*2.8. Secondary Metabolite Profiling and GC-MS Analysis*

The ADR1 metabolite extract was used at a concentration of 5 mg/mL for chemical profiling of the classes of metabolites present in the extract. The tests for the different class of metabolites, for example, anthraquinones, glycosides, terpenoids, flavonoids, tannins, alkaloids, saponins, sterols, anthocyanins, coumarins, tannins, lactones, terpenes, fatty acids, proteins/amino acids and carbohydrates were carried out by using standard methods reported earlier [38–40].

Further analysis of the metabolites was carried out by employing GC-MS (GC-MS-QP2010 plus; Shimadzu, Kyoto, Japan) as outlined below. A constant column flow rate of 1.21 mL/min with helium gas was maintained in RESTEK capillary column (30 m × 0.25 mm I.D. × 0.25 μm film thickness). Initial oven temperature was 100 ◦C for 3 min, which was increased to 250 ◦C for a hold time of 5 min, was further increased gradually to 280 ◦C where it was kept constant for 15 min. A total of 3 μL of sample (3 mg/mL) was injected in split mode (split ratio of 10.0) and linear velocity of the column was maintained at 40.9 cm/s. The mass fragmentation patterns (spectra) of the metabolites were obtained at electron ionization (EI) of 70 eV scanned over a m/z range of 40–650. The compounds detected were identified on the basis of comparison of the mass spectra with those available in the NIST14 and Wiley8 spectral library. The spectra having a match limit value lower than 700 were not considered.
