Antioxidant activities of the selected plants from the family Euphorbiaceae , Lauraceae , Malvaceae and Balsaminaceae

Extraction of nine plants selected from the family Euphorbiaceae, Lauraceae, Malvaceae and Balsaminaceae was done in petroleum ether, chloroform, ethyl acetate and methanol/n-butanol in order of increasing polarity using soxhlet apparatus. Total phenolic contents were determined with FolinCiocalteu reagent which ranged from 30.5 to 547.0 mg GAE/g of extract. Maximum phenolic contents were found in n-butanol extract of Ricinus communis. Antioxidant activities of these extracts were evaluated through DPPH radical scavenging, phosphomolybdate and ferric thiocyanate (FTC) methods. Methanolic extract of Cinnamomum zeylanicum and Cinnamomum tamala showed highest antiradical (96.8%) and phosphomolybdate (1.131) activity, respectively, while ethyl acetate extract of R. communis exhibited maximum lipid per-oxidation (FTC) activity (79.3%). IC50 value of chloroform extract of C. tamala (2.2 g/ml) was less than gallic acid (4.4 g/ml), while ethyl acetate and methanol extracts of Abutilon bidentatum, Impatiens bicolor and Impatiens edgeworthii exhibited the IC50 values in the range of 10.0 20.0 g/ml.

In longer term, plant species identified as having high levels of antioxidant activity in vitro may be of value in the design of further studies to unravel novel treatment strategies for disorders associated with free radicalsinduced tissue damage.A number of synthetic antioxidants, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT) and tertiary butylhydroquinone (TBHQ) have been added to foodstuffs but, are reported to cause liver disorders (Valentao et al., 2002).Therefore, attention has been directed towards the purification of natural antioxidants from botanical sources, especially edible plants.The study was conducted to evaluate the antioxidant activity of the nine medicinal plants from four different families using different antioxidant models (ferric thiocyanate (FTC), 2,2 -diphenyl-1-picrylhydrazyl (DPPH) and phosphomolybdate).

Preparation of plant extracts
Whole plants of family Euphorbiaceae and Malavaceae were collected from Lahore region; plant material of Balsaminaceae was collected from Ayubia park, Muree (Pakistan) and identified at the Department of Botany (GC University, Lahore), while the leaves of C. tamala and bark of C. zeylanicum (Lauraceae) were purchased from local market.All plant materials were dried, powdered and extracted in different solvents (petroleum ether, chloroform, ethyl acetate and methanol/ n-butanol) using soxhlet apparatus.Crude extracts were filtered and concentrated at reduced temperature using rotary evaporator.

Determination of total phenols
Total phenolic contents of all the extracts were determined by Folin-Ciocalteu reagent (Makkar et al., 1993).0.1 ml of extract was combined with 2.8 ml of 10% Na2CO3 and 0.1 ml of 2 N Folin-Ciocalteu reagent.After 40 min, absorbance was measured at 725 nm using UV-visible spectrophotometer (CECIL-7200).The results were determined as mg equivalent of gallic acid per gm of extract by computing with standard calibration curve (R 2 = 0.9909 value) constructed for different concentrations of gallic acid.

FTC assay
The antioxidant activity of different extracts on inhibition of linoleic acid per oxidation was assayed by ferric FTC (Osawa and Namiki, 1981).0.1 ml of the ethanolic solution of the extract (5 mg/ml) was mixed with 10 ml of absolute ethanol, 10 ml of 0.2 M phosphate buffer (pH 6.0) and 2 ml of 2% (v/v) linoleic acid.All the samples were incubated at 40°C.At regular intervals, (48 h) 5 ml ethanol, 0.1 ml 0.02 M ferrous chloride in 3.5% HCl and 0.1 ml of aq.20% Shahwar et al. 1087 ammonium thiocyanate was added in the above solution and absorbance was recorded at 500 nm.Gallic acid, BHT and -tocopherol were used as standard reference in 2 mg/ml concentration.

DPPH radical scavenging assay
The antioxidant activity of different extracts was measured in terms of radical scavenging ability by DPPH method (Erasto et al., 2004).Methanolic solution (1 ml) of different extracts at 100 g/ml concentration was added to 1 ml methanolic solution of DPPH (2 mg/ml).The absorbance was measured at 517 nm after 30 min.
The results were evaluated as percentage scavenging of radical (% scavenging of DPPH • = Abs. of blank -Abs. of sample/ Abs. of blank x 100).IC50 value (concentration of sample where absorbance of DPPH decreases 50% with respect to absorbance of blank) of extracts were determined.The results were compared with standards (gallic acid and BHT).

Phosphomolybdate assay
Total antioxidant activity of extracts was evaluated by the formation of phosphomolybdenum complex (Prieto et al., 1999).0.1 ml methanolic solution of extracts (100 µg/ml) was added to 1.9 ml of reagent solution (0.6 M H2SO4, 28 mM sodium phosphate and 4 mM ammonium molybdate).The blank solution contained only 2 ml of reagent solution.The absorbance was measured at 695 nm after 60 min.

Total phenolic contents
Phenolic compounds are commonly present in both edible and non-edible plants and exhibit multiple biological effects including antioxidant activity (Kahkonen et al., 1999).The phenolic contents of the selected plant extracts were determined by FC reagent and expressed as gallic acid equivalents in mg/g of crude extract.Total phenolic contents of different solvent extracts are given in Table 1.The yields ranged from 30.5 to 547.0 mg GAE/g of crude extracts.All the ethyl acetate and methanol/ nbutanol extracts were found rich in phenolics except the extract ERE of E. royleana and ChTB of C. tinctoria which showed 88.5 and 64.5 mg GAE/g of crude extract.Among the other extracts, n-butanol extract of R. Communis (RCB) demonstrated the highest phenolic content (547.0GAE/g of crude extract).

Antioxidant activities DPPH radical scavenging assay
Antioxidants react with DPPH • , which is a stable free radical and convert it to 1, 1 -diphenyl-2-picryl hydrazine (Fargere et al., 1995).The degree of decolourization of the purple coloured solution of DPPH • indicated the scavenging potential of the antioxidant compound.It was found that the radical scavenging activity of CZM, CTP,

Conclusion
In the past few years interest in the search of new natural antioxidants has grown because reactive oxygen species (ROS) production and oxidative stress is linked to many diseases.The use of synthetic antioxidants generally leads to problems of toxicity.In this study, antioxidant potential of nine extracts of four different plant families (Euphorbiaceae, Malvaceae, Lauraceae and Balsaminaceae) were assayed by FTC, DPPH and phosphormolybdate methods.The antioxidant activity of the extracts was also studied using linear regression analysis and found strongly correlated with total phenolic contents.These correlations suggested that polyphenols are mainly responsible for the antioxidant activity displayed by these extracts.Previous reported work on C. tinctoria, J. gossypifolia and R. communis revealed the presence of flavonoids with flavonol structure (Delazar et al., 2006;  Sankara et al., 1971;Upasani et al., 2003;Kang et al., 1985).Phytochemical studies of C. zeylanicum and C. tamala indicated three flavonoids namely quercetin, kaemferol and quercetrin (Nagendra et al., 2009)

Figure 1 .
Figure 1.Correlation between total phenols and DPPH activity of extracts of Euphorbiaceae family.

Figure 2 .Figure 3 .
Figure 2. Correlation between total phenols and total antioxidant activity of extracts of Euphorbiaceae family.

Table 1 .
% Scavenging of DPPH, % lipid per oxidation, total phenols and total antioxidant activity of the extracts of selected medicinal plants.