Cajanus Cajan Analysis

2.3. Extraction of the flavonoids fraction of Cajanus cajan (FFCC)
The Cajanus cajan L. seeds were air dried at 10% moisture, then powdered and preserved in a refrigerator at -80°C. Five hundred gm from the powdered seeds were defatted with hexane, then extracted with methanol (MeOH - 70%) four times, then concentrated under vacuum to yield 37 g. Polyamide and sephadex LH-20 chromatography columns were used to fraction the MeOH (70%) extract of Cajanus cajan. Seven flavonoid fractions were collected and identified as follows: apigenin, isorhamnetin, luteolin, orientin, quercetin, quercetin-3-O-B-D-glucopyranoside, vitexin. The pharmacological composition of the extracted flavonoids and their antioxidant activities were described in our coauthors
with colchicine for 2.5h before collecting bone-marrow cells or spermatocytes to assess chromosome abnormalities [25,26]. For chromosomal preparation from bone-marrow cells and spermatocytes, both types of cells were collected separately in isotonic solutions (0.9% NaCl or 2.2% tri-sodium citrate respectively), centrifuged at 500xg for 5min. The pellets were resuspended in hypotonic solutions (0.075M KCl or 1.1% tri-sodium citrate respectively) and incubated at 37°C for 20min prior to fixation with the Carnoy’s fixative (methanol : glacial acetic acid: 3:1, 3X). The fixed cells were spread over cold slides; air dried, and then stained with 7% Giemsa stain in phosphate buffer (pH6.8). A hundred well spread metaphases per animal were analyzed for chromosome aberrations in bone-marrow cells or spermatocytes separately. Metaphases with gap, break, deletion, fragment, centric fusion, and polyploidy were scored in bone-marrow cells; and metaphases with autosomal univalent, XY-univalent, fragment and chain IV aberrations were recorded in germ cells. Cells were scored at 1000X under a light microscope (Olympus, Saitama, Japan). The activity of FFCC to reduce chromosome aberrations induced by CP was assessed by the following inhibitory index formula
Mouse bone-marrow cells were collected from femurs with phosphate buffer saline, centrifuged to collect the pellet cells with 50µl of 0.7% low melting agarose. The resuspended cells were spread over 0.5% normal melting agarose pre-coated slides. Then, the slides were immersed in lysis buffer for at least 1hour prior to deep them in the electrophoresis chamber containing electrophoresis buffer for 20 min, followed by another 20min with running currency at 1.25V/cm and 300mA. Comets were stained with ethidium bromide (50µl: 10µg/ml), and were recorded by scoring 150 cells per each animal under fluorescent microscope (Zeiss, Germany) at 400X using Cool Cube 1-Metasystems camera GmbH (Altlußheim, Germany), and comet imager measurements software V2.2 (Metasystems, Germany), to record the DNA damage (%), tail length (µm) and tail moment