Total soluble sugars:

Total soluble sugars were determined by Riazi et al., (1985). Fresh leaves (1 g) were grounded in 10 mL of 80% methanol. Centrifuged at 10,000rpm and filtered the extract and supernatant was used for the estimation of sugars. Plant extract 0.3mL was taken in test tubes and 3 ml of anthrone reagent was added to …

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Total flavonoids:

Flavonoids were determined by using the AlCl3 method. 1ml of the sample was taken in cuvette and 0.3ml of NaNO2 was added. Before adding the next chemical shaken well and then added 0.3ml of AlCl3. After mixing well, 2ml of NaOH was added. The optical density was taken at 510nm via a spectrophotometer.

Total phenolics:

Following Julkenen-Titto (1985), fresh leaf 0.5g was ground in 10ml of 80% of methanol. Centrifugation was done at 10,000rpm for 10 minutes.  Then 0.25ml of plant extract, along with 1.25ml of Folin-Phenol Ciocalteus reagent was mixed. The addition of 1.25ml of sodium carbonate was the last step. The total phenolics in leaf samples were anticipated …

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Total free amino acids:

Total free amino acids were determined using Hamilaton & Slyke (1943) method. 0.5g of plant material was ground in 10ml of phosphate buffer using pestle and mortar that was placed on an ice tub. The phosphate buffer used was made of two salts K2HPO4 and KH2PO4 and the buffer pH was maintained at 7.8. The …

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Total soluble protein contents:

The total soluble proteins were determined by following Bradford’s (1976) techniques. 0.5g of plant leaf was ground in 10ml of the buffer while the pestle and mortar were placed on an ice plate. The extract was subjected to centrifugation at 10,000rpm for 10 minutes, while the temperature was maintained at 4C. The supernatant after centrifugation …

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Peroxide dismutase (POD):

The peroxidase activity was determined by Chance and Maehly (1954). The test tubes were added with 2.7ml of phosphate buffer, 0.1ml of guaiacol, and 0.1ml of enzyme extract and mixed well. The absorbance was taken at 470nm for 120 seconds, every 20 seconds. A change in absorbance of 0.01 units per minute defined one unit …

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Superoxide dismutase (SOD):

For the determination of SOD activity the method of Beauchamp and Fridovich (1971) was followed. The principal was to measure the absorbance at 560nm which determined the photoreduction of nitroblue tetrazolium (NBT). The reaction contained 650ul phosphate buffer and added with 100ul of methionine, then added 50ul of riboflavin along with 100 ul of enzyme …

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Catalase activity:

Chance and Maehly (1954) method was followed with minor changes for evaluating the catalase activity. The activity of catalase was determined by measuring the reduction in absorbance for 120 seconds, every 20 seconds at 240nm. The reaction mixture contained 2.8ml of phosphate buffer along with the addition of 0.1ml of H2O2, 0.1ml of enzyme extract …

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MDA contents:

MDA in the leaves was investigated by the method of Carmak & Horst (1991). This reaction was based on Thiobarbituric acid (TBA). Fresh leaves (0.5g) were ground properly in 10 ml of 0.1% tri-chloroacetic acid solution and centrifuged for 10 minutes at 12,000rpm. 1ml of the supernatant was reacted with 1 ml of 20% TCA …

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H2O2 contents:

Hydrogen peroxide concentration was estimated using the method of Velikova et al. (2000). 0.5g of the leaf was crushed with pestle mortar and plant material was homogenized in 10ml of 0.1% trichloroacetic acid on an ice bath. The extract was centrifuged at 12,000rpm for 10 minutes. The reaction mixture was added with 0.5ml supernatant, 0.5 …

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