The fresh leaves of Azadirachta indica were collected from BHU campus and then identified by Prof. NK Dubey (with a voucher number of Melia. 2019/1), Department of Botany, Institute of Science, Banaras Hindu University, Varanasi.
Preparation of Azadirachta indica ethanolic extract
Plant leaves were grind using mortar-pestle and subjected to alcoholic extraction by soxhlet apparatus. 5 gm of powdered leaves was taken in a cotton cloth and placed into flat-bottom beaker of the soxhlet containing 50 ml of 50% (v/v) ethanol and kept overnight. Soxhlated ethanolic extract so prepared was then transferred into Petri plates followed by their incubation at 37oC for 7-8 hours.
Swiss mice (18-28 gms) of either sex were randomly divided into 5 groups of 6 mice each (Table 1). The mice were housed in a plastic cage at room temperature (25 ± 2°C) for the experiment.
Grouping of animal model.
Neem extract (100mg/kg BW) induced smoker
Neem extract (400mg/kg BW) induced smoker
Preparation of Drugs
The stock solution of dexamethasone was prepared by dissolving 1 mg of it in 1 ml of distilled water of which 250 µl was used as a working solution. The ethanolic neem leaf extract was used in two different concentrations which were 400 mg/kg body weight and 100 mg/kg BW in two mice groups respectively (smoker and non-smoker) shown in Table 1.
Mice were placed in a smoke chamber (a rectangular box) with a partition wall dividing it into two halves, containing 16 holes in it. A cigarette puff plugged into a motor was adjusted on either side of the box for its ignition whose smoke was allowed to pass through these small holes towards the other side of the wall where mice were placed. Cigarette Smoke exposure was continued for 5-7 minutes after the drug administration which was given 45 minutes before the CS exposure. This treatment was given thrice in a week with an interval of two days and continued for 7 weeks.
Administration of drug into the animal model
Drugs from working solution were administered into mice intraperitoneally 45 minutes before cigarette smoke exposure. This was repeated thrice a week for 7 weeks with an interval of two days.
Test for tannins
2 ml of neem leaf extract was taken to which 2 ml distilled water was added followed by introduction of few drops of Ferric chloride to confirm the presence of tannins. Green precipitates showed the presence of tannins (Sivakumar and Gajalakshmi 2013).
Test for Saponins
Distilled water and neem leaf extract were added into test tube in 1:1 ratio and shaken vigorously. The persistence of foam indicated presence of saponins (Ijoma et al. 2017).
Test for sterols
1 mg of crude neem leaf extract was dissolved into 10 ml of chloroform followed by the addition of an equal volume of conc. sulphuric acid through wall of test tube. The upper layer turned red while the lower layer appeared yellowish in colour with green fluorescence (Saklani et al. 2012).
Test for detection of protein
Concentrated Nitric acid (HNO3) was added to neem leaf extract. The appearance of yellow colour confirmed the presence of proteins (Sati and Kumar 2015).
Test for phenols
Few drops of Ferric chloride (FeCl3) were added to the extract. Formation of bluish-black precipitates showed the presence of phenols (Ijoma et al. 2017).
Test for alkaloids
The extract was dissolved into diluted HCl. Subsequently the mixture was filtered and later few drops of Wagner’s reagent were added. Reddish-brown precipitates at the bottom of test tube indicated the presence of alkaloids (De et al. 2010).
Test for flavonoids
A few drops of NaOH solution were added to few ml of neem leaf extract followed by the addition of diluted acid. Intense yellow colour was observed on the addition of NaOH which turned colourless on the addition of diluted acid (Mohammed 2019).
Test for carbohydrate
5 ml of distilled water was added to neem leaf extract and the mixture was then subjected to filtration through Whatman filter paper No. 1. Benedict’s solution was added to the filterate so obtained. Orange-red precipitates gave positive results (Nadhiya et al. 2019).
Test for diterpenes
Neem leaf extract was added with water and few drops of freshly prepared copper acetate solution. Emerald green precipitates at the bottom of test tube confirmed the presence of diterpenes (Shahverdi et al. 2019).
Test for triterpenes
Chloroform was added to the neem leaf extract and mixure was then filtered. Conc. Sulphuric acid was added to the filterate. Golden-yellow precipitates appeared in the test tube as per the method (Kumar et al. 2015).
Analysis of Bodyweight of mice
The body weight of mice was noted every week to observe gain/reduction due to the administration of dexamethasone, neem extract, and cigarette smoke. It was observed from the first to the last day of the experiment. The differences in body weights of mice were calculated before sacrificing them.
Analysis of Mortality rate
Mortality rate was observed during the whole experiment.
Estimation of eosinophil peroxidase (EPO) in liver
100 mg of mice tissue was homogenized (1:1) into 1.9 ml PBS followed by its centrifugation at 12000 X g for 10 minutes. Supernatant was taken for enzymatic assay and equal amount of substrate (0.075 mmol/L Tris- HCl pH 8, 1.5 mmol/L O- phenylenediamine, and 6.6 mmol/L H2O2 in) was added. Further reaction was stopped with the addition of 50 µL of 1mol/L H2SO4, and absorbance was recorded at 490 nm (Adamko et al. 2004).
Histopathology of liver
The histopathology of liver was performed as per the protocol (Ragavan et al. 2006). Tissue from liver was dissected and placed into 10% NBF (Neutral Buffered Formalin) followed by its dehydration using different grades of alcohol which was transferred to the cassette filled with filtered paraffin wax and left for whole day. After microtomy, tissue was fixed on slides with DPX (Distyrene, Plasticiser, and Xylene) to observe under microscope.
Total Protein Content in liver tissue
Total protein concentrations were measured as µg/ml in serum by Folin’s assay (Lowry’s method) as per the previously defined procedure (Waterborg et al. 1994). Reagents were added into samples and observed using UV-spectrophotometry.
Liver function test by quantifying AST and ALT in serum
5 µl serum was diluted upto 10 µl with distilled water. A reagent mixture was prepared from Autospan liquid gold standard kit (Arkray Healthcare Pvt. Ltd.) and ELISA was performed for the estimation of AST and ALT. 10 µl of serum as a sample was mixed in 100 µl reagent prepared according to assay kit [SGPT (DST) (IFCC Method) Rechon Diagnostics P. Ltd.] and ELISA was performed followed by its observation at 390 nm. The enzyme activity is expressed as International Units/Litre (IU/L).
Estimation of Reactive Oxygen Species (ROS) in the liver
Tissue homogenate (1:1) was prepared as per method (Shin et al. 2016) and was centrifuged followed by the addition of PBS and DCFDA to the pellet. Observations were taken at an absorbance of 485 nm and emission at 535 nm.
Estimation of superoxide dismutase (SOD)
Phosphate Buffer was added for the homogenization of tissue and other reagents were mixed including L-methionine, hydroxylamine hydrochloride, hydrogen peroxide, and EDTA followed by incubation for 10 mins under the white fluorescent light inside an aluminum foil-wrapped box. Freshly prepared Griess reagent was added and absorbance was measured at 543 nm (Das et al. 2006).
All the experiments were repeated twice to compare results. Experimental data were reported as mean ± SEM of n= 5. Multiple group comparisons were determined using Student’s t-test.