Collection and authentication of crude drug
Bark of Aegle marmelos were collected from local market of Delhi, India in the month of September, 2010 and were identified by botanist Dr. H. B. Singh, HOD, Dept. of Botany at National Institute of Science Communication and Information Resources (NISCAIR) on 21st September, 2010 reference no NISCAIR/RHMDC/Consult/2010-11/1536/134, New Delhi, India.
Preparation of methanol extract
The bark (5.0 kg) was shade dried and pulverized in an electric grinder (Bajaj Bravo3, India); 2.0 kg of the powdered drug was extracted with 6.0 L of methanol for 48 h with occasional shaking. The filtrate (2.0 L) was concentrated under reduced pressure at 40°C to yield 60 g (6.0% w/w) of a brown soluble residue . The residue was further dried in an oven at 37°C to eliminate traces of methanol solvent and stored in a sealed dark airtight plastic container at 4–8°C, until use. The crude residue suspended in (0.5% CMC w/v) served as the doses form for experimentation.
Male albino wistar rats (200–250 g), procured from the animal house of Delhi Institute of Pharmaceutical Sciences and Research (DIPSAR), New Delhi, were used. Rats were housed in temperature controlled room; 25 ± 2°C with a 12-hour light/dark cycle and 57 ± 7% relative humidity under standard hygienic conditions and had free access to fresh tap water & pelleted diet. The animals were acclimatized for seven days prior to experimental use. The study was done with prior approval from institutional animal ethical committee (IAEC) vide protocol no. (IAEC/DIPSAR/20010-I/15) DIPSAR, New Delhi.
The animals were divided into five groups: Control group (n = 6 animals), Standard Drug (SD) 50 mg/rat/day/p.o Lonidamine  (n = 18 animals). Group I (G I), Group II (G II), Group III (G III), (n = 24 animals each), The animals that served as control, were treated with 2.0 ml of 0.5 percent CMC each day for 60 days G I, G II, G III were give methanolic bark extract at a dose of 200, 400 and 600 mg/kg b.w respectively. Dose selection was based upon earlier studies done on this plant extract to perform anti diabetic activity at a safe dose levels of 600 mg/kg as no reported LD50 on bark extract were available . Six animals were sacrificed from SD, G I, G II, and G III groups after 24 hours of 20th, 40th and 60th days of treatment.
After 60 days of the treatment schedule of methanolic bark extract at a dose of 200, 400 and 600 mg/kg b.w of RG I, II & III respectively (n = 6) were withdrawn from treatment for next 30 days. Animals were sacrificed and recovery was assessed.
Blood samples were collected by cardiac puncture and were used for routine hematology and serum clinical chemistry to evaluate the effect of treatment on the other body function and toxicity, if any.
Hematological parameters like red blood cell, pack cell volume, total leukocyte, & hemoglobin were done with the help of Coulter ACT diff (Beckman coulter India Pvt. ltd, Mumbai). erythrocyte sedimentation rate (ESR) done by wintrobe method.
Serum glutamate oxalate transaminase (SGOT), serum glutamate pyruvate transaminase (SGPT), Creatinine (CR), Blood Urea Nitrogen (BUN), Uric acid (UA) alkaline phosphatase (ALP), Cholesterol, Total Protein (TP) and Albumin (ALB) levels were estimated (Roche Hitachi 912), Chemistry Analyzer with Roche biochemical Kits. All tests were calibrated on two point and full calibration, as and when required, Precinorm and Precipath quality control serum were processed with each batch. Serum testosterone levels were estimated using Testosterone ELISA (RE52151) kit (Immuno Biological Laboratories, USA).
Body and organ weight
Body weight, weight of testis, epididymis, seminal vesicles and ventral prostates, excised free of adhering tissues, were evaluated periodically every 20th,40th,60th days during the treatment and 30th day of recovery period.
The cauda epididymis, wherein the spermatozoa mature and are stored, were dissected in 1000 μl of normal saline, and the clear fluid was used for the analysis of sperm concentration, motility, viability and abnormality, as per the procedures described in the WHO Laboratory Manual . Acrosomal integrity is measured using fluorescently labeled plant lectins as described in WHO Laboratory Manual with modification . Briefly acrosomal integrity of each sperm samples was smeared on glass slides and air-dried followed by fixation with 99% methanol. For staining, slides were incubated with (PSA–FITC, L0770, Sigma–Aldrich), at 37°C for 30 minutes, then washed with phosphate buffer saline and analyzed under microscope (Leica DMR fluorescence microscope) using an appropriate filter. The stained rat spermatozoa were classified into two groups; spermatozoa displaying intensively bright fluorescence in the acrosomal region were considered as intact acrosome and spermatozoa displaying weak, patchy, or no fluorescence in the acrosomal region were considered as damaged acrosome.
Libido and fertility tests
Libido and fertility were assessed using the procedure as described previously . Briefly at the termination of treatment schedule, three male rats from each group (randomly selected) were paired individually with two proven fertile females in estrous phase. Success of mating was confirmed by the presence of spermatozoa in the vaginal smear of the mated rats. The females were laparotomized on the 10th day of postcoitum, and the number of corpus luteum implantation were counted if any, and number of litter deliver are recorded. Percentage preimplantation, post implantation loss and antifertility activity.
The testis and other vital organ were used for histological studies at different stages of treatment and after recovery. For histology evaluation, testis were fixed in Bouin's fixative and other tissues in 10% formalin. Tissues were processed for wax embedding and embedded paraffin wax blocks were sectioned 5 μm thick and stained with haematoxylin and eosin.
Analysis of each data set was performed by student-t test, and one-way analysis of variance (ANOVA). Statistically significant effects were further evaluated with Newman-Keuls tests. Differences were considered significant at P < 0.01. Results were expressed as means ± SEM.