Ameliorative Effect of Ziziphus Mauritiana (Lamk.) Extract against
Transcription
Ameliorative Effect of Ziziphus Mauritiana (Lamk.) Extract against
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: [email protected]) 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 12 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. References Bajgar, J. (2004). 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