**3.1 Isolation of glucan**

*Ganoderma lucidum* were collected from Southern parts of India. The polysaccharides were isolated from the fruiting bodies by the method of Mizuno [20]. Purification of the compound was done by ion-exchange chromatography. Qualitative confirmation was done by anthrone [21] and phenol sulphuric acid reagent [22]. Further characterization of the compound was done by IR and NMR, mass spectra, gel filtration and acid hydrolysis.

#### **3.2 Comet assay**

 Comet assay was performed by the method of Singh with modifications [23]. DNA damage in blood leukocytes was estimated. Ten microliters of heparinised whole blood, is mixed with 200 μl of low melting point agarose at 37°C and layered on frosted slides pre-coated with 200 μl high melting point agarose. The slides were pre-chilled in lysing solution and the standard protocol was followed [24].

CASP software was used for the quantitation of the DNA strand breaks of the stored images by which the percentage DNA in tail, tail length, tail moment, and olive tail moment [25]. The tail length of comet specifies the extent of damage as the smaller molecules move faster on the agarose gel. The longer tails of the comets indicate that the strand breaks are more frequent. The tail moment normalizes the difference in the size of the nucleus studied, which is product of the percent DNA in the tail of the comet and tail length. Calculation of olive tail moment distance of centre of gravity of DNA is considered rather than usual tail length.

### **3.3 Metaphase preparation**

Six groups of six animals each were used. At 22 h after irradiation all the animals were injected i.p. with 0.025 colchicine and sacrificed 2 h later by cervical dislocation. Bone marrow from the femur was aspirated, washed in saline, treated hypotonically (0.565% KCl), at 37°C for 30 min, fixed in 3:1::methanol:acetic acid, spread on clean slides and stained with 4% Giemsa [26].

The aberrations were scored with the help of a light microscope. Per animal 500 metaphases were scored. Chromatid breaks, chromosome breaks, fragments, rings and dicentrics as well as cells showing polyploidy and severely damaged cells (SDC), cells with 10 or more aberrations of any type, the different types of aberrations were scored. In 'chromosome type' aberration, breaks involved both the chromatids and in 'chromatid type' aberration involved only one chromatid. Fragments are those deleted portion having no apparent relation to any particular chromosome [27]. Data are mean ± (S.E).

## **3.4 Treatment of animals**

Group I—double distilled water (DDW).

Group II—300 mg/kg body wt. of amifostine i.p. (30 min prior to irradiation). Group III—20 mg/kg body wt. of glucan orally (5 min after irradiation). Group IV—DDW + 4 Gy radiation (RT).

Group V—300 mg/kg body wt. of amifostine (30 min before irradiation) + RT 4 Gy. Group VI—RT 4 Gy + 20 mg/kg body wt. glucan orally (5 min after irradiation).
