Proline as one of the secondary metabolites was determined according to the method described by Bates et al

Proline as one of the secondary metabolites was determined according to the method described by Bates et al. (1973). Fresh leaf material (0.5 g) was homogenized in 10 ml of 3% aqueous sulfosalicylic acid and was centrifuged at 10,000 × g for 10 min. Afterward, 1 ml of the filtrated mixture was mixed with 1 ml of acid- Ninhydrin and 1 ml of glacial acetic acid in a test tube. The mixture was placed in a water bath for 1 h at 100° C. The reaction mixture was extracted with 4 ml toluene and the absorbance was measured at 520 nm with a spectrophotometer (Model Shimadzu UV- 1601, Shimadzu Corp., Japan).
Phenolic compounds as another one of secondary metabolites were assayed with Folin-Ciocalteu reagent as described by Chen et al. (2008). The calculations were based on a standard curve obtained with gallic acid. The content of phenolic was expressed as mg of gallic acid equivalent per g of fresh weight.

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