Experimental animals
Wistar albino rats weighing 80-100 g were procured from animal house, IPR, GLA University, Mathura. Four groups containing six animals were made. Animals of Group-II, III & IV were fed orally with 125, 250, 500 mg/kg body weight of HME of N. cadmba bark respectively while control group (Group-I) animals were orally fed with rat pellet and water ad libitum for 21 days.

Chemicals
Freund’s complete adjuvant (FCA), Freund’s incomplete adjuvant (FIA), Concanavalin A (Con A), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), trypan blue, were procured from Sigma chemicals Co, USA and Anti rat IgG-HRP conjugate antibody was obtained from Thermo Scientific, USA.

Plant material
Neolamarckia cadamba bark was gathered from Mathura (27^o31’2” N – 77^o39’26’’ E). Identification and authorization of plant material was done by Dr. A. S.Updadhye, Agharkar Research Institute, Pune with voucher deposition No. L-084. Dried bark powder was used in preparation of hydo-methanolic extract (HME) of N. cadamba.

Preparation of extract
Coarsely powdered bark (300-350 g) was taken in soxhlet apparatus in presence of hydro-methanolic (80:20, v/v) solvent and subjected to boil at 65°C for 8-10 hrs. Evaporation was done at controlled temperature and reduced pressure using rotary evaporator. Finally, dark brown crystals (% yield: 14) were obtained.

Experimental animal
Wistar albino rats (80-100 g) were taken from central animal house, GLA University, Mathura and used for immunomodulatory studies. Ethical clearance was obtained with IAEC approval vide GLAIPR/CPCSEA/IAEC/2014/Biotech/02. Four groups (Groups-I, II, III & IV) containing six animals each were made for assessment of in vivo humoral immune response.

Determination of safe and non-toxic dose of HME of N. cadamba
To determine the safe and non-toxic dose of hydro-methanolic extract (HME) of N. cadamba, four groups of Wistar albino rats, comprising six rats in each group were made. Safe dose level was determined according to Organization for Economic Co-operation and Development guidelines No. 423¹⁷. Animals of Group-II, III & IV were fed orally with 125, 250, 500 mg/kg b.wt respectively for twenty one days whereas animals of Group-I (control) were fed with rat pellet and water ad libitum. All the groups of albino rats were monitored for the development of clinical signs, changes in physical health conditions and body weight.

Apparent heath condition and toxic signs
Rats of test groups (HME fed rats and unfed rats) were kept separately and observed every day for any toxic sign / symptoms / abnormal changes throughout the experiment. The dose causing no adverse effect was taken as safe and nontoxic dose (NTD) for further studies.

Body weight gain/loss
Animals fed with different doses of HME of bark including control group were weighed at weekly interval and the mean body weight was recorded at 0 and 21st day. The mean body weight of rats of test groups was recorded and compared with the control group.

Measurement of antibody titre for determination of humoral immune response
Salmonella typhimurium ‘O’ antigen was prepared as per method suggested¹⁸. Smooth colonies of S. typhimurium grown on Tryptose agar medium were selected and inoculated in nutrient broth. Inoculated broth was incubated for 6-8 hours at 37°C and then boiled at 100°C for two hours thirty minutes. Heated S. typhimurium culture was used as ‘O’ antigen for determination of humoral immune response in rats. Antibodies were raised against animals of all groups subcutaneously immunized with Salmonella typhimurium ‘O’ antigen. On 1st day, all animals of Group-I (control), Group-II (125 mg/kg b.wt), Group-III (250 mg/kg b.wt) and Group-IV (500 mg/kg b.wt) were subcutaneously immunized with equal volume (0.5 mL) of S. typhimurium “O” antigen (2 mg/ml) and FCA. Then after at 7^th and 14^th day all groups including control were immunized with equal volume (0.5 mL) of S. typhimurium “O” antigen and FIA. At 21st day serum from all experimental animals was used for determination of humoral immune response. Antibody titer of experimental and control group animals was determined in accordance to protocol¹⁹. Assessment of humoral (antibody mediated) immune response was done by conventional indirect ELISA.

Determination of cell mediated immune response
Splenocyte preparation
Rat splenocytes preparation was carried out in accordance to method suggested ¹⁹. Spleen was macerated and filterated through nytex membrane to remove large particles. Cells were then resuspended in RPMI-1640 medium containing 10 % fetal calf serum and centrifuged at 2500 rpm for 10 minutes. Centrifuged cell were then treated with 0.15 M NH₄Cl (lysis buffer) for erythrocytes lysis and then washed with RPMI-1640 medium. Cell viability was determined by trypan blue dye (0.1% solution) and viable cell concentration was adjusted to 2 x 10⁶ cells/mL by haemocytometer and was used for splenocytes proliferation assay.

In vitro effect of HME of N. cadamba bark on splenocytes proliferation/inhibition
Efficacy of HME of N. cadamba bark over cell mediated immune response of Wistar albino rats was determined by studying the effect of HME over rat’s splenocytes. 200µL of 2×10⁶ spleen cells/ml in triplicate seeded with 10% FBS with 5µg/ml Con-A (optimum conc.) was cultured in RPMI-1640 medium. Different concentrations 20, 50, 100, 250, 500 µg/mL of HME of N. cadamba bark were added in triplicate to respective wells of plate. Incubation of culture plate was carried at 37°C for 72 hours using CO₂ incubator (5% CO₂). After that 20 µL of MTT solution (5 mg/mL) was added to each well. Formation of formazone crystals occurs due to reduction of MTT. Plate was further incubated at 37°C for 4 hours in CO₂ incubator (5% CO₂ and 80% relative humidity), followed by removal of supernatant. The plate was allowed to dry and formazone crystals were dissolved by adding 100µL DMSO. Mean OD values were determined with respect to control by ELISA reader.