**3. Techniques of isolation and purification of bioactive molecules from extracts**

#### **3.1 Fractionation of bioactive compound by flash chromatographic technique**

Flash chromatography instrument consisting of (Analytical technologies limited, Shanghai china) consisting of TBP2H02pump along with TBD2000 UV detector and automatic fraction collector was used for analysis. System equipped with Chromo station software was used for data monitoring during the analysis. The separation was carried out on OROCHEM OROFLO-4SiHPS column made up of silica particles [29, 30].

#### **3.2 Gas chromatography**

The gas chromatography used for estimation of residual class 3 ethyl acetate solvent in both crude extracts was performed using a Gas Chromatography system 7890 B with Agilent DB 624 column with helium gas at 1 ml/min flow mode reference solution tetrahydrofuran. GC temperature was set at 50 °C (hold for couple of min) to 250 °C at 20 °C/min. (hold up to 5 min) [31, 32].
