3.2. Pseudo-Ternary Phase Diagrams of the Blank Microemulsions
The pseudo-ternary phase diagrams obtained from IPM, water, and Smix including Labrasol®
(1:1) with Transcutol®
in the ratios of 1:1, 2:1, and 3:1 are shown in Figure 3
A–C, respectively. The microemulsion (isotropic) region was observed in all diagrams. As reported in Panapisal, et al. [15
], the isotropic region can be obtained when using IPM, water, and Smix of Labasol®
at a weight ratio of 0.5:0.5:1. However, in our study, the Smix ratios used varied from 0.5:0.5:1, 1:1:1, and 1.5:1.5:1, respectively. As the ratio of Labasol®
compared to that of Transcutol®
(cosurfactant) increased from 1:1 and 2:1 to 3:1, the isotropic region increased. Some studies reported that an increase in the cosurfactant ratio could stabilize the microemulsions and affect their formation [16
]. However, similar to this study, there have been reports that an increase in the surfactant:cosurfactant ratio could increase the isotropic region by reducing the interfacial tension, hence improving the fluidity at the interface of the colloidal solution and increasing the entropy of the system [18
In addition, other systems constructed using Tween 80 and Span 80 as surfactants and Transcutol® or PG: ethanol (1:1) as cosurfactants in the ratio of surfactant:cosurfactant at 1:1 were tested for comparison. Compared to the use of PG:ethanol, Transcutol® could increase the isotropic region, indicating that Transcutol® could better stabilize the microemulsion droplets. However, these two diagrams presented smaller isotropic regions than the region of the Smix of Labasol® and HCO-40®.
3.3. Preparation and Characterization of Blank Microemulsions and Microemulsions Containing GW Extract
Blank microemulsions and microemulsions containing GW extract were then prepared by selecting the IPM:Water:Smix ratio within the isotropic region. A total of nine formulations selected from the five diagrams were prepared and characterized.
The characterization results of the blank microemulsions are shown in Table 2
. The droplet sizes of the blank microemulsions were within the range of 6–43 nm. It can be observed that the microemulsions with a high water content (ME_W1–W4) had larger droplet sizes than the corresponding formulations with a high oil content (ME_O1–O4). Similar results were observed that increasing the water content led to an increase in the droplet size of microemulsions [21
]. All formulations showed zeta potential values near zero, indicating the neutral charge of the microemulsion droplets. Similar results have been found in other studies that contained nonionic surfactants as major components in microemulsions and these contribute to the neutral charge of the obtained microemulsion [23
]. The type of microemulsion was evaluated by electrical conductivity measurements. According to Djordjevic et al. [27
], the microemulsion type can be characterized by comparing the ratio of the oil (o) and water (w) phases, in which a higher ratio is specified as the external phase, in combination with the conductivity value (w/o 2.9–3.8 µS/cm, bicontinuous microemulsion 10.3–52.5 µS/cm, and o/w 80.5–94.3 µS/cm). Among the formulations prepared, the formulation using Labasol®
as surfactants with a high water content (ME_W1–W3) could be characterized as a bicontinuous phase (BP). For the formulations using Tween 80 and Span 80 (ME_W4 and W5), higher conductivities were observed than those of the Labasol®
formulations, and thus, could be characterized as o/w microemulsions. However, for the formulations with a high oil content (ME_O1–O4), lower conductivities were observed than their corresponding formulations and could be characterized as BP or w/o type.
shows the characteristics of nine formulations of microemulsion containing the GW extract. The droplet size of all formulations increased significantly in comparison to that of the blank microemulsions. A similar result was found in the incorporation of Sompoi in microemulsions, showing that the extract may be solubilized and enriched in the external phase or surfactant layer resulting in a larger droplet size [28
]. Unlike the blank microemulsions, the droplet size of the microemulsions containing GW extract with a high content of water (ME_W1–W4) and high content of oil (ME_O1–O4) were similar. The zeta potentials of these formulations were neutral. The importance of the highly negative charge of emulsion droplets for their stability has been reported [29
]. Nevertheless, some studies found that neutrally charged microemulsions were physically stable after 3 months of storage [24
]. As an evaluation of stability was not conducted in this study, which is a limitation of this study, the effect of neutral charge on the stability of microemulsions could not be determined. Therefore, further study on the stability of these microemulsions should be conducted in future work. The conductivity of all formulations increased, which might be owing to the ionized moieties of the phenolic compounds in the extract. Therefore, the conductivities in the formulations incorporating the GW extract were not counted as changes in the microstructure or type of microemulsions.
The viscosity and pH of all the blank microemulsions and microemulsions containing the GW extract are shown in Table 4
. The viscosities of the blank microemulsions were low, in the range of 48 to 383 cP. However, the addition of 1% GW extract resulted in an increase in viscosity as high as 212–2273 cP. The formulations with the highest content of Transcutol®
(ME_W1 and O1) had the lowest viscosity, and the viscosity increased as the cosurfactant content decreased. The explanation is that the presence of the cosurfactant molecules at the interfacial film could influence the fluidity of the interfacial film and thus reduce the apparent viscosity of the system [31
]. Furthermore, the viscosity of the formulations with low water content was lower than that of its corresponding high water content formulations, as the high volume of water caused swelling of the microemulsion droplets, leading to stronger interactions between the interfacial membranes [32
]. For pH, the addition of the GW extract caused a decrease in the pH of the corresponding blank microemulsions in all formulations, as it contained phenolic acids as the active compounds.
3.4. Skin Permeation and Disposition of Microemulsions Containing the GW Extract
The skin permeation profiles of all microemulsion formulations are shown in Figure 4
, and the steady-state flux of gallic acid and enhancement ratio are shown in Table 5
. The steady-state flux was calculated from 4–22 h, since the linear portion was observed during this period. In addition, at 24 h, the cumulative amount of gallic acid permeated slightly deviated from the linear line, indicating that the integrity of the skin might be changed. Among all formulations, ME_W1, which contained a high water content and the highest ratio of Transcutol®
, showed the highest permeation at 22 h, followed by ME_W3, W4, and gel (enhancement ratio = 1.78, 1.60, 1.28, and 1.00, respectively). However, in the formulations with high oil content (ME_O1–O4) and ME_W2, a lower permeation of gallic acid was observed than that in the gel. This result indicated that the hydrophilic phenomenon of the microemulsion is useful for the skin penetration of gallic acid. As previously reported, the increase in water content could affect the microstructure of the microemulsions and enhance the skin permeation of hydrophilic and lipophilic compounds [32
]. The explanations for this effect are the change in the thermodynamic activity of drugs, skin hydration, and loading of the skin penetration enhancers in the surfactant mixtures [35
]. In addition, water in the formulations could hydrate the skin, swell the SC, and increase the drug diffusivity in the skin, which resulted in an increase in drug flux [36
In addition to the water content, the compositions of the surfactants and cosurfactants used in this study might be a reason for the different levels of skin permeations. The results of this study indicated that 30% Transcutol®
(ME_W1) could provide the highest skin permeation of gallic acid. However, 20% Transcutol®
(ME_W2) decreased the skin permeation as compared to that with the 15% Transcutol®
(ME_W3). In most cases, Transcutol®
promoted higher drug permeation as its concentration increased, but an exception was found in some concentrations of Transcutol®
when combined with IPM or Miglyol 812 [37
]. Furthermore, because the concentration of Smix was fixed at 60%, the increase in Transcutol®
concentration of Smix decreased the concentration of the other surfactants used. Therefore, suitable ratios of surfactant:cosurfactant needed to be optimized, in which the highest permeation observed in this study was from the 1:1 ratio.
A comparison of skin permeation from ME_W4 and W5, which contained Tween80:Span80 but had different cosurfactants, showed that gallic acid permeated more with the ME_W4 formulation, which contained Transcutol® as a cosurfactant and is a skin penetration enhancer. For the formulations with a high oil content (ME_O1–O3), the skin permeation of gallic acid seemed to increase as the ratio of surfactant:cosurfactant increased (from 1:1 and 2:1 to 3:1), or reduced the concentration of Transcutol®, but with lower permeation than the gel.
For the skin disposition experiment, the amounts of gallic acid in SC and VED are presented in Figure 5
and Figure 6
, respectively. The highest amount of gallic acid accumulated in SC was observed in the formulation ME_W2, which provided low skin permeation compared to that with the other microemulsions containing high water content, followed by gel. Other formulations showed a lower amount of gallic acid accumulation than the control gel. Interestingly, ME_W1 gave the lowest gallic acid disposition among all formulations, in contrast to the skin permeation amount.
In the VED, the gel provided the highest gallic acid accumulation, followed by W5, W2, and W3. Although formulations W2 and W5 did not provide a high permeation of gallic acid through the skin, the accumulation in VED was high and suitable for use in dermal formulations. The enhancement in drug disposition, which is not concomitant to drug permeation, might be owing to the depot effect of some penetration enhancers. IPM, Transcutol®
, and propylene glycol monocaprylate have been reported to be retained in the skin and increase the retention of anthramycin [38
also caused swelling of the SC lipid and deposited the drug in the lipid [39
]. However, the reason for the depot effect of ME_W2 and W5 found in this study was complex and requires further investigation.
For formulations with a high content of oil, low amounts of gallic acid were observed to be accumulated in both the SC and VED compared to that in the control gel, similar to the skin permeation results.
As seen in this study, ME_W1, W3, W4, and the gel could serve as the transdermal carriers for enhancing the skin permeation of gallic acid. The gel, W2, and W5 could be better used in the skin formulation aimed to form the drug depot in the skin.
Characteristics and mechanisms of microemulsions to enhance skin permeation and disposition have been proposed—e.g., its small droplet size and large surface/volume ratio, the penetration enhancing effect of the combination of its components, increase in drug solubility and drug loading by microemulsions, and improved skin hydration and occlusion [35
]. The reason for the different skin permeation results of gallic acid from that of various microemulsion formulations observed in this study included the microstructure of the microemulsions, the optimal ratios of surfactant: cosurfactant, and the compositions. We observed that microemulsions with high water content could enhance the skin permeation of gallic acid. These microemulsions were characterized as an o/w type. In addition, we observed that the particular ratio of surfactant:cosurfactant could enhance the skin permeation of gallic acid compared to that of the gel, as previously described. Moreover, the choice of components used affects skin permeation and disposition.
The main components used in this study were non-ionic surfactants—i.e., Labrasol®
, Tween 80, and Span 80. These nonionic surfactants act mainly to form the interfacial film of the microemulsion droplets. Moreover, they enhance skin penetration by fluidizing or solubilizing the intercellular lipid, or its interaction with the keratin of corneocytes [41
The cosurfactant Transcutol®
has been studied for its skin penetration enhancing effects on various drugs, such as caffeine, ibuprofen, diclofenac, and metronidazole [40
]. Its mechanisms were proposed, including the effect on the thermodynamic driving force of drugs, increasing drug partitioning in the SC, hydrating the SC, and fluidizing the SC intercellular lipids [37
]. PEG and ethanol have also been used as cosurfactants in various microemulsion formulations. They decreased the interfacial tension and stabilized the microemulsions, as well as enhanced the skin permeation of drugs [42
Various mechanisms might be incorporated and influence the skin permeation and disposition of the microemulsions prepared in this study. These findings were beneficial in the development of suitable carriers for delivering phenolic antioxidant compounds from the GW extract for both transdermal and topical treatments.