HPTLC Analysis for Qualitative Estimation of linalool in Timur Oil (Indian Variety and Nepali Variety)

The separate applications of each oil were made on 5 cm × 10 cm chromatographic precoated silica gel plates (Merck, TLC grade) that served as the stationary phase. Toluene and ethyl acetate (95:5 *v*/*v*) were used as the mobile phase in a twin-trough glass chamber where the TLC plates were prepared. The plates were taken off after the solvent front had moved 15 cm away from the initial extract site, and they were then left to dry. When the spots on the produced plates had dried, they were examined under visible (white), short UV (254 nm), and long UV (366 nm) light.

GC Analysis of Timur Oil, Marketed Timur Oil, and Nepali Timur Oil for Quantification of Linalool Content

The oils were subjected to GC analysis on a Shimadzu 15A gas chromatograph with a split/splitless injector (250 ◦C). The DB-5 capillary column (30 m 0.25 mm, film thickness 0.32 µm) was used, and nitrogen was utilized as the carrier gas (1 mL min−<sup>1</sup> ). After maintaining a temperature of 60 ◦C for three minutes, the column was heated to 220 ◦C at a rate of 5 ◦C min−<sup>1</sup> and maintained at this temperature for five minutes. The relative percentage quantity was estimated using a Shimadzu C-R4A Chromatopac from the peak region [53].

### *4.3. Screening of Components for Preparation of Nanoemulsion*

The miscibility of the chosen oil served as the criterion for choosing the surfactant and co-surfactant. To make the stable nanoemulsion, the combination of a high HLB value with a low HLB value at the optimum temperature was used. Miscibility studies with oil were carried out by mixing Smix in a 5:5 ratio and vortexing for 5 min. Then, the mixtures were kept at room temperature for 24 h. After 24 h, an evaluation was performed based on the color change and phase separation [54].

### *4.4. Construction of Pseudoternary Phase Diagram*

After the selection of the surfactant and co-surfactant (Smix) based on the miscibility with the oil, Smix was mixed in different volume ratios (1:0, 1:1, 1:2, 1:3, 2:1, 3:1, and 4:1). Based on the titration chart, different ratios of Smix were developed. Based on the points obtained from the titration chart, the phase diagram was made. The phase diagram was studied in detail for the formulation of the nanoemulsion [54].

### *4.5. Method for Preparation of the Phase Diagram (Aqueous Titration Method)*

For the preparation of the phase diagram, the oil and different volume ratios of Smix were mixed. The ratios ranged from 1:9 to 9:1, and these were mixed to make 16 different combinations, which were 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3.5, 1:3, 3:7, 1:2, 4:6, 5:5, 6:4, 7:3, 8:2, and 9:1. The pseudoternary diagrams were developed based on the aqueous titration method. Each oil and Smix underwent a slow titration with the aqueous phase while being moderately stirred. Water was added in varying amounts, ranging from 5 to 95% of the total volume. The formulation was visually observed after the addition of the water to the volume [19].
