Vaccine …, 2011 - Am Soc Microbiol
... The effect of RVFHbαP on HeLa hVEC viability was determined
by the KineticBlue assay
(Krishgen Biosystems, India). This assay is based on the
reduction of resazurin dye into a pink-colored product,
resorufin, by dehydrogenase enzymes. ...
Abstract:
Antimicrobial peptides (AMPs) constitute a phylogenetically
ancient form of innate immunity that provides host defense at
various mucosal surfaces, including the vagina. Recently, we
have identified one such AMP, rabbit vaginal fluid hemoglobin
alpha peptide (RVFHbαP), from the vaginal lavage of rabbits (Oryctolagus
cuniculus).
The recent demonstration of a protective role of this peptide in
erythrocytes and vaginal cells led us to investigate (i) the
lipopolysaccharide (LPS) interactive domain in RVFHbαP and (ii)
whether RVFHbαP of rabbit origin modulates the cellular immune
responses of another species (humans) in vitro. HeLa-S3, a human
vaginal epithelial cell line (hVEC), was exposed to LPS alone
(10 μg/ml for 6 h), or LPS-induced cells were treated with
RVFHbαP (70.45 μM for 1 h) and cultured for 24 h, and the
results obtained were compared with the medium control. We show
here that RVFHbαP exerts an anti-inflammatory activity in hVECs,
as suggested by the prevention of LPS-induced production of
extracellular (supernatant) and intracellular (lysate) levels of
cytokines (interleukin 6 [IL-6] and IL-1α) and chemokines (IL-8
and monocyte chemoattractant protein 1 [MCP-1]). The
demonstration of Toll-like receptor 4 (TLR4) and NF-κB
expression in hVECs and the observations of RVFHbαP suppression
of human β-defensin-1 (hBD1) mRNA expression further support the
hypothesis of a genomic activity of RVFHbαP. Confocal microscopy
and flow cytometry results demonstrate that RVFHbαP inhibits
LPS-induced phagocytosis of Escherichia coli by macrophages. The
chemotaxis studies performed using the Boyden chamber Transwell
method showed the increased migration of U937 cells when
supernatants of LPS-induced hVECs were used, and this effect was
inhibited by RVFHbαP. In conclusion, our study proposes a novel
explanation for the protective role of RVFHbαP in
inflammation-associated infections, which not only may provide
the new cellular targets for the screening of RVFHbαP ligands
acting in the vaginal tissue but also has the potential to
develop RVFHbαP as a therapeutic agent for reproductive tract
infections.
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Evaluation of in
vitro toxicity of Rumalaya liniment using mouse embryonic
fibroblasts and human keratinocytes
Varma SR, Godavarthi A, Vidyashankar S, Nandakumar KS, Patki PS.
International Journal of Green Pharmacy, 2011;5:1-5
Abstract :
The skin irritation potential of topical formulations is
investigated prior to human exposure to identify the chemicals
which might induce adverse skin reactions. Rumalaya liniment (RL)
a novel formulation using natural oils and plant extracts is
used for reducing inflammations associated with musculoskeletal
disorders. Preclinical studies on RL are needed prior to skin
application. The aim of the study was to evaluate the possible
cytotoxic effects of RL and a commercial sample (CS) on mouse
embryo fibroblasts and human keratinocytes using neutral red
uptake, (3-(4,5- Dimethylthiazol -2-yl)-2,5-di phenyl
tetrazolium bromide (MTT) and resazurin assay. The CTC 50 values
obtained for RL was significantly higher than that of CS, which
revealed that RL is less toxic to CS. RL was less toxic (<17%)
on both cell lines at 400 μg/ml and was nontoxic at further
lower concentrations, whereas the toxicity of CS was above 59%
even at 400 μg/ml. It was observed from the present study that
by using three different assay methods and two different cell
lines, the toxicity of RL was significantly lower than that
observed with CS. From the study, it could be concluded that RL
could be safer to skin due to their low cytotoxicity as compared
with CS.
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Identification and
characterization of anti-microbial peptides from rabbit vaginal
fluid
Mandar
Patgaonkar, Clara Aranha, Gauri Bhonde, K.V.R. Reddy ,
Division of Molecular Immunology, National Institute for
Research in Reproductive Health (NIRRH), Indian Council of
Medical Research (ICMR), Jehangir Merwanji Street, Parel, Mumbai
400 012, Maharashtra, India
Veterinary Immunology and Immunopathology, Volume 139, Issues
2-4, 15 February 2011, Pages 176-186, Received 16 July 2010;
revised 6 September 2010; Accepted 5 October 2010. Available
online 13 October 2010.
Abstract:
Antimicrobial peptides (AMPs) serve as a first line of host
defense and represent an important, though poorly understood
components of the innate immune system. The present study was an
attempt to identify and characterize the major molecules having
anti-bacterial activities from the vaginal fluid of rabbit,
Oryctologus cuniculus. AMPs from the rabbit vaginal fluid (RVF)
were identified in the acid extracts of pooled RVF samples after
RP-HPLC purification. The protein, RVFAMP was effective against
Gram negative (Escherichia coli and Pseudomonas aeruginosa) and
Gram positive (Staphylococcus aureus and Streptococcus pyogenes)
bacteria. The results of acid urea-PAGE-gel overlay assay
(AU-PAGE-GOA) demonstrated clear zone of growth inhibition of E.
coli corresponding to 6 and 15 kDa protein bands. LC–MS data of
these proteins indicated that 15 kDa protein consists of
lysozyme, lipopolysaccharide binding protein (LBP), hemoglobin-α
and β subunits (Hb-α/β), whereas 9 kDa protein band consists of
transthyretin and calcyclin while uteroglobulin and neutrophil
antibacterial peptide-5 (NAMP-5) are present in the 6 kDa
protein band. Of the eight proteins, Hb-α derived protein was
further characterized, as it showed the highest Probability
Based Mowse Score (PBMS) of 288. A 25 mer peptide, RVFHbαp was
active against several clinical pathogens as demonstrated by
minimum inhibitory concentration (MIC) and radial diffusion
assays (RDA). The interaction of RVFHbαP with bacterial liposome
membrane was assessed by calcein dye leakage assay. RVFHbαP did
not show cytotoxicity against human endocervical cells
(End1/E6E7) or erythrocytes. RT-PCR and immunofluorescence
results revealed the expression of RVFHbαP mRNA and protein in
rabbit vaginal tissue. To the best our knowledge, this is the
first report describing the detection of AMPs in RVFs. In
conclusion, these studies indicated that vaginal epithelial
cells (VEC) derived RVFHbαP may have therapeutic potential in
the management of reproductive well being of rabbits. The major
reason for undertaking this study in rabbits is that, it forms
an excellent in vivo model system for human's studies.
-----------------------------------------------------------------------------------------------------------------------------------------------
Antibacterial
activity of a synthetic peptide that mimics the LPS binding
domain of Indian mud crab, Scylla serrata anti-lipopolysaccharide
factor (SsALF) also involved in the modulation of vaginal immune
functions through NF-kB signaling.
Sharma S, Yedery RD, Patgaonkar MS, Selvaakumar C, Reddy KV.
Division of Molecular Immunology, National Institute for
Research in Reproductive Health (NIRRH), Indian Council of
Medical Research (ICMR), Jehangir Merwanji Street, Parel, Mumbai
400012, India.
Micro Pathog. 2011 Mar-Apr;50(3-4):179-91. Epub 2010 Dec 30.
Abstract:
Recently the cDNA coding for anti-lipopolysaccharide factor
(ALF) has been identified from the Indian mud crab, Scylla
serrata and has been named S. serrata anti-lipopolysaccharide
factor (SsALF). SsALF protein sequence demonstrated the presence
of two highly conserved cystine residues between which the
putative lipopolysaccharide (LPS) binding domain is known to be
located. In this study, we have designed and synthesized a 24
amino acid linear (lSsALF24) and a cyclic (cSsALF24) peptides
based on this putative LPS binding domain and demonstrated the
ability of these peptides to bind to LPS. The peptides were
active against vaginal pathogens demonstrated by MIC, CFU and
phagocytosis assays. cSsALF24 did not show toxicity to human
vaginal epithelial cells (HeLa-S3), macrophages and rabbit
erythrocytes even at high concentration (64.64 μM). Flow
cytometry results demonstrated that cSsALF24 peptide suppressed
LPS induced phagocytosis of FITC labeled E. coli. HeLa cells
were stimulated with LPS (10 μg/ml) alone for 6 h or after two
washings with PBS, treated for 1 h with cSsALF24 (64.64 μM).
After washing, the cells were cultured for 24 h in fresh media.
The spent media as well as cells were collected for the
determination of cytokine/chemokine levels such as interleukin-6
(IL-6) interleukin-8 (IL-8), monocyte chemotactic protein-1
(MCP-1) and interleukin-1α (IL-1α) using ELISA and RT-PCR
respectively. Similar results were obtained with LPS stimulated
cells treated with c/nSsALF24 or unstimulated cells treated with
c/nSsALF24. The expression of cytokine/chemokines and mRNA's
coding these proteins were unaffected in c/nSsALF24 treated
cells. In contrast, in LPS stimulated cells, the expression
levels of these molecules were up-regulated via the induction of
nuclear factor kappa-B (NF-kB) levels. However, the expression
of these pro-inflammatory markers was decreased in LPS
stimulated cells following the treatment with cSsALF24,
attributing anti-inflammatory potential of the peptide.
Collectively, these findings suggest that cSsALF24 might
regulate the vaginal epithelial cell immune responses indirectly
through modulation of LPS-TLR4 binding in NF-kB pathway.
Copyright © 2010 Elsevier Ltd. All rights reserved.
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