w/w) and acetone insoluble fraction

w/w) and acetone insoluble fraction (CCAIS; yield: 67.19%, w/w). The methanol extract (CCM) and these three broad fractions i.e. CCES, CCAS and CCAIS were subjected to phytochemical and phar- macological screening.

2.4. Phytochemical screening (Stahl, 1969; Harbone, 1976; Wagner et al., 1984)

The CCM extract and its broad fractions were screened for the presence of tannins, saponins, unsaturated sterols, triter- penes, alkaloids, flavonoids, lactones/esters, protein/amino acids and carbohydrates and/or glycosides with thin layer chromatog- raphy (TLC). Thin layer plates precoated with silica gel G (Merck,
0.25 mm thickness) were used. Development was carried out with different solvent systems such as ethyl acetate:methanol:water (100:13.5:10, v/v/v), ethyl acetate:formic acid:acetic acid:water (100:11:11:26, v/v/v/v), chloroform:methanol:water (70:30:4, v/v/v), toluene:ethyl acetate:diethylamine (70:20:10, v/v/v) and ethyl acetate:methanol:water:acetic acid (65:15:15:10, v/v/v/v). After development of chromatogram in the solvents the plates were dried and sprayed with Dragendorff’s, AlCl3 , hydroxylamine-ferric
chloride, ninhydrin and antimony trichloride for the detection of
alkaloids, flavonoids, lactones/esters, protein/amino acids, unsat- urated sterols and triterpenes respectively. While detection of anthraquinones, saponins, tannins, carbohydrate and/or glyco- sides is carried out using KOH, anisaldehyde-sulphuric acid reagent, ferric chloride and naphthoresorcinol reagent respectively and visualization is carried out under visible and UV light ( :
366 nm).
CCM extract and its fractions were also quantified for pres- ence of important secondary metabolites such as total polyphenol, flavonoid and flavonone compounds using following spectroscopic methods.

2.4.1. Determination of total polyphenol compounds (TP), flavonoids (TFA) and total flavanones (TFO)
Total polyphenol content was measured using Folin–Ciocalteu colorimetric method (Singleton and Rossi, 1965; Singlenton et al.,
1999). Gallic acid was used as a reference for constructing the stan- dard curve (10–100 mg/ml). The results were expressed as mg of gallic acid equivalents (GAE)/g of extract.
Flavonoid content was determined by the aluminum chloride method (Jin-Yuarn and Ching-Yin, 2007; Stanojevic´ et al., 2009). While the modified 2,4-dinitrophenylhydrazine method was used for determination of flavanones (Nagy and Grancai, 1996). Rutin and naringin were used as a reference standard and results were