Essential oils spectroscopy
For spectrophotometric data, the calibration curves of the essential oils containing B. persicum and T. ammi were plotted in the isopropyl solvent and PBS. These were shown as y = 0.0179 x + 0.1686 and y = 0.025x - 0.6296 with a correlation coefficient (r2) of 0.9962 and 0.9947 for isopropyl solvent and PBS, respectively (Fig. 1S and 2S).
The line diagram equation in the calibration of essential oil containing T. ammi in isopropyl solvent and PBS was y = 4.278 x+ 0.1473 and y = 3.6511 x + 0.0362, respectively. The regression coefficient (R2) of the essential oil containing T. ammi in the isopropyl solvent and PBS was 0.9985 and 0.9996, respectively (Fig. 2S, 3S, and 4S).
The loading rate of essential oils in the nano-liposomal systems
Based on the calibration diagram of the essential oils containing B. persicum and T. ammi in isopropyl (Fig. 1S and 3S) and the loading rate of both essential oils in the liposomal systems, were 51.64±1.24 and 40.12 ±2.71%, respectively.
Patterns of release of essential oils from the nano-liposomal systems
The release charts for essential oils containing B. persicum and T. ammi from the liposomal system were plotted during 24 hours at 35°C and pH = 6 based on the calibration chart. Examination of the essential oil release pattern of the liposomal system (Table 2 and 3) shows that the release of the essential oil in the first hours (at 6 hours after release) and the sustained release of the essential oil in the last hours (interval 12 within 24 hours after release), nano-liposomal essential oils containing B. persicum and T. ammi were present in this study. In addition, the release charts for the two types of essential oils were removed from both nano-liposomal systems in 24 hours (100% release in 24 hours). The release results were presented in Figure 1, Tables 2 and 3 with standard deviations.
Characterization of the size and zeta potential of essential oils in the nano-liposomal systems
The DLS results show that the particle size in the nano-liposomal system of essential oils containing B. persicum and T. ammi, 121 nm and 111 nm, respectively (Figures 2 and 3). In addition, the surface charge (zeta potential) of the nano-liposomal systems of essential oils containing B. persicum and T. ammi was calculated to be -16.7 mV and -9 mV, respectively (Figures 4 and 5).
The present study led to the production in the nano-liposomal systems of essential oils containing B. persicum and T. ammi. The results showed that the load of essential oils containing B. persicum and T. ammi in the nano-liposomal systems was 51.64±1.24 and 40.12 ±2.71%, respectively.
The particle size and zeta potential were also calculated for the nano-liposomal systems containing T. ammi were 111 nm and -9 mV, whereas for B. persicum they were 121 nm and -16.7 mV, respectively. The release of essential oils of B. persicum and T. ammi from the liposomal system was slow in 24 hours. The essential oils from both nano-liposomal systems were released at the end of this period (24 hours).
Nano-liposomal systems containing essential oils morphology
The morphology on the nano-liposomal systems of both essential oils containing B. persicum and T. ammi was investigated by the Atomic Force Microscope (AFM), Scanning Electron Microscope (SEM), and the formation of liposome was confirmed (Figures 6 and 7). The SEM image shows that the morphology of the constituent particles are liposome systems containing essential oil, are spherical with smooth surface and average particle size from 25 to 40 nm.
In addition to electron microscopy images, it also confirms the formation of liposomal systems, spherical morphology and the appropriate constituent particles of liposomal systems. Cellular studies also showed that the nano-liposomal systems had low toxicity for HFF cells, and the nano-liposomal systems of essential oils containing T. ammi and B. persicum for 24 and 48 hours showed toxicity for the parasite T. vaginalis and were further prevented its growth.
So far, previous studies have been performed on the production of lipid systems containing drugs and essential oils, each of which investigates different physicochemical factors of lipid systems, including loading efficiency, drug release pattern, particle size and surface charge (zeta potential) [31-32].
This physicochemical index depends on several factors, including the size of the constituent system, the type and molar percentage of phospholipids used in the structure of the liposomal system, the molar percentage of cholesterol used in the structure of the liposomal system and the nature of the loaded material.
In the present study, the amount of essential oils containing B. persicum and T. ammi in the liposomal system was measured as 40.12 ±2.71 and 51.64±1.24%, respectively. Other studies have yielded similar results, for example, Majdizadeh et al., 2018, reported loading of essential oil of Mentha piperita by 61.38% . In addition, Haghiralsadat et al, 2016 reported the loading rate of essential oil of T. ammi in the liposomal system as 35.6± 7.4 . Other features studied in liposomal systems are the patterns of drug release. The present study showed that the release of essential oils of B. persicum and T. ammi from liposomal systems was slow in 24 hours.
The pattern of release of these essential oils from the liposomal systems shows that, due to the high concentration of essential oils between the liposomal systems and the buffer that surrounds them, the release of essential oils from the system was explosive in the first hours and decreased over with time. This difference in concentration decreased the release of the essential oil and caused the slope of its release chart to tilt to zero .
Another important feature evaluated in this study was the level of superficial charge of the liposomal systems, which is one of the factors that influence the stability of the liposome. There is a direct relationship between the surface charge and the chance/stability of the liposomal systems. Therefore, the greater the surface load of the liposomal system, the greater the chance that these liposomal systems will form and aggregate, increasing their stability [35-37].
Cytotoxicity studies by MTT assay on Human foreskin fibroblasts (HFF) cell line
The evaluation of the effect of the essential oil-free liposomal system on human foreskin fibroblast (HFF) cells showed that the viability of these cells in the presence of 10 and 100 μg/mL of an empty liposomal system is 96.7% and 97.2%, respectively. Thus, the empty liposomal system has little toxicity to HFF cell lines.
Nowadays, the use of herbal compounds for therapeutic purposes has been widespread and numerous studies have been conducted on the effects of herbal compounds on health and treatment. In the present study, after the development of liposomal systems containing essential oils of B. persicum and T. ammi, the cytotoxic effects of the vacuolar system on the HFF cell line and the cytotoxic effects of essential oil systems in T. vaginalis were investigated, with minor effect on the HFF cell line.
IC50 of nano-liposomes containing the essential oil against T. vaginalis
The IC50 of B. persicum-containing liposome essential oil was calculated to be 45.19 µg/mL and 14.41 µg/mL in T. vaginalis after 12 and 24 hours, respectively. The IC50 value of the nano-carrier containing T. ammi essential oil was determined after 12 and 24 hours against the parasite, T. vaginalis, at 25.81 µg/mL and 8.08 µg/mL, respectively (Fig. 8 and 9). The value of SI (Selectivity Index) were 3.1 for T. ammi and 2.49 B. persicum.
Cellular Uptake Study
Highly successful nano-liposomes cell extract of containing essential oils without fluorescent dyes DIL was synthesized by fluorescent microscopy. According to the Fig. 10, the intensity of green fluorescence in healthy HFF skin cells indicates the cellular uptake of nano-liposomes essential oils, nano-liposomes are well absorbed by the cell. Core-shaped with DAPI color (blue) and colored DIL nanoliposomes (green) were painted.