## 3 Results and Discussion
The release profiles of MTC from transdermal film formulations (TF-NR, TF-EU, TF-TP and TF-LM) through the cellulose membrane are shown in Figure 5. There was no significant difference in transdermal films containing nerolidol (TF-NR, 1.12 \(\pm\) 0.32 mg/cm\({}^{2}\)), eucalypt (TF-EU, 1.20 \(\pm\) 0.25 mg/cm\({}^{2}\)) and dl-limonene (TF-LM, 0.98 \(\pm\) 0.19 mg/cm\({}^{2}\)) in terms of the released amount of drug (\(p>0.05\)) whereas transdermal film containing terpionlene (TF-TP, 1.63 \(\pm\) 0.42 mg/cm\({}^{2}\)) had significantly higher drug release rate than those of other formulations (\(p<0.05\)). This significant increase in the extent of drug release was observed with transdermal film composed of terpionlene can be attributable to the lower solubility capacity of terpionlene than those of other terpen and this could be explained by the fact that MTC showed reduced affinity for the vehicle. The release mechanism of the drug from transdermal films was examined by fitting the obtained release data into zero order, first order and Higuchi matrix model [32, 43]. Our results indicate that MTC release from transdermal films followed Higuchi model (\(r^{2}\) = 0.98-0.99). This observation implies that the drug released from all transdermal films prepared follows matrix diffusion mechanism, in which the rate controlling step is the process of diffusion through matrix polymer base [44, 45, 46]. The regular release of MTC from all formulations led to prove the homogeneous physical structure of the films. Figure 4: _In vitro_ adhesion force of transdermal films. ATR-FTIR spectroscopy measurements were performed to investigate the effects of sodium alginate based film formulations containing different terpenes on _in vitro_ conformational order of _stratum corneum_ intercellular lipids. Excised pig skin was used as model membrane which is accepted possessing similar properties to human skin. The absorbance frequency shift, area and height of C-H symmetric absorbance peaks were analysed and the results are given in Figure 6a,b. Figure 5: _In vitro_ release of MTC from transdermal film formulations. Figure 6: (**a**) Peak positions; (**b**) peak height and areas of skin lipids C–H symmetric absorbances after application of MTC transdermal films. Chemical enhancers are commonly incorporated in transdermal drug delivery systems in order to improve the diffusion kinetics of the drugs administered [47]. Terpenes act to alter the lipid or protein structures in the _stratum corneum_ by modifying the intercellular packing, disrupting the order of lipids, and/or increasing drug diffusivity into the tissue [20; 21; 48]. It has been reported that the C-H symmetric stretching band is more susceptible for the introduction of conformational disorder and a blue shift of C-H symmetric band to higher frequencies is characteristic of a high content of gauche conformers indicating higher lipids disorder [33; 49; 50]. For untreated skin the C-H symmetric frequency was observed at 2850.6 \(\pm\) 0.2 cm\({}^{-1}\). ATR-FTIR results showed a more fluidized _stratum corneum_ lipid state in the presence of terpenes as enhancer (\(p>0.05\)). In general, a decrease in peak height and area of C-H stretching is attributed to reduced lipid content in skin [48; 50; 51]. As shown in Figure 6b, transdermal film formulations TF-NR, TF-EU, TF-TP, and TF-LM produced a greater decrease in peak heights and areas for C-H symmetric stretching absorbances in comparison with untreated skin and the skin treated with TF-CONT. It was reported that the presence of various co-solvents can affect the interaction of terpenes with _stratum corneum_ and the combination of limonene in propylene glycol has been found to produce a synergistic action and thereby resulting in higher _stratum corneum_ lipid extraction [51; 52; 53].