To determine the total quantity of phytochemicals (terpenes and oxygenated compounds) present in the LO, quantitative analysis was done using Gas chromatography-mass spectrometry (GC-MS). GC-MS was performed on Agilent Technology (Thailand) 6890 series gas chromatography (GC) system equipped with 5973 mass spectrometry (MS) detector and 7683 series auto-injector. The compounds were separated on HP5 column (30 m × 0.25 mm, 0.25 µm filter). The column temperature was 70˚C for 2 min, and 130˚C at 30˚C/min and changes the gradient to 230˚C with 10˚C/min and held at 230˚C for 6 min with the total run time of 20 min. An electron ionization (EI) system with ionization energy 70 eV was used for detection. The temperature of ion source was set at 230˚C, detector voltage was 2 kV and the interface temperature were 250˚C. The mass spectrum was acquired in full scan mode at a rate of 0.98 scan/sec (mass range of 20 - 800 amu). The measurement was performed in duplicate for each sample with solvent delay for 2 min [10] [11].

The fabrication and optimization of varied excipients in colloidal solution was performed by Spontaneous Emulsification method. For the preparation of LO-NE, LO was selected as an oil phase, Tween 80 and Ethanol as surfactant and co-surfactant, respectively with distilled water as an aqueous phase. Further, these excipients were blended together in 7 different S_mix (surfactant and cosurfactant) ratios ranging from 1:0 to 1:6 and 16 varied combinations of oil:S_mix (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) ratios. Thereafter, these prepared NE samples were kept on shaker at 37˚C for 72 h to achieve equilibrium and then centrifuged at 2800 g for 15 min and analyzed for clear titration zones with no phase separation in the colloidal solution. Finally, the formulations were measured spectrophotometrically for the presence of flavonoid and phenolic content and the combinations ratios were plotted on ternary phase Triplot software [12] [13].

Phenolics are considered amongst the class of most effective free radical scavengers that can interact with the biological systems and play role in anti-inflammatory, antimicrobial, anti-carcinogenic and antioxidant activities [14]. The TPC was performed by the Folin-Ciocalteu method with Gallic acid (GA) as a standard. Briefly, LO was dissolved in deionized water (10% v/v) and 0.5 mL of Folin-Ciocalteu (1 N) was added followed by vortexing and incubation for 5 min at 37˚C. Thereafter, 0.12 mL of 5% sodium bicarbonate was added to all the tubes and the reaction mixture was incubated for 40 min in dark, then absorbance was recorded at 725 nm subsequently. The mean of three replications were recorded and results were expressed as mg of Gallic acid equivalents (GAE) per mL of LO [15] [16].

Flavonoids are a strong antioxidant and show a radical scavenging activity and appear to lower the risk of chronic diseases. The TFC of LO was determined by aluminium chloride method against Quercetin standard (1 mg/mL). LO was dissolved in deionized water (10% v/v) followed by addition of 150 μL of 5% sodium nitrite and the solution was vortexed for 5 min. 150 μL of 10% aluminum chloride was added and incubated for 5 min. Lastly, 2 mL of 4% sodium hydroxide was added and absorbance was measured at 510 nm. The samples were prepared in triplicate and the same procedure was repeated for the standard solution. The TFC was expressed as Quercetin equivalents in per mL of LO [17].

Ternary Phase diagrams were constructed to study the interfacial tension between oil and water, stabilized by adding surfactant and co-surfactant in to the colloidal system. Pseudo-ternary phase diagrams of the prepared LO-NE with clear titration zones and different surfactant/cosurfactant ratios were plotted by using Triplot 4.1.2 software tool. Consequently, the existence zones representing the physical state of NE were determined by plotting the pseudo ternary phase diagram with three axis, one presenting the oil, another showing a constant S_mix ratio and the third one representing the aqueous phase. It provides the insight into the behavior and compositional structure of the prepared NE system [18] [19].

The prepared NE formulations were analyzed for stability by subjecting them to varied alternative temperature and stress conditions. Firstly, they were incubated at 4˚C for 3 days followed by 45˚C for another 3 days, repeating the same cycle for 3 times. Thereafter, NEs were centrifuged at 2800 g for 30 min and the formulations that cleared the previous steps were exposed to freeze-thaw cycles by storing them at −20˚C (3 days) followed by 25˚C (3 days). Lastly, all those NEs that sustained through all the tests were dispersed in water (2 mL) and checked for any creaming, coalescence and phase separations issues, if any. The ones that cleared all the tests and remained stable were further taken for characterization [20].

The rheological parameters that were studied for the optimized formulation (LO-NE) included pH, density, conductivity and viscosity. pH is the logarithmic concentration of hydrogen ion in the dispersed sample and the hydrogen bond interaction between the NE particles and their viscosity were found to be a key factor in influencing the process of agglomeration between them. The pH was measured using a pH meter (Thermo Orion, 420A+) and viscosity was determined by Brookfield viscometer. Similarly, electrical conductivity is known as a measure of electrical charge present on the dispersed particles in relation to dispersion medium, which was further analyzed by the conductometer (CM 180, Elico, India) at the frequency of 1 Hz [21].

NE can be characterized by two ways—determination of physical properties (like shape, size, and dispersity) and chemical characteristics (like the presence of chemical bonding of ligands and other conjugated molecules, zeta potential). The techniques that were employed to characterize physical and chemical aspects of the NE were: Transmission electron microscopy, Particle size analysis; and Fourier transform infrared spectroscopy.

In order to assess the release of LO from the prepared NE system, Franz diffusion cell facilitated release kinetics study was performed using pre-treated dialysis membrane (D9652, Sigma Aldrich, Singapore) and the receiver compartment was filled with phosphate buffer saline (PBS, 20 mL, pH 7.4). The formulations were added through the donor membrane and 2 mL of test samples (LO and LO-NE) were taken out after every 30 min followed by addition of equal volume of PBS to maintain the equilibrium. The experiment was conducted for 12 hours and the measure of active phytoconstituents was done by GC-MS analysis as explained earlier [32].

The stability of LO and LO-NE was tested by the method described by Basheer et al, 2013. Herein, we tested the stability of LO and LO-NE at the highest concentration (10 µl/mL) in terms of their ability to scavenge DPPH radical after storing the samples (LO, LO-NE) at 37˚C for 6 months and then comparing with the fresh prepared samples [33] [34].

All the data is expressed as mean ± standard deviation and tested using one-way ANOVA. The values were considered significant at p < 0.01.

LO is known to possess a high content of terpenes including monoterpenes, diterpenes, sesquiterpenes and oxygenated compounds such as carbonyl, alcohols and phenols, all of them reported to be highly volatile of varied phytoconstituents. Therefore, to determine the total quantity present in the standard extract of LO A high throughput GC-MS method for EO analysis [35] was used. The predominant compound in LO was Limonene (Retention time, RT 17.66 min), accounting for the peak area of 40.93% followed by Alpha-Pinene and Linalyl acetate with the intensity of 29.24% (RT-12.98 min) and 6.05% (RT-28.71 min) as represented in Figure 1. Table 1 summarizes the compound name, RT and chemical structures of all the identified compounds in LO found in this study.

Phase diagrams (Figures 2(a)-(e)) were constructed individually for varied S_mix ratios (1:1, 1:2, 1:3, 1:4 and 1:5) to attain O/W ME regions (Table 2). It was observed from the graphs that as the contents of surfactant were increased in the S_mix ratio, there was the decline in isotropic region and it shifted more towards S_mix axis (Figure 2). Further, this micelle behaviour and the degree of solubilisation of the optimised NE indicated the homogenous colloidal solution.

The total phenolics content in LO was 70 ± 2.5 GAE/ml of sample and 20 ± 1.56 mg Quercitin equivalent/mL of sample.

The thermodynamic stability testing of all the five combinations of LO-NE was done to analyse their physiological stability [36]. They were subjected for heating-cooling cycle followed by centrifugation tests and then freeze thaw cycle, lastly dispersibility test and during the entire tenure of testing time the samples were checked for constantly any kind of creaming, cracking, coagulation, phase separation or turbidity issues [37] [38]. After completing all the cycles of thermodynamic stability testing the most suitable formulation preparation (S_mix ratio: 1:1 and LO: S_mix ratio: 1:9) was selected as final optimized formulation based on its visual inspection and categorization as grade “A”. This selected formulation was then taken further for all other characterization tests to study more about its structural and biological properties [39].

The pH for LO and LO-NE was found to be 3.5 ± 0.5 and 5.3 ± 0.27 respectively (Table 3), which suggested that the formulation is much more suitable for transdermal application and safe as per the prescribed GRAS (Generally Regarded as Safe) limits, avoiding skin irritation. Consequently, the density of LO (1.6 ± 0.4 g/mL) was recorded slightly higher than LO-NE (1.05 ± 0.2 g/mL), much closer to the density of water (1 g/ml) making it safe and suitable to impregnate the dermal layers and reach much deeper layers through transcellular route. Then the conductivity of LO-NE (452.8 ± 0.6 mS/cm) was found to be closer to the conductivity of deionized water, sustaining an ideal action potential inside the biological system and enhancing the permeation of LO-NE. Lastly, the viscosity of LO-NE was observed to be 35 ± 0.2 cps regarded as safe for dermal usage [40] [41].

DPPH analysis is considered as one of the accurate methods for the antioxidant evaluation. This assay depicts the stable radical scavenging rate of DPPH by the presence of antioxidative compounds in the LO-NE. The IC₅₀ value is the measure of the potency of a substance in inhibiting the scavenging activity by 50%. LO-NE showed IC₅₀ of 4.74 ± 0.93 µl/mL whereas for LO it was 5.98 ± 0.65 µl/mL (Table 4). This clearly indicated that the LO-NE has higher ability to scavenge the free radicals present at a lower concentration in comparison to LO alone. The maximum antioxidative ability was observed at a concentration of 10 µl/mL where the corresponding activity was 93% ± 0.91% in LO-NE which was much more than the LO (75.99% ± 0.91%) (Figure 6(a)). Therefore, the order for inhibition was in the following order: LO-NE > LO > control. Similarly, in case of ABTS, the antioxidant activity increased with increase in the concentration of test samples. For LO-NE the scavenging was 97.66% ± 1.02% whereas, for LO, it was 81.36% ± 1.92% at 10 µl/mL (Figure 6(b)). Furthermore, the IC₅₀ value was 4.53 ± 0.59µl/mL in case of LO-NE and 6.51 ± 0.52 µl/mL for LO. This proved that LO-NE exhibited higher scavenging ability than LO and control. Among all the reactive oxygen species present, hydrogen peroxide is the most reactive one which has the ability to generate the radical molecule during a reaction. LO-NE has the ability to scavenge out OH⁻ free radicals in a dose dependent manner. The highest activity of scavenging was observed at a concentration of 10 µl/mL which corresponded to 88.72% ± 1.09% and 92 ± 1.25 from Hydrogen peroxide and Superoxide radical scavenging assays, respectively (Figure 6(c) and Figure 6(d)). Therefore, from all the antioxidant assay conducted we can conclude that the LO-NE possess higher free radical scavenging activity when compared to that of LO [45] [46].

The Franz diffusion setup for evaluating compound release kinetics was run for 12 hours and the readings were recorded at the interval of every hour. The pattern of compound release showed that 80% of the drug was released in initial 6 hours and only 20% was released in the second half of the experiment. The graph of percentage cumulative compound release versus time can be studied to validate the sustained release of the compound with time (Figure 7). The pattern of release for LO and LO-NE evidently showed the improvement in release profile of LO in the form of NE in contrast to the pure extract form. The analysis of data and verification from the standard release kinetics models (Figure 8) exhibits that the data best fits with Higuchi’s model, giving the highest value for correlation coefficient (R² = 0.9812 for LO-NE) (Table 5). The Higuchi’s model for release kinetics analysis is considered as the most well-known equation for controlled release [47] [48].

In the current study the stability of the LO-NE, LO was tested in terms of antioxidant activity after 6 months at 517 nm by DPPH assay. LO-NE was found to be stable in comparison to LO at the temperature of 37˚C (Figure 9). For the 10 µl/mL LO, the antioxidant activity was 75.99 ± 1.97% which degraded within the span of 6 months and reduced to 60 ± 2.54% which can be attributed to the volatile nature of LO. On the contrary, the antioxidant activity of LO-NE was estimated to be 93 ± 1.51% initially which after 6 months with a minor degradation reduced to 85 ± 1.61%. Therefore, the results suggested that, the LO-NE have higher stability percentage index than that of the pure LO alone [49].