Rutin was obtained from Acros organics, New Jersy, USA. DPPH (1, 1-diphenyl, 2-picryl hydrazyl), NBT (Nitro blue tetrazolium), NADH (Nicotinamide adenine dinucleotide phosphate reduced), PMS (Phenazine methosulphate), TCA (Trichloro acetic acid), Ferric chloride and BHT (Butylated hydroxy toluene) were obtained from Sigma chemical co USA. Ascorbic acid, curcumin and Vitamin E were obtained from SD Fine chem. Ltd, Biosar, India. TBA (Thiobarbituric acid) and pyridine were obtained from Loba chemie, Mumbai, India. EDTA (Ethylene diamine tetra acetic acid) and Hydrogen peroxide (H₂O₂) were obtained from Qualigens Fine chemicals, Mumbai, India. Naphthyl ethylene diamine dihydrochloride was obtained from Roch-light ltd, Suffolk, England. Sodium nitro prusside was obtained from Ranbaxy lab, Mohali, India. Pottassium ferric cyanide was obtained from May and Backer, Dagenham, UK. 2-deoxy-2- ribose was obtained from Fluka (Buchs, Switzerland).

Aerial parts of Cytisus scoparius was collected in Nilgiri hills, Tamilnadu region and authenticated through Government Arts College, Ooty. Voucher specimen (SNPS-011/ 2003–2004) of this plant material has been retained in the School of Natural Product Studies, Jadavpur University, India.

The aerial part of Cytisus scoparius plant was dried at room temperature and reduced to coarse powder. This powder was extracted with mixture of ethanol: water (7:3 ratio) for 48 hours. The solvent was completely removed by rotary evaporator and further removal of water was carried out by freeze drying. The dried extract was stored in vacuum desiccator for further use.

The free radical scavenging capacity of the extracts was determined using DPPH. A methanol DPPH solution (0.15%) was mixed with serial dilutions (0.5 μg to 8 μg) of Cytisus scoparius extracts and after 10 min, the absorbance was read at 515 nm using a spectrophotometer (Perkin – Elmer Lambda 20 UV – visible spectrophotometer). Vitamin C used as a standard. The inhibition curve was plotted and IC₅₀ values obtained 21.

Nitric oxide radical inhibition can be estimated by the use of Griess Illosvoy reaction 22. In this investigation, Griess Illosvoy reagent was modified by using naphthyl ethylene diamine dihydrochloride (0.1% w/v) instead of 1-napthylamine (5%). The reaction mixture (3 ml) containing sodium nitroprusside (10 mM, 2 ml), phosphate buffer saline (0.5 ml) and Cytisus scoparius extract (10 μg to 160 μg) or standard solution (rutin, 0.5 ml) was incubated at 25°C for 150 min. After incubation, 0.5 ml of the reaction mixture mixed with 1 ml of sulfanilic acid reagent (0.33% in 20% glacial acetic acid) and allowed to stand for 5 min for completing diazotization. Then, 1 ml of naphthyl ethylene diamine dihydrochloride was added, mixed and allowed to stand for 30 min at 25°C. A pink coloured chromophore is formed in diffused light. The absorbance of these solutions was measured at 540 nm against the corresponding blank solutions. Rutin used as a standard.

Measurement of superoxide anion scavenging activity of Cytisus scoparius was done based on the Nishimiki method 23. About 1 ml of nitroblue tetrazolium (NBT) solution (156 μM NBT in 100 mM phosphate buffer, pH 7.4) 1 ml NADH solution (468 μM in 100 mM phosphate buffer, pH 7.4) and 0.1 ml of sample solution of Cytisus scoparius (1.25 μg to 10 μg) in water were mixed. The reaction started by adding 100 μl of phenazine methosulphate (PMS) solution (60 μM PMS in 100 mM phosphate buffer, pH 7.4) to the mixture. The reaction mixture was incubated at 25°C for 5 min, and the absorbance at 560 nm was measured against blank samples. Decreased absorbance of the reaction mixture indicated increased superoxide anion scavenging activity. Curcumin was used as a positive control.

The rat liver microsomal fraction was prepared by the method of Bouchet et al 24. The reaction mixture contained in a final volume of 1.0 ml, 500 μl of liver microsomal fraction, 300 μl buffer containing the plant extract (50–150 μg), 100 μl of FeCl₃ (1 mM) and 100 μl ascorbic acid (1 mM) to start peroxidation. Samples were incubated at 37°C for 1 hour, after that lipid peroxidation was measured using the reaction with thiobarbituric acid (TBA). Thiobarbituric acid reactive substances were determined by the methods of Houghton and Aruoma 2526. The absorbance of the organic layer was measured at 532 nm. All reactions were carried out in triplicate. Vitamin E used as a standard.

The assay was performed as described by Halliwell method 27 with minor changes. All solutions were prepared freshly. 1.0 ml of the reaction mixture contained 100 μl of 28 mM 2-deoxy-2-ribose (dissolved in phosphate buffer, pH 7.4), 500 μl solution of various concentrations of the Cytisus scoparius (10 to 80 μg), 200 μl of 200 μM FeCl₃ and 1.04 mM EDTA (1:1 v/v), 100 μl H₂O₂ (1.0 mM) and 100 μl ascorbic acid (1.0 mM). After an incubation period of 1 hour at 37°C the extent of deoxyribose degradation was measured by the TBA reaction 2324. Measure the absorbance at about 532 nm against the blank solution. Vitamin E was used as a positive control.

The reducing power of Cytisus scoparius was determined according to the Oyaizu method 28. Different concentration of Cytisus scoparius extract (100 μg – 1000 μg) in 1 ml of distilled water was mixed with phosphate buffer (2.5 ml, 0.2 M, pH 6.6) and potassium ferricyanide [K₃Fe(CN)₆] (2.5 ml, 1%). The mixture was incubated at 50°C for 20 min. A portion (2.5 ml) of trichloroacetic acid (10%) was added to the mixture, which was then centrifuged at 3000 rpm for 10 min. The upper layer of the solution (2.5 ml) was mixed with distilled water (2.5 ml) and FeCl₃ (0.5 ml. 0.1%) and the absorbance was measured at 700 nm. Increased absorbance of the reaction mixture indicated increased reducing power. Butylated hydroxy toluene used as a standard.

Total soluble phenolic in the aqueous extract of Cytisus scoparius were determined with Folin-Ciocalteu reagent according to the standard method 29 using pyrocatechol as a standard. Briefly, 0.1 ml of extract solution (contains 1000 μg extract) in a volumetric flask diluted distilled water (46 ml). About 1 ml of Folin – Ciocalteu reagent was added and the contents of the flask mixed thoroughly. After 3 min, 3 ml of Na₂Co₃(2%) was added, then the mixture was allowed to stand for 2 hour with intermittent shaking. The absorbance was measured at 760 nm. The concentration of total phenolic compounds in the Cytisus scoparius determined as microgram of pyrocatechol equivalent by using an equation that was obtained from Gulcin et al 30. The equation is given below:

Absorbance = 0.001 x Pyrocatechol (μg) + 0.0033

All the invitro experimental results were mean ± S.D of five parallel measurements.

The hydro alcoholic extract of Cytisus scoparius exhibited a significant dose dependent inhibition of DPPH activity, with a 50% inhibition (IC₅₀) at a concentration of 1.5 μg/ml. The result was mentioned in figure 1. The IC₅₀ value of the extract was found to be lesser than the standard, vitamin C (IC₅₀ 3.0 μg/ml).

The scavenging of nitric oxide by plant extract was increased in a dose-dependent manner as illustrated in figure 2. At concentration of 116.0 μg/ml of extract 50% of nitric oxide generated by incubation was scavenged. This IC₅₀ value of extract found to be lesser than the standard, rutin (IC₅₀ 160.0 μg/ml).

The superoxide anion derived from dissolved oxygen by Phenazine methosulphate/NADH coupling reaction reduces nitro blue tetrazolium. The decrease the absorbance at 560 nm with the plant extract thus indicates the consumption of superoxide anion in the reaction mixture. As mentioned in figure 3, the plant extract as well as curcumin showed the scavenging activity; IC₅₀ values, 4.7 μg/ml and 5.84 μg/ml, respectively.

Activity of plant extract against non-enzymatic lipid peroxidation in rat liver microsomes has been shown in figure 4. Addition of Fe ²⁺/ascorbate to the liver microsomes cause increase in lipid peroxidation. The extract showed inhibition of peroxidation effect in all concentrations, which showed 50% inhibition effect at 104.0 μg/ml. The extract inhibition value was found to be lesser than the standard, vitamin E (IC₅₀ 120.5 μg/ml)

To attack the substrate deoxyribose hydroxyl radicals were generated by reaction of Ferric-EDTA together with H₂O₂ and ascorbic acid. When the plant extract were incubated with the above reaction mixture, it could prevent the damage against sugar. The results are shown in figure 5, the concentrations of 50% inhibition were found to be 27.0 μg/ml and 32.5 μg/ml for the extract and standard of vitamin E, respectively. The extract inhibition value was found to be lesser than the standard

Figure 6 shows the reductive capabilities of the plant extract compared to butylated hydroxy toluene. The reducing power of extract of Cytisus scoparius was very potent and the power of the extract was increased with quantity of sample. The plant extract could reduce the most Fe³⁺ ions, which had a lesser reductive activity than the standard of butylated hydroxy toluene.

The total phenolic contents of hydro alcoholic extract of Cytisus scoparius was 0.0589 μg pyrocatechol equivalent /mg.