1. health and fitness
2. disease

Sciknow Publications Ltd.
FBLS 2015, 3(1):9-13
DOI: 10.12966/fbls.03.02.2015
Frontiers of Biological and Life Sciences
©Attribution 3.0 Unported (CC BY 3.0)
Ameliorative Effect of Ziziphus Mauritiana (Lamk.) Extract
against DDVP Induced Immunotoxicity
Shankarjit Singh , and Aruna Bhatia*
Department of Biotechnology, Punjabi University, Patiala, India
*Corresponding author (Email: aruna_bhatia@rediffmail.com)
Abstract - The aim of the present study was to find out the protective nature of biotherapeutic property of Ziziphus mauritiana
(Lamk.) seed (ZMS) extract against Dichlorvos (DDVP) induced immunotoxicity. Lymphocytes were collected from pig spleen
and incubated with DDVP (100 µg/ml) and ZMS (400 µg/ml) separately and also in combination of DDVP+ZMS treatment. In
case of DDVP treatment, activity of Nitroblue tetrazolium (NBT) reduction, inducible nitric oxide synthestase (iNOS) and
Phagocytosis was decreased in comparison of control value, but reverse is observed in case of ZMS treatment. The result of the
present study suggests that ZMS is an effective immunopotentiator for DDVP immunotoxicity burden. It can be concluded that
DDVP induced immunotoxicity in pig spleen lymphocytes and ZMS has ameliorated these ill-effects.
Keywords - Ziziphus Mauritiana (Lamk.), DDVP, Immunotoxicity, NBT, iNOS, Phagocytosis
1. Introduction
The use of pesticides has become indispensible in the modern
life to get better produce and hence in turn is released into the
environment deliberately. Pesticides are a very important
group of environmental pollutants used intensively in
agriculture for protection against diseases and pests. A
pesticide is any substance or mixture of substances intended
for preventing, destroying, repelling, or mitigating any pest.
Though different classes of pesticide exist but
Organophosphorous pesticides (OPs) account for up to 50%
in all insecticides worldwide (Casiada & Quistad, 2004).
Moreover the ban on Organochlorine pesticides has resulted
in popularity of OPs. Though OPs are permissible for their use
but their ill effects have also been reported in literature. The
major toxicity of organophosphorus pesticides includes
neurotoxicity, caused by the inhibition of acetylcholinesterase
(Pope, 1999; Bajgar, 2004). It has been reported that OPs
affect immune response including effects on neutrophil
function (Hermanowicz & Kossman, 1984), macrophage
(Rodgers, Imamura, & Devens, 1985; Rodgers & Ellefson,
1988; Rodgers & Ellefson, 1990; Crittenden, Carr, & Pruett,
1998), antibody production (Casale et al., 1983; Johnson et al.,
2002), IL-2 production (Pruett & Chambers, 1988), serum
complement (Casale et al., 1989), and T cell proliferation
induced by IL-2 (Casale et al., 1993), concanavalin A and
phytohemagglutinin (Blakley et al.,1999).
Dichlorvos
(O-O-dimethyl-O-2,
2-dichloro-vinyl
phosphate; DDVP) is one of the widely used organophosphate
insecticides. The mechanism for the toxicity of
organophosphates
is
mainly
by
blocking
of
acetylcholinesterase – an enzyme which decomposes
acetylcholine (Harlin & Dellinger, 1993). Dichlorvos also
causes disturbances in the flow of ions through membranes by
inhibition of enzymes which regulate this flow (Gallichio et
al., 1987). DDVP significantly decreased human NK, LAK
and CTL activities in-vitro. DDVP inhibits the enzymatic
activity of granzymes (Li et al., 2002). It also inhibits the
expression of granzymes, granulysin and perforin in human
NK cells, as well as induction of degranulation of NK cells (Li
et al., 2005). Other study supports a role of reactive oxygen
species (ROS) in the mechanism of dichlorvos toxicity
(Sharma & Singh, 2012). Excessive generation of ROS causes
irreversible impairment of DNA, damage to membrane lipids
leading to the production of Malondialdehyde (MDA) (Gawel
et al., 2004).
The living cells have different mechanisms to alleviate
oxidative stress and immunotoxicity which is offered by
antioxidants and immunopotentiator agents. Plants produce
an extensive variety of antioxidant and immunopotentiator
compounds which make them suitable nutritional
supplements against toxic effects. Extracts of many plants as
well as their products have been found to possess
biotherapeutic potential. Z. mauritiana (Lamk.) commonly
known as Indian jujube is a fruit tree belonging to family
Rhamnaceae. The whole plant and leaves of Z. mauritiana
have been employed in traditional medicine as a tonic. The
extracts from fruits (Ndhala et al., 2006), leaves (Dahiru et al.,
2005; Dahiru & Obidoa, 2007), and seeds (Bhatia & Mishra,
2009) of Z. mauritiana have been reported to exhibit
10
Frontiers of Biological and Life Sciences (2015) 9-13
antioxidant activity. Z. mauritiana (Lamk.) has shown
ameliorative effects against Chlorpyrifos induced oxidative
stress (Singh & Bhatia, 2013).
Keeping in view the very vast traditional and medicinal
uses of Z. mauritiana we decided to evaluate ameliorative
immunopotentiator activity of the aqueous- ethanolic seed
extract against DDVP induced immunotoxicity in pig spleen
lymphocytes (in-vitro).
extract was added in test set and non-treated lymphocytes in
control set. The control and test was incubated at 37 °C for 20
minutes. 0.1 N HCl was added to mixture and centrifuged at
4000 rpm for 3 min. 5 ml of dioxan was added to both the test
and control sets and incubated at 70 °C for 20 min. Then
mixture was centrifuged at 4000 rpm for 5 min. Absorbance
of supernatant was noted at 520 nm, using dioxan as blank.
The % NBT reduction was calculated using the following
formulae:
2. Materials and Methods
The Pig spleen was used to assess the in vitro
immunomodulation studies. Spleen was excised aseptically
and used as source of lymphocytes. Spleen was teased in
MEM (Minimum Essential Media) and cells were collected
after centrifugation (400 ×g for 10 minutes at 4°C). The pellet
was then resuspended in MEM and lysed the cells by ACK
lyses buffer. Lymphocytes obtained were washed thrice in
PBS and count was adjusted to 2×10 6 cells / ml in MEM and
was used for the different parameters: NBT reduction, iNOS
activity, Phagocytosis activity.
2.1. Extract Preparation
Fruits of Z. mauritiana variety Umran were collected from
Botanical Gardens of Punjabi University Patiala, Punjab,
India and authenticated by Department of Botany, Punjabi
University Patiala, Punjab, India. The seeds of Z. mauritiana
were shade dried at room temperature and reduced to coarse
powder. The 10 g powder was extracted with 100 mL mixture
of ethanol: water (1:1). The solvent was completely removed
using rotary evaporator (Heidolph, Germany) under reduced
pressure at 50±5°C to yield the ZMS 11-12% (w/w).
2.2. Treatment of Lymphocytes
Lymphocytes adjusted to 2×10 6 cells/ ml in MEM were
incubated with ZMS (400 µg /ml), DDVP (100 µg/ml),
mixture of ZMS and DDVP (ZMS + DDVP) at 37 °C for 24 h
in humified CO2 chamber.
2.3. Chemicals and Reagents
DDVP provided by Hindustan Insecticide Limited, Bathinda,
Punjab, India. MEM, Nutrient broth and Nutrient agar
purchased from Hi-media, Mumbai. NBT, dioxin and
L-arginine purchased from Merck. All the other chemicals
used were of analytical grade.
2.4. Determination of Nitroblue Tetrazolium (NBT)
Reduction
NBT reduction is based upon the principle that whenever a
particle is ingested by a phagocyte, a respiratory burst is
induced. The nitroblue tetrazolium (NBT) reduction test is an
indirect marker of the oxygen dependent bactericidal activity
of the phagocytes. NBT reduction was measured using
spectrophotometric method (Mishra and Bhatia, 2010). For
each test sample, two sets of test tubes were taken one as
control as other as test. 1ml of lymphocytes treated with
2.5. Determination of Inducible Nitric Oxide Synthestase
(iNOS)
iNOS activity is based on the principle that when macrophage
are activated they express iNOS which oxidizes L-arginine to
citrulline and nitric oxide. The coloured citrulline is
extractable with Griess reagent. iNOS activity was assayed
spectrophotometricaly (Stucher & Marleta, 1987). Briefly,
1ml of treated lymphocytes was taken in test set and
non-treated lymphocytes in control set. Freshly prepared 1ml
MEM and 20µl L-arginine solution was added to both control
and test sets and incubated at 37 °C for 24 h in humified CO 2
chamber. The mixture was centrifuged and supernatant was
treated with Griess reagent and kept for 10 min in dark. O.D
was taken at 540 nm against Greiss reagent as standard. The %
iNOS activity was calculated using the following formulae:
2.6. Determination of Phagocytosis Activity
Phagocytosis activity of immunocytes was measured by the
plate count method (Reghuramulu et al., 1983). Bacterial
culture (Escherichia Coli) was inoculated in 50 ml of nutrient
broth and incubated at 37 °C for 16-18 hours. The bacterial
culture was centrifuged and pellet was washed twice with
KRP buffer again centrifuged and supernatant was discarded.
The washed pellet was suspended in saline and number of
cells was adjusted to 1×106 cells per ml. To the test set, 0.5 ml
of treated lymphocytes, 80 µl of E.coli (1×10 6 cells per ml)
were added. In case of control set only 80 µl of E.coli (1×106
cells per ml) was added. Volume of both sets was made 2 ml
with KRP buffer. The mixture was incubated at 37°C for 1 h
and 0.02 ml of reaction mixture was taken from both the test
tubes. To the reaction mixtures 0.8 ml of sterile distilled water
was added to lyse the lymphocytes. The mixtures were further
diluted to 108 times with normal saline and number of viable
E.Coli cells were counted by plating method. The %
Phagocytic activity was calculated using the following
formulae:
Frontiers of Biological and Life Sciences (2015) 9-13
2.7. Phytochemical Analysis
To preliminarily assess the different constituents of ZMS,
phytochemical analysis was done as described by the method
of Harborne (1973).
3. Results
3.1. NBT Reduction
The level of NBT reduction was in increasing order of DDVP
< Control < DDVP+ ZMS< ZMS. In the case of DDVP
11
treatment NBT reduction was 17.14% less as compared to
control. The result shows significant decrease in macrophage
activity. The NBT reduction was increased to 9.26% in case
of mixture of DDVP+ ZMS treatment as compared to control
and came in normal range (Fig.1). ZMS alone showed 39.52%
more reduction in NBT as compared to control. Results
indicated that the presence of plant extract (ZMS) have
significantly mitigated the reduction of macrophage activity
caused by DDVP.
b
60
% NBT reduction
50
40
30
c
a
20
a
10
0
Control
P.E.
DDVP
P.E.+ DDVP
Fig. 1. NBT reduction in pig spleenocytes of the treatment groups. P values: a≤0.001, b≤0.05 and c≤0.01
3.2. iNOS Activity
The percentage iNOS activity for control, ZMS, DDVP,
combination of both DDVP and ZMS was 20.24%, 59.25%,
5.2 % and 34.3% respectively (Fig. 2). So the maximum
percentage iNOS activity was observed in ZMS and minimum
in DDVP. In case of DDVP in combination of ZMS, the iNOS
activity was increased than control.
a
60
% iNOS activity
50
40
c
30
a
20
10
b
0
Control
P.E.
DDVP
P.E.+ DDVP
Fig. 2. iNOS activity in pig spleenocytes of the treatment groups. P values: a≤0.05, b≤0.001 and c≤0.01
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Frontiers of Biological and Life Sciences (2015) 9-13
3.3. Phagocytosis
The percentage phagocytotic activity for control, ZMS,
DDVP, combination of both DDVP and ZMS was observed to
be 21.4%, 57.5%, 4.1%, 32.24% respectively (Fig.3). So the
increasing order of percentage phagocytotic activity was
DDVP < Control < DDVP+ ZMS< ZMS. The results revealed
that DDVP shows minimum phagocytosis but the presence of
plant extract (ZMS) has significantly increased the
bactericidal activity as compared to control. ZMS has
mitigated the effects of DDVP on bactericidal activity.
a
60
% Phagocytic activity
50
40
c
30
a
20
10
b
0
Control
P.E.
DDVP
P.E.+ DDVP
Fig. 3. Phagocytosis activity in pig spleenocytes of the treatment groups. P values: a≤0.05, b≤0.001 and c≤0.01.
3.4. Phytochemical Analysis
Table1 shows the qualitative determination of flavonoids ,
alkaloids, terpenoids, proteins, saponins and carbohydrates.
Table 1. Phytochemical analysis of ZMS
Phytochemicals
Flavonoids
Presence
++++
Alkaloids
Tarpenoids
Proteins
Saponins
Tannins
Carbohydrates
+++
+++
++
+
+
++
4. Disscussion
The immune system is a complex biological system of
regulatory genes, hormones, antibodies and cells that has
evolved in organisms for the defensive mechanism against
foreign
pathogens
and/or
environmental
agents.
Environmental pollutants can impede with the normal
function of the immune system leading to a broad range of
disorders, diseases and dysfunction of immune system itself
i.e. immunotoxicity. Immunotoxicity may include decreased
humoral and/or cell mediated immunity, altered non-specific
immunity, decreased host resistance, pathology of immune
organs, hypersensitivity, autoimmunity etc.(Handy &
Gallowy, 2003). DDVP may lead to in vitro immunotoxicity
by decreasing human NK, LAK or by inhibiting activities of
CTL, granzymes etc. (Gallicho et al., 1987; Li et al., 2002). In
the present study, we have evaluated the ameliorative effect of
ZMS extract against DDVP induced immunotoxicity in
lymphocytes. The immunotoxic effects in lymphocytes were
studied by measuring the level of NBT reduction, iNOS test
and phagocytosis activity.
The results of the present study have revealed that the
NBT reduction was significantly decreased in pig
lymphocytes treated with DDVP as compared to control. The
decreased level of NBT reduction has indicated that DDVP
induced immunotoxicity. The results of the present study have
revealed that the iNOS activity was significantly decreased in
pig spleenocytes treated with DDVP as compared to control.
The decreased level of iNOS activity has indicated that DDVP
induced immunotoxicity. The bactericidal activity of pig
spleenocytes was also reduced by the DDVP treatment.
Biotherapeutic potentials of medicinal plants against
pesticides induced immunotoxicity remain an area that needs
extensive scientific research. The results of the present study
clearly indicated that ZMS extract has found to be effective in
improving the immune activity of pig spleenocytes. The
supplementation of ZMS to DDVP has increased the levels of
NBT reduction, iNOS activity, and phagocytosis as compared
to control. So far, therapeutic potential of Z. mauritiana has
been evaluated against several chemical toxicants (Dahiru et
al., 2005; Dahiru and Obidoa, 2007; Bhatia and Mishra, 2009).
Bhatia and Mishra (2010) studied the protective effect of Z.
mauritiana against alcohol induced oxidative stress. The
recent studies revealed the ameliorative effects of Z.
mauritiana against Chlorpyrifos induced toxicity (Singh and
Bhatia, 2013). Hitherto, no similar experimental scientific
Frontiers of Biological and Life Sciences (2015) 9-13
study has been reported for mitigation of DDVP induced
immunotoxicity by plant extract.
It has been argued that phytochemicals possess
antioxidant potential (Krishnaiah et al., 2007) and
antioxidants improve the immune system (Victor & De la
Funete, 2002). The immune cell functions are greatly
influenced by the antioxidant – oxidant balance. The
oxidative stress caused by DDVP may lead to immunotoxicity
and the stabilizing antioxidative and immunostimulatory
effects of phytochemical such as flavonoids and terpenes
present in ZMS may be responsible for its potent ameliorative
activity. For the precise mechanism of the observed protective
effect a detailed study is required to explore it.
5. Conclusion
In view of the data of the present investigation, it can be
concluded that DDVP has induced immunotoxicity in pig
spleenocytes but supplementation of ZMS has ameliorated
these effects. This study clearly indicates that Z. mauritiana
seeds inhibit the immunotoxic effects and can be used as good
source of immunostimulatory diet supplement.
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