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.