Studies on Immunomodulatory Potential of Flavonoids Ph.D ... · 2 Carbon Clearance Test The...
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Studies on Immunomodulatory Potential of Flavonoids
Ph.D. Synopsis
Submitted To
Gujarat Technological University
For The Degree
of
Doctor of Philosophy
In
Pharmacy
By
Aditya Ganeshpurkar
Enrollment No: 149997390001
(Pharmacy)
Guided By
Dr. Ajay K. Saluja,
Professor & Principal,
A. R. College of Pharmacy,
Vallabh Vidya Nagar, Gujarat
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Brief description on the state of the art of the research topic
Immune system is remarkably a sophisticated defense system within vertebrates, to protect
them from invading agents. It can generate varieties of cells and molecules and is capable of
recognizing and eliminating limitless varieties of foreign and undesirable agents. Immunity
refers to the ability of the body to identify and resist microorganisms that are potentially
harmful. After maturation, most immune cells circulate into the body and exert specific effect [1]
.Cell-mediated immunity is the result of the activity of many leukocyte actions, reactions,
interactions that range from simple to complex. This type of immunity is dependent on the
actions of the T (Thymus) lymphocytes, which are responsible for a delayed type of immune
response. The T lymphocyte becomes sensitized by its first contact with a specific antigen. In
humoral immunity special lymphocytes (white blood cells), called B (Bone cell)
lymphocytes, produce circulating antibodies to act against a foreign substance. This type of
immunity is based on the antigen–antibody response. Immunopharmacology is the
intersection of immunology and pharmacology. The most well-known immunopharmacology
agents include anti-rejection drugs and vaccines [2]
.Immunostimulants are drugs which can be
used to stimulate the immune system. In addition to drugs themselves, several vitamins,
minerals, and other chemicals are known to boost the efficacy of the immune system. While
immunosuppressant drugs have been studied more extensively than immunostimulants, this
latter class of therapeutic agents has so far shown some promise in the treatment of primary
immunodeficiencies and cancers as well as HIV and AIDS. Immunopharmacological
research is not only limited to the discovery of new drugs. It is also dedicated to examine how
the immune system functions, with a view of discovering new drug targets [3,4]
Definition of the problem
One of the most important factors explaining the mechanism, by which these pathologies
develop, involves oxidative modification of critical molecules. So, ROS can react with
proteins, lipids, carbohydrates and nucleic acids, altering gene expression and promoting an
inflammatory response [5–7]
. Scientific evidences justify the fact that diet influences the health
as well as controls and modulates many functions of body and thus plays an important role in
the maintenance of good health; this homeostasis produced therefore, decreases the chances
of many chronic diseases [8]
.
Objective and Scope of work
Flavonoids belong to a group of natural substances with variable phenolic structures and are
found in fruit, vegetables, grains, bark, roots, stems, flowers, tea, and wine [9]
. Flavonoids are
polyphenolic compounds that occur ubiquitously in plants having a variety of biological
effects both in vitro and in vivo. Anthocyanin pigment present in flowers provide colour to it
which contributes to pollination. Flavonoids present in leaves promote physiological survival
of plant by protecting it from fungal infections and UV radiations. In addition, flavonoids are
involved in photosensitization, energy transfer, respiration and control of photosynthesis,
morphogenesis, sex-determination, energy transfer [10]
. However as far as talking about
therapeutic potential, flavonoids have been found to have antimicrobial, antiviral, anti-
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ulcerogenic, cytotoxic, anti-neoplastic, mutagenic, antioxidant, antihepatotoxic,
antihypertensive, hypolipidemic, antiplatelet and anti-inflammatory activities. They have
been found to have anti-inflammatory activity in both proliferative and exudative phases of
inflammation [11]
. A number of plants rich in flavonoids are known to show
immunomodulatory potential. However, no systematic attempts to study effect of flavonoids
on immune system have been done so far. Thus, the aim of present study is to explore
immunomodulatory potential of rutin, catechin and hesperidin.
To evaluate the effect of flavonoid administration on delaved type type hypersensitivity
To evaluate the effect of flavonoid administration on clearance of foreign particles
To evaluate the effect of flavonoid administration on WBC and antibody production.
To evaluate the effect of flavonoid administration on production of IL-1Β, IL-6, TNF-α
and NO production.
Plan of Work
1. Procurement of Plant Flavonoids
2. Experimental
In vivo studies
Delayed type hypersensitivity response
Carbon clearance test
Cyclophosphamide induced myelosuppression
Neutrophil adhesion test
Humoral antibody titre
Effect on serum immunoglobulin
In vitro cell culture studies
Cell line: Murine macrophage/ Peritoneal macrophage
Parameters
A. Assessment of Cell viability using MTT assay
B. Determination of following mediators after treatment with bacterial liposaccharides
Interleukin 1β
Interleukin 6
Tumor Necrosis Factor α
Inducible Nitric Oxide synthase
3. Compilation and Presentation of Scientific Data.
Original contribution by the thesis
The work is original in terms of use of selected flavonoids for immunomodulation in the
current experimental models of depression. The studies conducted have been published in
peer reviewed journals
Methodology of research, Results/Comparisons
1. Procurement and analysis of Test flavonoids
Test flavonoids (rutin, catechin and hesperidin) were purchased from Central Drug House,
Mumbai, India. FTIR study was performed for structural confirmation of the compounds.
2. Animals
Wistar rats (180-200 g) of either sex were housed in polypropylene cages, maintained under
standardized conditions (12 h light/dark cycles, 28±2°) were used in the study. Animals were
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provided with standard pellet food and had free access to drinking water. All the animal study
protocols were duly approved by Institutional Animal Ethics Committee.
3. Selection of dose
As per studies performed by Shenbagam & Nalini, 2011 [12]
, Hamaishi et al. [13]
Hemanth
Kumar et al.[14]
, rutin, catechin and hesperidin in the dose of 25, 50, and 100 mg/kg of body
weight were used in the study.
Table1: Dosing protocol for studies
Treatment Dose
Group I Normal control
Group II Cyclophosphamide (30 g/ kg)
Group III Rutin (25 mg/ kg)
Group IV Rutin (50 mg/ kg)
Group V Rutin (100 mg/ kg)
Group VI Catechin (25 mg/ kg)
Group VII Catechin (50 mg/ kg)
Group VIII Catechin (100 mg/ kg)
Group IX Hesperidin (25 mg/ kg)
Group X Hesperidin (50 mg/ kg)
Group XI Hesperidin (100 mg/ kg)
Animals from Group III to Group XI received Cyclophosphamide (30 mg/ kg) once during the starting of
experiment except in cyclophosphamide induced neutropenia. In this protocol (cyclophosphamide induced
neutropenia) anilams from Group II to Group XI received Cyclophosphamide (100 mg/ kg) once during the
starting of experiment.
Study protocols
In vivo studies
1 Hypersensitivity reaction (delayed type)
On day 0, all the groups were immunized by subcutaneous administration of 1 ml of SRBC
cell suspension into the right hind footpad. On day 15, all groups were challenged by
subcutaneously injecting 0.5 mL of SRBC cell suspension into the left hind footpad[15]
, and
the thickness of the left hind footpadwas measured after 24 and 48 h of a challenge using
vernier calipers. The difference in the thickness of the right hind paw and the left hind paw
was used as a measure of DTH reaction and was expressed as a mean percent increment in
thickness/edema.
2 Carbon Clearance Test
The phagocytic activity of the reticulo-endothelial system (RES) was assessed by carbon
clearance method. [16,17]
.
3 Cyclophosphamide-induced neutropenia
The method described by Manjrekar et al. [18]
was adopted. Determination of total white blood
cells was carried out.
4 Neutrophil Adhesion test
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The rats were treated orally with standard and test compounds (rutin catechin and hesperidin)
for 14 days. On day 14, blood samples were collected from the retro-orbital plexus into
heparinized vials and analyzed for total leukocyte count and (TLC) differential leukocyte
count (DLC) [19,20]
.
The product of total leukocyte count and % neutrophil known as the neutrophil index was
determined for each of the respective groups[21]
. The % neutrophil adhesion for each of the
test groups was determined as follows:
% Neutrophil adhesion=(Difference in neutrophil count in untreated and fiber treated blood
)/(Neutrophil count of untreated blood) ×100
5 Indirect hemagglutination test
Rats were pretreated with the test drugs (rutin catechin and hesperidin) for 14 days, and each
rat was immunized with 0.5×109 sheep red blood cells (SRBCs) intraperitoneally, including
control rats [22]
.
6 Serum immunoglobulin
Animals were treated with standard/ test drug orally for 21 days as mentioned in table. Six
hours after the last dose, blood samples were collected, and the serum was separated The
standard BaSO4 solution was prepared by adding 3 ml of barium chloride solution (1.15%
w/v) to 97 ml of 0.2 N sulphuric acid. The turbidity obtained with this solution was expressed
as 20 zinc sulfate turbidity (ZST) units[23]
.
In vitro studies
1. Isolation of rat peritoneal-exudate macrophages
For induction of inflammatory responses, rats were injected intraperitoneally with 1 ml of 3%
thioglycollate broth, and peritoneal exudate was extracted 5 days later. After gentle
abdominal massage, about 30 ml of peritoneal fluid was extracted using the same syringe and
transferred to 50-ml sterile polypropylene tubes on ice. A 20 µl aliquot was then extracted for
cell counting in a hemocytometer. Aliquots of 100 µl of the cell suspension were added to the
wells of 96- well microculture plates (Corning, USA) or placed on microscope slides, and left
for 90 min in a humidified incubator (37 ˹C, 5% CO2) to allow adhesion. Nonadherent cells
were then removed by gently washing with HBSS[24]
.
2. Determination of the inflammatory cytokines and chemokines (TNF-α, IL-1β, IL-6)
The concentrations of inflammatory cytokines and chemokines in cell culture medium in
presence and absence of lipopolysaccharides (from E. coli) was determined by using
Krishgen ELISA based kits as per manufacturer’s instructions.
3. Determination of Nitric oxide
After stimulation of macrophages (1 × 105 cells) with LPS (1 μg/mL) for 24 h in the presence
of various concentrations of each drug, nitrite levels in the conditioned medium were
determined using Griess reagent [25]
. Nitrite concentrations in culture supernatants were
measured to assess NO production in macrophages. NaNO2 was used as standard to
calculated nitrite concentrations [26]
.
In silico studies
1. Software
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Python 2.7- language was downloaded from www.python.com, Molecular graphics
laboratory (MGL) tools and AutoDock 4.2 was downloaded from www.scripps.edu,
Discovery Studio visualizer 4.1 was downloaded from www.accelerys.com.
2. Protein preparation
The three-dimensional crystalline structures of 5 targeted proteins (Table1) were retrieved
from the Protein Data Bank (http://www.rcsb.org/). The retrieved protein were TNF-α (PDB
ID: 2AZ5), IL-1β (PDB ID: 2NVH), IL-6 (PDB ID: 1P9M) and NOs (PDB ID: 1NSI). The
coordinates of the structures were complexed with water molecules, and other atoms which
are responsible for increased resolution and therefore the water molecules and het-atoms were
removed using discovery studios and saved in. pdb format.
3. Docking analysis
The three-dimensional crystalline structures of 4 proteins were obtained from Protein Data
Bank (http://www.rcsb.org/). These protein were TNF-α (PDB ID: 2AZ5), IL 1β (PDB ID:
2NVH), IL 6 (PDB ID: 1P9M) and NOs (PDB ID: 5UO1). The 2D and 3Dchemical
structures of rutin, catechin and hesperidin was retrieved (http://pubchem.ncbi.nlm.nih.gov/).
These .sdf and .mol files obtained from PubChem were converted into .pdb files using
Marwin Sketch (http://www.chemaxon.com/marvin/ sketch/index.jsp). These .pdb files were
converted to .pdbqt using ligand preparation module of autodock tools 1.5.6. The docking
analysis of hesperidin was carried out using the Autodock tools (ADT) v1.5.4 and autodock v
4.2 programs. Rutin, catechin and hesperidin were docked to all the target protein complexes
with the molecule considered as a rigid body. The search was carried out with the
Lamarckian Genetic Algorithm; populations of 100 individuals with a mutation rate of 0.02
have been evolved for ten generations. The remaining parameters were set as default. The
Docked structure was then visualised using Discovery Studio 2016 for obtaining the binding
interactions
Statistical analysis
For all in vivo studies, all the values were expressed as mean±SEM. Statistics was applied
using Graph Pad Prism version 5.0 for Windows, Graph Pad software, San Diego, California,
USA. One-way ANOVA followed by Dunnet’s comparison test was used to determine the
statistical significance between various groups. Differences were considered to be statistically
significant when *p < 0.05; **p< 0.01, ***p < 0.001. For in vitro cell culture studies, the data
is expressed as Mean ± SD and statistical analysis was carried out employing the one-way
ANOVA followed by Bonferroni multiple comparison test. p values < 0.05 are being taken as
statistically significant.
Results
Hypersensitivity reaction
Treatment with Rutin. Catechin in the dose of 25, 50 and 100 mg/kg/day showed the footpad
thickness 4.81±0.12 mm, 5.03±0.11 mm and 5.06±0.13 mm respectively after 24 h.
Hesperidin in the dose of 25, 50 and 100 mg/kg/day demonstrated the foot pad thickness
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4.91±0.07 mm, 5.01±0.10 mm and 5.36±0.08 mm respectively after 24 h corresponding value
of 4.68 ± 0.16 mm for group II. (Figure 1).
Figure 1: Effect of test flavonoid (rutin, catechin and hesperidin) administration on delayed type
hypersensitivity response in rats. Results are given as mean ± SEM of six animals in each group.
Significance at *p < 0.05; **p< 0.01; ***p < 0.001 when compared to Group I;
Carbon clearance Test
Administration of Rutin in the dose of 50 mg/kg and 100 mg/ kg, per oral produced increase
in clearance of carbon particles from blood as indicated by a significant increase in
phagocytic index (*p<0.01, **p<0.001). Catechin administration, especially in the dose of
100 mg/ kg, caused increased clearance of carbon particles from blood (***p < 0.001)
(Figure 2). Hesperidin administration especially in the dose of 100 mg/ kg, caused increased
clearance of carbon particles from blood (***p < 0.001)(Figure 2).
Figure 2: Effect of test flavonoid (rutin, catechin and hesperidin) administration on phagocytic index
in rats. Results are given as mean ± SEM of six animals in each group.Significance at *p < 0.05; **p<
0.01; ***p < 0.001 when compared to Group I
Cyclophosphamide Induced Neutropenia
Administration of Rutin in dose of 25 and 50 mg/ kg, per oral produced decrease in
neutropenia condition as indicated by a significant increase in total leucocyte count
(***p<0.001, ***p<0.001). Catechin administration in a dose of 25, 50 and 100 mg/kg/day,
per oral caused a decrease in condition of neutropenia (***p<0.001). Hesperidin
*** *** *** *** *** *** *** *** *** *** *** *** ** *** ** * * * *
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Group I Group II Group III Group IV Group V Group VI Group VII Group VIII Group IX Group X Group XI
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Group I Group II Group III Group IV Group V Group VI Group VII Group VIII Group IX Group X Group XI
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administration in a dose of 25, 50 and 100 mg/kg/day, per oral also caused a decrease in
neutropenia (***p<0.001, ###p<0.001)(Figure 3).
Figure 3: Effect of test flavonoid (rutin, catechin and hesperidin) administration on WBC counts in
rats. Results are given as mean ± SEM of six animals in each group.Significance at *p < 0.05; **p<
0.01; ***p < 0.001 when compared to Group I
Neutrophil adhesion
Rutin administration showed significant increase in the neutrophil adhesion
(**p<0.01, **p<0.01, ***p<0.001) when compared to control. Lower dose of rutin was
more effective than high dose. A significant increase in neutrophil adhesion was observed in
Group VI and Group VII animals as compared to Group II animals. Administration of
hesperidin caused significant increase in the neutrophil adhesion (***p<0.001;
###p<0.001) when compared to control (Table 2).
Table 2: Effect of Hesperidin administration on leucocyte mobilization/ migration in rats.
Treatment TLC % Neutrophils Neutrophil Index % Neutrophil
adhesion UTB TB UTB TB UTB TB
Group I 6.20±0.12 5.64±0.10 45.50±1.05 36.66±1.38 282.46±12.68 206.83±14.65 9.13±0.14
Group II 4.76±0.28 4.40±0.25 40.66±0.49 34.82±0.65 193.81±13.89 153.54±16.81 7.51±0.24****
Group II 6.10±0.16 5.36±0.09 47.32±1.02 37.83±1.01 289.11±17.17 203.01±9.22 12.14±0.77***
Group IV 6.21±0.12 5.43±0.05 49.34±1.08 39.33±1.30 306.65±13.78 213.69±6.67 12.59±0.76***
Group V 6.33±0.07 5.64±0.24 50.33±0.76 40.16±0.70 318.76±16.01 226.58±16.87 10.92±0.61***
Group VI 5.81±0.21 5.26±0.70 47.16±0.70 36.83±0.71 274.31±14.78 193.66±4.94 9.59±0.44***
Group VII 5.96±0.14 5.33±0.98 48.33±0.88 38.66±0.99 288.37±12.97 206.18±9.78 10.61±0.56***
Group VIII 6.01±0.11 5.53±0.65 49.50±0.92 39.16±0.65 297.41±9.81 216.69±4.28 7.90±0.36ns
Group IX 6.14±0.17 5.03±0.42 46.33±0.56 36.66±0.42 284.51±13.26 184.71±1.82 17.95±0.20***
Group X 6.33±0.18 5.46±1.19 49.16±0.65 38.16±1.20 311.35±11.60 208.64±14.28 13.68±0.22***
Group XI 6.41±0.08 5.65±1.15 50.83±0.87 39.66±1.50 326.16±7.46 224.41±22.50 11.81±0.36ns
Haemagglutination reaction
Rutin demonstrated a significant increase in the antibody titer at low dose levels. The dose of
25 mg/kg/day showed response 96.00 ± 35.05 (*p<0.05) in comparison to group I (192.00
±28.62). There was a significant change in HA titer levels in animals treated with
cyclophosphamide (***p<0.001; 53.33 ± 16.52). Catechin administration demonstrated a
***
*** ***
*** *** ***
***
2000
4000
6000
Group I Group II Group III Group IV Group V Group VI Group VII Group VIII Group IX Group X Group XI
WB
C C
ou
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(th
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significant increase in the antibody titer at low dose levels. Hesperidin in the dose of 25
mg/kg/day showed response 106.66 ± 13.49 (**p<0.01) and dose of 50 mg/kg/day showed
response 138.66 ± 10.66 (***p<0.001) in comparison to group I (192.00 ±28.62)(Figure 4).
Figure 4: Effect of test flavonoid (rutin, catechin and hesperidin) administration on HA antibody titer
in rats. Results are given as mean ± SEM of six animals in each group.Significance at *p < 0.05; **p<
0.01; ***p < 0.001 when compared to Group I
Serum immunoglobulin
Figure 5 revealed a significant increase in the serum immunoglobulin levels in rutin
treated group (group III 10.94±0.56, ***p < 0.001; group IV 11.82±0.72, **p< 0.01)
Hesperidin also caused a significant increase in the serum immunoglobulin (group ix
10.12 ±0.38, ***p < 0.001; group X 11.19 ±0.39, ***p < 0.001, ###p < 0.001; group XI
13.01±0.28, ***p < 0.001,) was seen; whereas group II animals revealed lower serum
immunoglobulin levels (8.61±1.22) compared to vehicle control Group I animals
(14.59±0.48) (Figure 5).
Figure 5: Effect of test flavonoid (rutin, catechin and hesperidin) administration on serum
immunoglobulin levels in rats. Results are given as mean ± SEM of six animals in each
group.Significance at *p < 0.05; **p< 0.01; ***p < 0.001 when compared to Group I
In vitro studies
Cell viability
MTT assay is widely used protocol for measurement of mitochondrial enzyme succinate
dehydrogenase. This assay is widely used to determine the toxicity of chemicals from natural
and synthetic origin. The MTT assay was used in the present study to determine the effect of
***
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Group I Group II Group III Group IV Group V Group VI Group VII Group VIII Group IX Group X Group XI
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rutin on viability of rat macrophage. Cell viability was not altered by maintained due to Rutin
(98%) catechin (97%) and hesperidin (98%) treatment.
Effect on cytokine levels
The results indicated a decrease in TNF-α levels in the cells treated with test flavonoids
(rutin, catechin and hesperidin) as compared to control (LPS only) in a concentration
dependent manner. There was a significant decrease in level of cytokine at 1 µM (p < 0.001)
(Figure 6). Rutin and hesperidin (10 µM) caused a significant (p < 0.001) decrease in IL-1β as
compared to control (Figure 7). Results indicated that the rutin inhibited the release of IL-6
from macrophages in a concentration dependent manner whereby maximum inhibition was
observed at concentration 10 µM (p < 0.05) (Figure 8).
Figure 6: Effects of the test flavonoid (rutin, catechin and hesperidin) on the production of TNF-α
in rat macrophages. Data are expressed as the mean ± S.D. of three individual experiments.
Statistically significant change of cytokine release (*p < 0.05, **p < 0.01, ***p < 0.001), as
compared with the group treated without LPS treatment.
Figure 7: Effects of the test flavonoid (rutin, catechin and hesperidin) on the production of IL-1β
in rat macrophages. Data are expressed as the mean ± S.D. of three individual experiments.
Statistically significant change of cytokine release (*p < 0.05, **p < 0.01, ***p < 0.001), as
compared with the group treated without LPS treatment.
### ***
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Figure 8: Effects of the test flavonoid (rutin, catechin and hesperidin) on the production of IL-6
in rat macrophages. Data are expressed as the mean ± S.D. of three individual experiments.
Statistically significant change of cytokine release (*p < 0.05, **p < 0.01, ***p < 0.001), as
compared with the group treated without LPS treatment.
Effect on NO production
Rutin catechin and hesperidin inhibited the NO production in a concentration dependent
manner at a dose range of 0.1-100 µM. The maximum inhibition by these test flavonoids was
observed in the range 0.1-10 µM compared to control (p < 0.001).
Figure 9: Effects of the test flavonoid (rutin, catechin and hesperidin) on the production of NO in
rat macrophages. Data are expressed as the mean ± S.D. of three individual experiments.
Statistically significant change of cytokine release (*p < 0.05, **p < 0.01, ***p < 0.001), as
compared with the group treated without LPS treatment.
Docking studies
The four crystal structures of proteins were retrieved from protein databank. A docking was
performed to identify the precise binding sites on various immunomodulatory targets.
Molecular docking is an effective approach that helps to envisage the principal ‘binding
modes’ of the ligand with the ‘protein/ receptor/ enzyme’ having known ‘three-dimensional
structure’. In the present study, we carried out docking studies on rutin, catechin and
hesperidin to various inflammatory and immunomodulatory targets, with the purpose to study
and analyse in silico interaction of former on later. The docking scores and analysis of the
###
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***
*** ***
*** ***
00.010.020.030.040.050.060.070.080.09
Nit
rate
(µ
M)
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interactions of rutin, catechin and hesperidin suggest the ability of these flavonoids to bind to
multiple targets involved in inflammation and immunomodulation.
Table 3: Molecular docking studies of rutin, catechin and hesperidin on some chemokines
and enzymes
Target Resolution PDB
ID
`Rutin Catechin Hesperidin
Binding
energy
Inhibitory
constant
(µM)
Binding
energy
Inhibitory
constant
(µM)
Binding
energy
Inhibitory
constant
(µM)
Tumor Necrosis Factor-α 2.10Å 2AZ5 -6.25 25.62 -5.79 57.14 -6.96 230.93
Interleukin 1β 2.50Å 1ITB -4.24 774.3 -7.50 3.16 -6.64 394.91
Interleukin 6 3.65 Å 1P9M -5.96 42.43 -6.20 20.69 -7.07 191.74
Nitric oxide synthase 1.90 Å 5UO1 -5.71 64.88 -7.44 3.45 -6.83 288.42
Rutin, catechin and hesperidin interacted with various chemokines and inflammatory
mediators’ viz. TNF-α, IL-1β, IL-6 and NOs. With each target, rutin, catechin and hesperidin
demonstrated a noteworthy affinity for binding. Findings from the present study show that
rutin, catechin and hesperidin may interact with several chemokines and inflammatory
mediators.
Achievements with respect to objectives
Immunomodulators are the agents that amend the activity of immune system and have dual
effects. A few of them may stimulate the immune system at a lower level while the others
may have inhibitory effects on normal or activated immune response [27]
. Immunomodulators
are likely to be used for the reintegration of ‘immune deficiency.' Plant extracts and
phytochemicals have been extensively investigated for possible immunomodulatory effects [28]
. The present study aimed to explore the immunomodulatory effect of test flavonoid (rutin,
catechin and hesperidin) against various immunological challenges. Rutin, cahechin and
hesperidin were studied for possible protective effects for hypersensitivity reaction, carbon
clearance, cyclophosphamide-induced neutropenia, and neutrophil adhesion. Studies were
also performed to determine haemagglutination titer and serum immunoglobulin levels.In the
present study, test flavonoid (rutin, catechin and hesperidin)administration in rats caused a
significant increase in the delayed type of hypersensitivity . Phagocytic index of test flavonoid
(rutin, catechin and hesperidin) treated animals was significantly more as compared to the
cyclophosphamide-treated group (Group II). The administration of test flavonoid (rutin,
catechin and hesperidin) to animals (Group III-XI) resulted in a significant increment (***p <
0.001) in the levels of white blood cells. Such an effects might be contributed due to
cytoprotective [29]
and antioxidant [30]
effect of catechin over the biological system.In the
present study, there was a dose-dependent intensification of neutrophil adhesion to nylon
fibers which suggest intense migration of neutrophil to ‘foreign bodies.' As discussed above,
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13
such an effect might be due to antioxidant potential of catechin. Neutrophil adhesion to
‘nylon fibers’ represents the movement of neutrophils towards the site of ‘inflammation’[20]
.
In the present study, SRBCs immunization to catechin trerated group resulted in noteworthy
antibody production. [31]
. Along with HA titer studies, antibody production were also
calculated by zinc sulfate turbidity method. A notable increase in the levels of serum
immunoglobulin production due to the administration of rutin, catechin and hesperidin was
seen. In the present study, immunomodulatory effect of rutin was determined on macrophage
activity. The decrease in concentration of TNF-α and IL-1 β due to test flavonoid (rutin,
catechin and hesperidin)has been identified previously in other cells/ tissues [32]
. The decrease
in IL-6 levels due to treatment with rutin, catechin and hesperidin could be one of the
important reasons for its down regulation. The outcomes of present study clearly revealed the
decrease in production of NO synthesis due to test flavonoid (rutin, catechin and hesperidin)
which could be predisposed due to inhibition of underlying enzyme (nitric oxide synthase).
Similar interactions were seen in docking studies.
Conclusion
In conclusion, test flavonoid (rutin, catechin and hesperidin) modulated the immune system by
acting on cellular and humoral levels in experimental models of immunity in animals. test
flavonoid (rutin, catechin and hesperidin)also decreased cyclophosphamide induced
myelosuppression. There was an increased reduction of foreign particles, antibody titer, and
immunoglobulins. There was suppression of proinflammatory cytokines and NO production.
Outcomes of the present study established these flavonoid (rutin, catechin and hesperidin) to be
an immunomodulatory agent. Rutin showed significant immunomodulatory effect, followed
by hesperidin and catechin. Additional studies on them are necessary to establish its
therapeutic effects in autoimmune diseases.
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List of Publications
Review Articles
1. Ganeshpurkar, A., Saluja, A.K., The Pharmacological Potential of Rutin. Saudi
Pharmaceutical Journal 2016. doi:10.1016/j.jsps.2016.04.025. Impact Factor 2.611.
(Cited 101 times till date)
2. Ganeshpurkar A, Saluja, AK. Experimental Animal Models Used for Evaluation of
Potential Immunomodulators: A mini review. Bulletin of Faculty of Pharmacy, Cairo
University 2017; 55; 211-216.
Research Articles
1. Ganeshpurkar A, Saluja AK. 2017. Protective effect of rutin on humoral and cell
mediated immunity in rat model. Chemico-Biological Interactions. 2017;273:154-159.
Impact Factor 3.143.
2. Ganeshpurkar A, Saluja AK. 2017. Protective effect of catechin on humoral and cell
mediated immunity in rat model. International Immunopharmacology. 2018;273:261-
266. Impact Factor 3.067.
3. Ganeshpurkar A, Saluja AK. In silico interaction of Rutin with some
immunomodulatory targets: A Docking analysis. Indian Journal of Biochemistry &
Biophysics. 2018; 55 :88-94 (Impact Factor 0.385)
4. Ganeshpurkar A, Saluja AK. In silico interaction of Catechin with some
immunomodulatory targets: A Docking analysis. Indian Journal of Biotechnology.
2018; 17: 626-631 (Impact Factor 0.368)
5. Ganeshpurkar A, Saluja AK. In silico interaction of Hesperidin with some
immunomodulatory targets: A Docking analysis. Indian Journal of Biochemistry &
Biophysics (Gallay Proof) (Impact Factor 0.385)
Conferences/ Seminars
Conference: 32 th
M.P. Young Scientist Congress
Venue: Vigyan Bhawan, Madhya Pradesh Council of Science and Technology,
Bhopal
Place: Bhopal, Madhya Pradesh
Date: March, 10-11, 2017
Title of the paper: Modulatory effect of rutin on humoral and cell mediated immunity
in rat model.
Conference: International Conference on Emerging Trends in Drug Discovery and
Development (ETDDD)
Venue: Department of Pharmaceutical Engineering and Technology, Indian Institute
of Technology, Varanasi
Place: Varanasi, Uttar Pradesh
Date: January 18-20, 2018
Title of the paper: Modulatory effect of catechin on humoral and cell mediated
immunity in rat model.