Immunomodulatory Efficiency of Abelmoschus esculentus in Swiss Albino Mice

Modulation of the immense system denotes to change in the immune response that can involve induction, expression, amplification or inhibition of any part or phase of immune response. Modern immunology aims to modify the immune system to prevent or control diseases using immuno modulatory tool. Interaction between foreign particle and immune cells was initiated by way of differentiation in immune cells in mice due to administration of plant extract (Goldsby et al., 2002). Excessive application of antibiotic instead of controls the diseases, develop antibiotic resistant microbes and cause various side effects (Dutta et al., 1998). 
       
Immunomodulation cause immunostimulation of cells or production of their metabolic inducers or by inhibiting the immunity limiting factors (Agarwal and Singh, 1999). Medicinal plants serve as therapeutic alternatives, safe choices, or some cases, as the only effective treatment. Secretary proteins present in plant act as an immuno modulator in immuno -suppressed animals and help to develop immunity. The plant extract administered mice have immuno-enhansive property due to plant extract proteins (Szalai et al., 1996). A large number of these plants and their isolated constituents have shown beneficial therapeutic values, including anti-oxidant, anti-inflammatory, anti-cancer, anti-microbial and immunomodulatory effects (Miller et al., 2004). In the present investigation analysed immuno modulatory response of A. esculentus using Swiss albino mice.

In the present study the plant A. esculentus samples were collected in the early morning during summer season of April, 2015 for doctoral research purpose and experiments were carried out in Prathyusha Engineering College, Tiruvallur, Tamilnadu, India. Immunomodulatory effect of the plant A. esculentuson the dynamics of lymphocyte and DTH response were assessed in a mice (Swiss albino) model. Either sex of laboratory breed mice (Two months of age; weighing 25-30g) were used to evaluate the immuno modulatory activity of different organic extracts of the tender fruits of A. esculentus. Mouse was housed in polyvinyl cage littered with paddy husk under standard condition and fed with balanced pellet diet (Lipton, India Ltd) and tap water ad libitum.
       
The extract (Weighted quantity) was dissolved in sterilized distilled water, and the concentration of 100 g/kg/day was prepared. The plant extract was dissolved in water and fed to the mice along with drinking water using a special feeding bottle. Cyclophosphamide (Khandelwal Laboratories, India) was used as a standard immuno-suppressant drug. Proimmu (Envin-bioceuticals, Shorapur, India) was used as standard immune potentiating drug. Cellular antigens such as sheep erythrocytes were obtained from fresh blood of sheep sacrificed in the local slaughterhouse. Sheep red blood cells (SRBC) was prepared by washing sheep blood in phosphate buffer saline thrice by· centrifuging at 3000 rpm for 10 minutes. Packed volume of SRBC is resuspended to get a concentration of 0.1 ml containing 1 x10⁸ cells for immunization and challenge.
 
Experimental design
 
A total of nineteen groups (each group containing six mice) of mice were experimented for immunological studies with an inclusion of control and immunised control. Drugs were administered to various groups of mice in the following manner. After treatment, the blood sample was drawn from each group of animals with a time interval of 7 days upto 21 days.
 
Group I -          Control (sterile water)
Group II -         Immunised control
Group III -        A. esculentus hexane extract treated group (dose levels of 100 mg/kg)
Group IV -        A. esculentusbutanol extract treated group (dose levels of 100 mg/kg)
Group V -         A. esculentus ethanol extract treated group (dose levels of 100 mg/kg)
Group VI -        A. esculentus chloroform extract treated group (dose levels of 100 mg/kg)
Group VII -       A. esculentus water extract treated group (dose levels of 100mg/kg)
Group VIII -      A. esculentus hexane extract treated group (dose levels of 100 mg/kg)and cyclophosph-amide (30mg/kg)treated group
Group IX -        A. esculentusbutanol extract treated group (dose levels of 100 mg/kg) and cyclophosph-amide (30mg/kg)treated group
Group X -         A. esculentus ethanol extract treated group (dose levels of 100 mg/kg) and cyclophosph-amide (30mg/kg)treated group
Group XI -       A. esculentus chloroform extract treated group (dose levels of 100 mg/kg) and cyclophosph -amide (30mg/kg)treated groupmg/kg)
Group XII -       A. esculentus water extract treated group (dose levels of 100 mg/kg)and cyclophosphamide (30mg/kg)treated group
Group XIII -      A. esculentus hexane extract treated group (dose levels of 100 mg/kg)and Proimmu treated group (30 mg/kg)
Group XIV -     A. esculentusbutanol extract treated group (dose levels of 100 mg/kg)and Proimmu treated group   (30 mg/kg)
Group XV -      A. esculentus ethanol extract treated group (dose levels of 100mg/kg) and Proimmu treated group (30 mg/kg)
Group XVI -     A. esculentus chloroform extract treated group (dose levels of 100 mg/kg) and Proimmu treated group (30 mg/kg)
Group XVII -    A. esculentus water extract treated group (dose levels of 100 mg/kg) and Proimmu treated group (30 mg/kg)
Group XVIII -   Cyclophosphamide alone treated group (30 mg/kg)
Group XIX -       Proimmu treated group (30 mg/kg)
 
B and T cell E rosette assay
 
Blood was collected from treated and control mice as previous mentioned using a heparin pretreated vials. B and T cell count in the blood samples were carried out by the following method. 5-10ml of blood was collected and it was introduced into sterile conical flask/ beaker containing sterile 4 to 5 glass beads. It was then continuously swirled until no sounds were heard from the beads. This indicates that all the fibrins have adhered to the beads. This blood was considered as de-fibrinated blood. This de-fibrinated blood was taken and diluted with equal volume of physiological saline 3 ml of the lymphoprep solution was taken in a centrifuge like. The tube was kept in slanting position and 9 ml of diluted blood was slowly added along the side of the centrifuge tube using Pasteur pipette care was taken so that the FICON layer of the lymphoprep solution present in the centrifuge tube was not disturbed the content of the centrifuge tube was then centrifuged at 1600 rpm for 20min. the interphase (containing lymphocytes) was removed using pipette. The cells were washed with 1 ml saline and excess FICON was removed. The sample was again washed with 1ml of saline after centrifugation the supernatant was decanted by inverting the tube over a filter paper after all saline was drained; the pellet was then resuspended in 300ml of RPM1 1640 medium.
       
Resupended lymphocytes were loaded into the activated nylon wool column. Then the column was held vertically above an eppendorf tube, now hot saline (at out 60°C) was slowly dripped into the column. The hot saline passing out of the column was collected in the eppendorf tube, which contain T lymphocytes. 2.0ml of the saline containing T cell) was taken in a separate eppendorf tube. To this 0.2ml of 1% SRBC was added and then the mixture was centrifuged for 12 minutes at 1600 rpm. After centrifugation the sample were incubated in an ice box or refrigerator (at 4°C) for 5 minutes. After cold incubation, the pellet in the eppendorf till was re-suspended by gentle flushing with a Pasteur pipette. Then a drop of it was taken in a clean dry slide, observed and enumerated T cell under the microscope (20x/40x) for rosettes. Number of T cell rosettes formed were observed among hundred lymphocytes observed was tabulated. After hot saline elution, cold saline was added to separate B lymphocytes. The column in gently squeezed to released the adhered B cells (repeat twice) the cold saline dripping in another eppendorf tube. 0.2ml of the saline containing B lymphocyte (from the eppendorf tube containing B cell) was taken in a separate eppendorf tube. To this 0.2ml of 1% SRBC was added and then the mixture was centrifuged for 12 minutes at 1600rpm. After centrifugation the sample were incubated in an icebox or refrigerator (at 4°C) for 5 minutes. After cold incubation, the pellet in the eppendorf till was re-suspended by gentle flushing with a Pasteur pipette. Then a drop of it was taken in a clean dry slide, observed and enumerated T cell under the microscope (20x/40x) for rosettes. Number of T cell rosettes formed were observed among hundred lymphocytes observed was tabulated.
 
Delayed type hypersensitivity
 
Mice were sensitized by subcutaneous injection in the intranasal region with 0.5 ml of Freunds adjuvant.  Intradermal injection with 500 mg of DNCB after diluted with sterile phosphate buffer was carried out in foot paw of mice. Thickness of the swelling was measured with a Vernier caliper prior to challenge, i.e. 0^th, 3^rd and 24^th hour post challenge, each with three readings.

B-cell production of control and treated animals were estimated by rosette forming assay and recorded in Table 1.  The result showed remarkable changes in all kind of treated animals when compared to control. The increment in ‘B’ lymphocyte number was much pronounced in mice by the administration of A. esculentusin combination with immunoenhansive drug than the lone effect of immuno enhansive drug. B cell decrement was pronounced in mice treated with immunosuppressive drug while a moderate decrement was noticed due to A. esculentus in combination with immunosuppressive drug. The increment in ‘B’ cell count may be due to the impact of plant drug on the synthesis, proliferation and activation of ‘B’ cells in treated animals. Similar results were observed by Dhasarathan et al., (2010) in mice administered with plant extracts. The decrease in ‘B’ cell count in animal administered with immuno suppressive agent might be due to excess oxygen free radical production, increased rapid peroxidation, damage to membrane DNA fragmentation and apotosis due to immunosuppressive drugs thereby suppressed the functioning of immune system (Trebeden Negre et al., 2003).
       
The presence of saponins and flavanoids in the plant A.esculuntusobserved in the present phytochemical analysis may bind with receptor of naive immune cells, which augmented the humoral response by stimulating the macrophages and B-lymphocytes involved in antibody synthesis. Similar immno enhancement was reported in mice due to the influence of terpenoids found in Achillea wilhelmsii (Raphael and Kuttan, 2003).
 

 
The mice exposed to plant extract developed antibody against plant extract that antibody in turn destruct the antigen. This may be the reason for the increased T-Cell production in mice administered with plant extracts (Sharififar et al., 2009).
       
T cell production of control and treated animals were estimated by rosette forming assay and recorded in Table 2.  The result showed remarkable changes in all kind of treated animals when compared to control. The increment in ‘T’ lymphocyte number was much pronounced in mice by the administration of A. esculentus in combination with immuno-enhansive drug than the lone effect of immuno-enhansive drug. ‘T’ cell decrement was pronounced in mice treated with immunosuppressive drug while a moderate decrement was noticed due to A. esculentus in combination with immunosuppressive drug (Table 3). The increment in ‘T’ cell count may be due to the impact of plant drug on the synthesis, proliferation and activation of ‘T’ cells in treated animals. Similar results were observed by Paulsi and Dhasarathan (2011) in mice administered with plant extracts. The decrease in ‘T’ cell count in animal administered with immunosuppressive agent might be due to excess oxygen free radical production, increased rapid peroxidation, damage to membrane DNA fragmentation and apotosis due to immunosuppressive drugs thereby suppressed the functioning of immune system (Trebeden Negre et al., 2003).
 

 

 
The present investigation suggested that plant extract stimulate both the cellular and the humoral immunity. In DTH responses directly correlated with cell mediated immunity and were found to be highest at the maximum dose (100 mg/Kg) of plant extracts A. esculuntus. When challenged by the antigen, they were converted to lymphoblast and secreted a variety of molecules including pro-inflammatory lymphokines, attracting more scavenger cells to the site of reaction. The mechanism involved in the elevation of DTH during the CMI responses could be due to sensitized T lymphocytes. The increased DTH response indicated a stimulatory effect of the plant which has concerned on the lymphocytes and accessory cell types required for the expression of this reaction (Mitra et al., 1999).
       
The plant extract altered the DTH responses in mice exposed to antigen. Injection at 24 hour maximum enhancement of DTH response to antigen was observed with plant extract and immunoenhansive drugs. The immunosuppressive drug administered mice showed less DTH responses against antigen (Table 4 and 5). From the results it was clear that the plant extract induced immunomodulating potential of the test animal. On administration of plant extract an enhanced and visible DTH responses were observed. The suppressing DTH responses by maximum dose of plant extracts (100mg/kg) of the present study coincided with the work of Kannan et al., (2007) who had reported a suppressor of DTH responses in mice when maximum doses of plant drugs were used.
 

 
Delayed type hypersensitivity (DTH) is a part of the process of graft rejection, tumour, intracellular disease causing microorganism, especially those causing chronic disease such as tuberculosis. DTH requires the specific recognition of a given antigen by activated T lymphocytes which subsequently proliferate and release cytokines. These, inturn, increase vascular permeability induce vasodilation macrophage accumulation (Descotes, 1999) and activation promoting increased phagocytic activity and  increased concentration of lytic enzymes for more effective killing were used to elicit contact hypersensitivity reactions.
       
According to Tiwari et al., (2004) the active principle, sesquiterapene lactone present in the plant Tridax procumbens assisted in cell mediated immune response and enhanced DTH reaction, which was reflected in the increased foot pad thickness due to heightened infiltration of macrophages to the inflammatory site as seen in the present study.  Similarly, Jolly et al., (1997) reported that the active fraction c1 - 1 protein in the plant pigeon tea, Cajananuscajan enhanced DTH response significantly in rats on SRBC antigenic challenge. Histological investigation by (Siheyla et al., 2003) on the inflammatory site showed perivascular cuffing with mononuclear cells followed by a more extensive exudation of mono and poly morphonuclear cells. The effectors cells that promote DTH reactions (T DTH cells) caused the activation of macrophages, infiltration of polymorphonuclear cells, increased vascular permeability and edema, thereby it induced T-cell mediated response also observed an elevation in DTH response in rat treated with the plant extract (Patwardhan, 2000).
       
Mice treated with cyclophosphamide (30mg/kg) of the present study had a suppressed foot pad thickness indicating the suppressive act on cell mediated immunity. According to Gutali et al., (2002) the DTH response was associated with T cells and sensitized T-cells release mediators to promote inflammatory processes. The possible mechanism behind DTH reactions, included, activation of complements releasing of mediators by activated most cells; kinin reactive oxygen or nitrogen species by benzofuranone metabolites histamine and pro-inflammatory cytokines (Khatune et al., 2005). The results of the present study provided unequivocal evidence of the immunostimulant activity in the active compound of A.esculentus. The active principles found in the plant were influenced through the non binding interactions and it may be well connected with receptors of immune system and which inturn elevated immune response.
       
Immunomodulation using medicinal plants can provide an alternative to conventional chemotheraphy for a variety of diseases, especially when host defense mechanism has to be activated when the conditions of impaired immune response or when a selective immunosuppresion is despaired in situations like auto immune disorders. There is a great potential for discovery of more specific immuno modulators which mimic antagonize the biological effect (Goldsby et al., 2002 and Sujatha et al., 2010). Immunomodulator  effect exhibited by flavonoids, cetaurein and its aglycone isolated from butanol subfraction of Bidenspilosa (Alamgir and Uddin, 2010) coincided with the report of the present study relating to the immunomodulator efficiency of A.esculentus.
       
Immunomodulation using medicinal plants can provide an alternative to conventional chemotherapy for a variety of diseases, especially when host defence mechanism has to be activated under the conditions of impaired immune response or when a selective immunosuppression is desired insituations like autoimmune disorders. There is great potential for the discovery of more specific immunomodulators which mimic or antagonize the biological effects of cytokines and interleukins, and the refinement of assays for these mediators will create specific and sensitive screens. Natural remedies should be revisited as important sources of novel ligands capable of targeting specific cellular receptors (Alamgir and Uddin, 2010). 

Modern immunology aims to modify the immune system to prevent or control diseases using immunomodulatory tools. Medicinal plants are rich source of substances which are claimed to induce para immunity, the non specific immunomodulation of essentially granulocyte macrophage, natural killer cells and complement functions.The results of B and T cells showed significant changes in all kind of treated animals, when compared to control. In five kinds of treatment, the increment in ‘B’ lymphocyte number was much pronounced in combination of plant extract with immuno-enhansive drug followed by immuno-enhansive drug. In DTH responses which directly correlate with cell mediated immunity were found to be highest at the maximum dose, 100 mg/kg of tested plant extract. Immunomodulation using medicinal plants can provide an alternative to conventional chemotheraphy for a variety of diseases, especially when host defense mechanism has to be activated when the conditions of impaired immune response or when a selective immunosuppresion is despaired in situations like auto immune disorders. Further, the results obtained with ethanol extract of A.esculentus have  close similarity with standard drugs particularly with standard antibiotic Streptomycine and immunomodulatory ability with Cortisol. From the overall investigation it is concluded that the ethanol extract of tender fruit of A.esculentus at the concentration of 10mg/kg is safe and it shows immunomodulatory effect and thereby it may be recommended for the preparation of plant based drug.