B. persicum and T. ammi were collected from the mountain Bahabad, located in the Province of Yazd, in Iran, and their species were identified and authenticated in the botanical section of the Yazd Agricultural Research Center, with the voucher numbers 1141 and 24985, respectively. A total of 100 g of seeds of B. persicum and T. ammi were harvested, dried, and mechanically powdered using an electrical blender. The whole dry of two plants was dissolved in distillated water (250 mL) overnight at room temperature using Clevenger apparatus and after 4 hours the yield was used.
Standard calibration curves of B. persicum and T. ammi (Essential Oils)
To perform the calibration curve, 1 mg/mL of B. persicum and T. ammi was prepared in methanol. Then, different dilutions of essential oils in isopropyl and PBS buffer (phosphate buffered saline) were prepared. The absorption spectra of each dilution were measured by a spectrophotometer (Carry 100 Bio Co. Varian, Victoria, Australia). The experiment was repeated three times, the standard diagram of the essential oils of B. persicum and T. ammi was drawn and then the calibration chart of essential oils of B. persicum and T. ammi was drawn in isopropyl solvent and PBS.
Preparation of liposomal systems containing essential oils of B. persicum and T. ammi
The liposomal system containing essential oils was prepared by thin-layer coating with a combination of soybean phosphatidylcholine (70%), and cholesterol (30%). Soybean phosphatidylcholine choline, cholesterol and essential oil were dissolved in a chloroform solvent at 45°C on a rotary (Heidolph Instruments GmbH & Co. KG, Schwabach, Germany) and dried under vacuum. Hydration was then performed by adding sterile distilled water for one hour at 55°C. The prepared nano-liposomes were then reduced in size using a bath sonicator for 40 minutes. At the end of the mixture, the size reduction was filtered using 0.45 and 0.20 μm filters to homogenize the constituent particles.
Determination of essential oil loading percentage in liposomal systems
Liposomes containing essential oils were inserted into the dialysis bag after size reduction and filtration and incubated with distilled water for 4 hours at 4°C to remove the free and uninspired essential oil. Liposomes were mixed with 1:20 isopropyl to break the lipid wall around the essential oil and release the essential oil. The amount of essential oil absorbed in the liposomal system was determined by a wavelength spectrophotometer. Finally, using the following relationship, the percentage of B. persicum and T. ammi load of essential oils in liposomal systems was determined separately.
Essential oils release from liposomal systems
The release of essential oils from liposome system was performed by the dialysis bag method. In this method, liposomes containing the essential oils were poured into the dialysis bag and placed in the vicinity of the PBS buffer at 35°C and pH = 6 for 24 hours. The absorbance was read by spectrophotometer (570 nm) and based on this, the release chart of liposome essential oils was drawn. All steps were performed separately to release from the liposome system containing B. persicum and T. ammi. The release results were analysed according to the standard deviations.
Size and zeta potential characterization of essential oils in liposomal systems
The particle size distribution range, as well as particle size peak, was determined using Dynamic Light Scattering (DLS), by nanosizer (Brookhaven Instruments Corp, NY, USA). Nano-liposomes were measured at an angle of 90 degrees and laser light irradiation at 657 nm at 25°C was used. The sample was prepared diluted to 0.1 mg/mL. Measurements were performed immediately after preparation. Samples were measured 3 times and each time for 30 seconds. In addition, surface charges (zeta potential) of nano-liposomes containing essential oils were measured by Zeta Sizer (Brookhaven Instruments Corp, NY, USA).
Morphology of liposomal systems containing essential oils
The liposomal system was imaged using an Atomic Force Microscope (AFM: Nano wizard II, JPK instruments, Berlin, Germany) to confirm the formation of essential oil-bearing nano-liposomes containing essential oil. In addition, 25 µL of liposome sample was poured onto a slide and air dried. Samples were then coated with gold for a few seconds to be conductive. At the end, the surface morphology of the nano-carriers (roughness, shape, smoothing and mass) was investigated using a Scanning Electron Microscope (SEM: EM3200, KYKY Technology Development Ltd, Beijing, China).
Cytotoxicity studies by MTT assay
The cytotoxicity of the empty liposomal system was determined by the MTT assay. To measure toxicity, cells were cultured separately at 1×104 cells in a 96-well plate for 24 hours. Healthy human foreskin fibroblast (HFF) cells were then treated with the same volume of fresh culture medium. Then, HFF fibroblasts were cultured in four replicates at 100 and 10 μg/mL, respectively, and incubated again for 48 hours. Then, 20 µL of MTT solution at a concentration of 5 mg/mL were added to each well and incubated for 3 hours. The supernatant was then removed and 150 μL of DMSO was added to dissolve the Formosan crystals. At each step, centrifugation was performed to remove the liquid. The absorbance was recorded at 570 nm using a spectrophotometer. Finally, cell viability was calculated according to the relationship below:
Determination of IC50 nano-liposomes containing essential oil on T. vaginalis
The strain of T. vaginalis was isolated from the vaginal discharge of a 34-year-old woman with a confirmed diagnosis of trichomoniasis. This study was approved by the ethical committee of the University of Medical Sciences of Isfahan, Isfahan Province, Iran (Ref. No. 1396.851) to obtain human biological samples. The parasite isolate was cultured in TYI-S-33 medium and incubated at 37°C until reaching the number of parasites in the logarithmic phase. Smears were prepared to determine the number of parasites. The parasitic strain of the logarithmic phase (105 parasites) was then exposed to different concentrations of B. persicum and T. ammi nano-liposomes at a concentration of 100,000 per mL (grouped according to Table 1).
All groups were kept at 37°C and after 12 and 24 hours the number of live parasites was counted by Trypan blue using a Neubauer smear and light microscope (Haemocytometer method). Growth inhibition was calculated for each of the different concentrations (1.95- 1000 µg/mL) of liposome-containing essential oils in the parasite strain by the formula GI = (a-b/a). Where a: the number of live parasites in the negative control sample, b: the number of live parasites in the sample containing B. persicum and T. ammi nanoliposomes. Then, SigmaPlot™ 13 software was used to determine IC50 (50% inhibitory concentration) of the above nano-liposomes. It should be noted that all experiments were performed in three replicates separately for liposomal nano-carriers containing essential oils of B. persicum and T. ammi and the results were shown in an average level. Selectivity Index (SI) is obtained from the IC50 value of liposomal nano-carriers containing essential oils of B. persicum and T. ammi against HFF cells divided by the IC50 value of T. vaginalis. Selective activities of the compounds were calculated as follows:
Selectivity Index (SI) = IC50 Cells HFF/IC50 Cells T. vaginalis
Cellular uptake study
A total of 105 HFF cells in 6-well plates were grown in a monolayer for 24 hours. Then, the cells with the optimal nano-liposomes formula contained DIL color without essential oil. For the fluorescence of the Newsome system and, to a large extent, 0.1% M in the organic phase to dry the structure, the additives were followed by the steps of the thin film method, as described by Alemi A et al, 2018 . Subsequently, fluorescent colored nano-liposomes attached to the DIL. These were incubated with essential oils for 3 hours and washed with PBS. Subsequently, the 95% alcoholic solution was stabilized and the nuclei were stained with DAPI (1 mg/mL) for 15 minutes. The cells were harvested and then examined under a fluorescent microscopy (Olympus, Okayama-shi Okayama, Japan).
In this study, all data were recorded, edited, and entered in the Excel software to plot the results which were reported as mean and standard deviation. Meanwhile, the SigmaPlot ™ 13 software (Systat Software Inc., San Jose, CA, USA) was also used to calculate IC50.