ISOLATION OF IMMUNOMODULATORY LECTIN FROM SEEDS OF ZIZYPHUS MAURITIANA

Ashwin B. Butle 1 and Mandakini B. Patil 2* . 1. Research Scholar, Department of Biochemistry, RTM Nagpur University, Nagpur, LIT Premises, Amravati Road, Nagpur 440033, India. 2. Professor and Head, Department of Biochemistry, RTM Nagpur University, Nagpur, LIT Premises, Amravati Road, Nagpur 440033, India. ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript History

Immunomodulation is the process in which immune response is altered either due to poor nutrition or infectious diseases. Usually the synthetic compounds as immunomodulators are employed to induce or suppress the immune system but they appear to have many adverse effects. Therefore, the use of natural compounds as immunomodulators with less or no side effects is the requirement of time. Plant lectins have been shown to have immunomodulatory activitiy. In this context, in the present study an isolated lectin from seeds of Zizyphus mauritiana was tested for its immunomodulatory activity in vitro. The lectin was isolated and purified by the standard procedures. Immunomoduatory activity was tested with sheep blood lymphocytes including MTT cytotoxicity assay, lymphocyte proliferation assay with XTT, phagocytic activity assay and lysosomal enzyme activity assay. The purified lectin was found to be tripeptide in nature. It was found that the lectin has little or no cytotoxicity towards sheep lymphocytes with maximum concentration up to 200 µg/ml (p<0.0001) and effective dose was found to be 12.5 µg/ml (p<0.0001). Lymphocyte proliferation with effective dose was found to be significantly increased as compared to controls (p<0.0001) and it was found to be more than the LPS (p<0.0001) and that of Con-A (p<0.05) as standard mitogens. Increased phagocytic and lysosomal enzyme activity of the macrophages was observed with purified lectin (12.5 µg/ml, p<0.0001) as compared to the control. Atropine solution, used as negative control, did not show any phagocytic activity (p<0.0001) and lysosomal enzyme activity (p<0.001). From these findings it appears that the lectin of Zizyphus mauritiana can be employed as natural immunomodulatory agent to advance human health. 2002 suppression of immune system. Immunostimulants are the agents that stimulate the response of immune system while immunosuppressants are those agents which suppress an immune response. Immunomodulators are natural or synthetic substances which help to regulate or normalize the immune system (Patil et al., 2012).
The immune system is the most versatile biological system of body. The basic role of immune system is to differentiate between the self from non-self in host body (Patchen et al., 1987). The immune responses of human body are of two types, an innate (natural or non-specific) and the other is adaptive (acquired or specific). Both these responses have two components each, viz. cellular and humoral (Benny and Vanitha, 2004). The leukocytes in innate response act as weapons of an immune system keeping them in a highly prepared state for encounter with non-self. Leukocytes are of two types: granulocytes (neutrophils, eosinophils and basophils) and agranulocytes (Tlymphocytes, B-lymphocytes and monocytes) (Abbas et al., 2000). The innate responses use phagocytic cells: neutrophils, monocytes, and macrophages, cells that release inflammatory mediators: basophils, mast cells, and eosinophils and natural killer cells. The molecular components of innate response include complement, acute-phase proteins and cytokines such as the interferons. Acquired immune responses involve the proliferation of B and T cells, which occur when the antigen binds to the surface receptors of these cells ( Delves and Roitt, 2000).
Phagocytosis by the leukocytes is major event that occurs as immune response shown by neutrophils or macrophages. These phagocytes are having specialized receptors to recognize specific antigen; this complex machinery mediates degradative mechanisms that terminate in killing and disposal of engulfed antigens. Elimination of antigen in phagolysosome takes place by reactive oxygen species, reactive nitrogen species, anti microbial proteins (proteases, elastases, lysozyme), anti microbial peptides (defensins form pore in bacterial membrane), binding protein (lactoferrin), acidification. Another method of magrophage to kill pathogens is opsonization in which antibody functions as an opsonin, a molecule that binds to both antigen and macrophage and enhances phagocytosis with the help of lysosomal enzymes (Kindt et al., 2007;Lee et al., 2003).

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Immunomodulators are natural or synthetic molecules that help to normalize the host immune system. Natural immunomodulators are generally less effective than synthetic immunomodulators and are also less likely to cause side effects than synthetic compounds (Patil et al., 2012). Therefore, there is a need of most potent natural immunomodulatory molecules having minor or no adverse effects in the coming decades of 21 st century. In the presented work the lectin was isolated by the standard protocols from the seeds of Zizyphus mauritiana and tested for the in vitro immunomodulatory activity (Butle and Patil, 2016).

Blood Samples:-
Rabbit blood samples were collected for the estimation of hemagglutination activity of lectin with rabbit erythrocytes. Rabbit blood was collected (following instructions of CPCSEA, Govt. of India) from Departmental Animal and Breeding House, University Department of Biochemistry; RTM Nagpur University, Nagpur (MS), India.
Sheep blood (for the isolation of lymphocytes) was obtained from the Animal Husbandry Department of Veterinary Collage, Nagpur (India). Blood was collected in heparinized vials for isolation of lymphocytes.

Isolation and Purification of lectin:-
Isolation and purification of lectin from cotyledons of Zizyphus mauritiana was done as described earlier (Butle and Patil, 2016). Cotyledons were crushed in mortar pestle using liquid nitrogen treatment and homogenized with 0.02M sodium phosphate buffered saline (PBS) at pH 7. Proteins in crude extract were precipitated by ammonium sulphate at 0 to 35% concentration, solubilized in small volume of extraction medium and dialyzed against same for 14 h at 4 o C and designated as ammonium sulphate fraction (ASF) (Appukuttan et al.,1977). Three ml ASF was loaded on affinity column (3 g guar gum packed in 10 *0.5CM long column). The bound proteins were eluted from the column by the extraction solution containing 0.4 M galactose and dialyzed as previously described (Appukuttan et al.,1977).

Methods for in vitro immunomodulatory activity:-2.2.2.1 Isolation of lymphocyte from sheep blood:-
Five ml of sheep peripheral blood collected into heparin-containing bottles was mixed in the ratio of 1:1 with complete RPMI-1640 medium (containing 10% heat inactivated fetal calf serum (FCS) supplemented with antibiotic-antimycotic solution). The diluted blood was then layered over Histopaque1077 gradient (Sigma, St. Louis, M.O.), and centrifuged at 400 g for 40 min. Lymphocytes (PBLs) were harvested from interphase white layer, and washed thrice with complete RPMI medium. The lymphocyte concentration was adjusted to1x10 6 cells/ml. The suspension was considered as macrophage suspension for phagocytic and lysosomal enzyme activity assay. Viability of cells was determined by trypan blue dye exclusion, and was consistently greater than 96% (Kainthala et al., 2006).

MTT assay for effective concentration of lectin:-
The MTT assay was performed to assess the cytotoxic effects of the lectin on isolated lymphocytes. This assay evaluates cell viability based on the reduction of MTT by mitochondrial succinate dehydrogenase enzyme of the viable cells to a purple formazan product (Mosmann, 1983). The cells were suspended in growth medium (complete RPMI 1640) and seeded in 96-well plates at a density of 2 × 10 6 cells/well and incubated at 37 o C for 24 h to attain 80% confluence. Stock concentration of lectin dissolved in PBS and made to a final concentration ranging from 1.5 to 200 g/mL was prepared. Cells were exposed to the standard concentrations LPS and Con-A (10 g/mL and 5 g/mL respectively) serving as a standard control and PBS with cells as a positive control (PC).The plates were incubated with 5% CO 2 and 40% humidity at 37 o C for 24, 48, and 72h in CO 2 incubator (Thermo Scientific, Class-100). After incubation, a volume of 50 L of MTT (5mg/mL) was added in every well and and the plates were incubated at 37 o C for 4 h. The media was aspirated and cells were washed with phosphate-buffered saline (PBS). The cells were dissolved in 100 L DMSO and absorbance was measured at 570 nm using a microtiter-plate reader (Thermo electron corp., 358) (Frehney, 1987). The viability of cells expressed as lymphocyte proliferation stimulation index (LSI), was calculated as: LSI = O. D. of Experimental / O. D. of control

XTT assay for proliferation of lymphocytes:-
Effect of lectin on lymphocyte proliferation was tested by XTT assay (Kainthala et al., 2006). Lymphocyte suspension (1x10 6 cells/well) suspended in complete RPMI 1640 medium was treated with double diluted different concentration of lectin from the effective dose dissolved in PBS. The PBS was used as a control while atropine solution was used as a negative control (NC). Two groups were treated with 10 µg/ml of LPS and 5 µg/ml of Con-A individually and considered as standards (a powerful known mitogens). After incubation for 48 h at 37 o C in 5% CO 2 humidified atmosphere, the medium was removed and the adherent lymphocytes were washed twice with RPMI medium. A mixture of XTT and PMS was then introduced along with RPMI 1640 medium. Cells were then incubated for 4 h at 37 o C in 5% CO 2 humidified atmosphere. Absorbance was measured at 450 nm using microplate reader. Lymphocyte proliferation stimulation index (LSI) was determined as: LSI = O. D. of Experimental / O. D. of control

Phagocytic activity assay:-
Capacity of lectin to stimulate phagocytosis was tested by NBT dye reduction assay (Rainard, 1986). Macrophages (1x10 6 cells/well) suspended in complete RPMI 1640 medium were treated with different double diluted concentration of effective dose of lectin dissolved in PBS. Control was run with PBS (without lectin, designated as C) and atropine solution was used as a negative control (NC). After incubation for 24h at 37 o C in 5% CO 2 humidified atmosphere, medium was removed and adherent macrophages were washed twice with RPMI medium. Zymosan -A (1µg/ml in PBS) was introduced along with NBT solution (1.5 mg/ml of PBS) and cells were incubated for 60 min at 37 o C in 5% CO 2 humidified atmosphere. After 60 min, medium was removed and cells were washed twice with PBS and air dried. Finally, 2 M KOH was added and absorbance was measured at 570 nm using micro plate reader. Phagocytic index (PI) was calculated by following equation

Lysosomal enzyme activity assay:-
Ability of lectin to stimulate lysosomal enzyme activity (acid phosphatase) was tested using pNPP assay (Suzuki et al., 1988). Macrophages (1x10 6 cells/well) suspended in complete RPMI 1640 medium and were treated with different concentration of lectin from the observed effective dose in MTT assay dissolved in PBS. Control was run with PBS (without lectins) and atropine solution was used as NC. After incubation for 24h at 37 o C in 5% CO 2 humidified atmosphere, medium was removed and adherent macrophages were washed twice with RPMI medium. 1 % Triton X-100, 10 mM pNPP and 0.1 M citrate buffer (pH 5.0) were then introduced and cells were incubated for 37 o C in 5% CO 2 humidified atmosphere. After 30 min, 0.2 M borate buffer (pH 9.8) was added and absorbance was measured at 405 nm using microplate reader. Lysosomal enzyme activity index (LI) was calculated according to equation: LI = O. D. of Experimental / O. D. of control

Statistical analysis:-
Each experiment with lymphocytes was run in triplicate, and the results are expressed as their mean±SD (standard deviation). The data was compared and analyzed using student's 't' test. P ≤ 0.0001 was considered as significant. All the graphs were plotted by 'Sigma Plot 10.0' software and statistical analysis was done with 'MedCalc 10' software.

Isolation and Purification of lectin:-
The lectin was purified from 0-35% ammonium sulphate fraction on guar gum was principally based on affinity binding towards the ligand D-galactose. The 0.20M concentration was found to be suitable for elution of lectin on DEAE-cellulose on ion-exchange chromatography and gives maximum agglutination and single band on non denaturing PAGE confirming purity of lectin. Table 1 show that the lectin was purified in the good yield (22%) with specific activity 17066 with 20480 HAU/ml. The positive phenol sulphuric acid test of the isolated lectin shows attached carbohydrate moiety in the structure of the lectin indicating glycoprotein nature of the purified lectin. The homogeneity of isolated lectin was checked on 10% Non denaturing PAGE. Figure

Isolation of lymphocyte from sheep blood:-
White interphase layer of sheep blood was separated from density gradient medium and washed thrice with complete RPMI 1640 medium. Concentration of cells was calculated by 'Improved Neubauer Chamber'. The reactivity of the trypan blue is based on the fact that the chromophore is negatively charged and does not interact with the cells unless the membrane is damaged, therefore all the cells which exclude the dye are viable (Freshney, 1987). Trypan blue is a diazo dye and used as a vital stain specifically to color dead cells ('Trypan blue', www.wickipedia.com). Viability of cells by trypan blue dye exclusion was observed consistently greater than 96%. Maximum viability of cells has been found in medium indicates better isolation and suitable concentration of ingredients in complete RPMI 1640 medium for lymphocyte growth.

MTT assay for effective concentration of lectin:-
Very few lectins from plants possess the unique capacity to induce inactive lymphocytes to grow and divide, a phenomenon known as mitogenic stimulation (Kilpatrick, 1999). Mitogenic lectins copy the action of antigens on lymphocytes, except that they activate a large proportion (as much as 70-80 %) of the cells, whereas antigens stimulate only specific clones, each of which cooperate a minute proportion, 0.1% or less, of the total lymphocytes (Ashraf and Khan, 2003). The isolated lectin was found to be fairly effective at dose 12.5 µg/ml (*p<0.0001) in stimulating lymphocyte proliferation. In figure 2 it was observed that the little or no cytotoxic effect by the isolated lectin above the effective dose (25 -200 µg/ml, *p<0.0001). The proliferation index at higher concentration was not increased as the standard mitogens (LPS and Con-A, *p<0.0001) but comparatively more than PC. The proliferation index of lymphocyte at lower concentration of lectin than the effective dose (6.15 -1.5 µg/ml, *p<0.0001, #p>0.05) was found to give dose dependent (Fig 2). Related study was done with lectin of Phaseolus vulgaris possessing an ability to stimulate lymphocytes to undergo mitosis (Sharon, 2007).

XTT assay for proliferation of lymphocytes:-
In the XTT assay (Fig 3), it was observed that the effective dose of isolated lectin significantly proliferate the lymphocytes and comparable with PC (*p<0.0001). The proliferation index of effective dose of lectin gives increased proliferation than standard known mitogens (LPS and Con-A, *p<0.0001, #p<0.05 respectively). Dose dependent decreased proliferation index was observed with double diluted concentration of lectin (@p=0.001, Purified lectin from seeds of Zizyphus mauritiana was explored for its phagocytosis stimulating activity at different concentrations by dissolving it in PBS. It was observed that the effective concentration has shown its maximum activity (*p<0.0001, Fig 4). Atropine, a standard alkaloid used as negative control did not show any considerable significant activity. From the result of phagocytic activity assay it was confirmed that the lectin from seeds of the Zizyphus mauritiana causes an increase in the phagocytic activity. Similarly lectin isolated from Bauhinia variegata seeds has been considered as potent chemotactic agents for neutrophils through the release of cytokines (Neto et al., 2011).

Lysosomal enzyme acticity assay:-
The lectin shows dose dependent increase in the activity of lysosomal enzymes. At an effective concentration, the lysosomal enzyme activity has shown an increase of more than double as compared to controls PC and NC (*p<0.0001, #p>0.01, Fig 5). The Atropine used as a negative control shows no effect on enzyme activity. It was studied that enzymes and mediators of inflammation usually sequestered within lysosomes are released from viable polymorphonuclear leukocytes (PMNs) during phagocytosis (Goldstein et al., 1973). Galactose binding non toxic plant lectins was found to be very effective for cells of several organs of body in lysosomal storage disease (Radin and Jonesboro, 2013). Glycan binding lectins like galectin-8 also found to activate lysosomal enzymes in the antigen degradative pathways of the cell (Lichtenstin and Rabinovich ., 2013).

Conclusion:-
As per the results presented above lectin from the seeds of Zizyphus mauritiana was isolated and purified to homogeneity. The isolated lectin was tripeptide and did not show cytotoxic effect with lymphocytes. This lectin was shown to have great impact in stimulating phagocytosis in a dose dependent manner. These results were supported by lysosomal enzyme activity assay which indicate an immunostimulation in positive direction. The isolated lectin was found to be efficient and being natural, the chances of side effect are less if employed as an immunostimulating agent in vivo.