Nanoemulsions of Jasminum humile L. and Jasminum grandiflorum L. Essential Oils: An Approach to Enhance Their Cytotoxic and Antiviral Effects

Unprecedented nanoemulsion formulations (NE) of Jasminum humile and Jasminum grandiflorum essential oils (EO) were prepared, and examined for their cytotoxic and antiviral activities. NE characterization and stability examination tests were performed to ensure formula stability. The antiviral activity was determined against hepatitis A (HAV) and herpes simplex type-1 (HSV-1) viruses using MTT assay, while the cytotoxic potential was determined against liver (HepG-2), breast (MCF-7), leukemia (THP-1) cancer cell lines and normal Vero cells. Statistical significance was determined in comparison with doxorubicin as cytotoxic and acyclovir as antiviral standard drugs. GC-MS analysis indicated twenty four compounds in the EO of J. humile and seventeen compounds in the EO of J. grandiflorum. Biological investigations of pure EOs revealed weak cytotoxic and antiviral effects. Nevertheless, their NE formulations exhibited high biological value as cytotoxic and antiviral agents. NE formulations also showed feasible selectivity index for the viral-infected and cancer cells (especially HepG-2) than normal Vero cells. Both nanoemulsions showed lower IC50 than standard doxorubicin against HepG-2 (26.65 and 22.58 vs. 33.96 μg/mL) and MCF-7 (36.09 and 36.19 vs. 52.73 μg/mL), respectively. The study results showed the dramatic effect of nanoemulsion preparation on the biological activity of EOs and other liposoluble phytopharmaceuticals.


Introduction
Jasminum is a genus of flowering plants (approximately 600 species), belonging to family Oleaceae, usually used in perfume industries and ornamental purposes due to their bright colored flowers and characteristic fragrances. Traditionally, the essential oils (EOs) of different Jasminum species were used in aromatherapy for the treatment of diarrhea, fever, abdominal spasms, conjunctivitis, skin inflammations, bronchial asthma and uterine hemorrhage [1]. Nowadays, several reports investigated the biological activities of the EOs of several Jasminum species. Jaminum sambac EO was reported to exhibit antibacterial activity and to suppress puerperal lactation [2] in addition to its antidepressant and mood uplifting properties [3]. Jasminum officinale EO was reported to have antiviral activity against hepatitis B virus [4]. Only few studies investigated the biological activities of Jasminum grandiflorum EO, much less Jasminum humile EO. The EO of Jasminum grandiflorum was examined for its antimicrobial activity against Gram-positive and Gram-negative bacteria as well as candidiasis [5]. Jasminum grandiflorum was reported to have cytotoxic activity against brain cancer cell line [6].
In recent times, there has been more interest in the nanoscience and nanotechnologies applications. Nanotechnology modifies and develops the properties of drugs by converting them into their nanoparticles. It has numerous applications including drug delivery

Plant Materials
The fresh flowers of Jasminum humile L. and Jasminum grandiflorum L. (250 gm, each) were collected in the early morning during May 2021 from the botanical garden of Kafr EL-Sheikh university, Egypt and were attested by Dr. Ibrahim Mashaly, Professor of Ecology, Faculty of Science, Mansoura University, Egypt. The essential oils (EOs) were extracted by hydro-distillation for almost 8 h; according to the European pharmacopoeia (European Pharmacopoeia, 1975) using Clevenger's cohobation apparatus to produce 2 mL and 2.5 mL, respectively of yellow colored EOs. The collected oils were dehydrated over anhydrous sodium sulfate and then they were stored in sealed vials at low temperature for further (GC/MS) analysis and biological investigations.

Capillary Gas Chromatography-Mass Spectrometry (GC/MS) Analysis
The GC/MS analysis was carried out at the faculty of postgraduate studies for advanced sciences (the central laboratory), Beni Suef. Egypt. Identification of the components was confirmed by comparing their mass spectral fragmentation patterns and retention indices with the previously reported literature [13][14][15][16][17] as well as the mass spectral NIST/ChemStation database.

Preparation of Nanoemulsion Formulations
JhEO-NE and JgEO-NE formula were prepared by mixing 1% w/w of each EO with 8% w/w glycerol monoacetate (GMA). Tween 80 (surfactant) was mixed with labrosol (co-surfactant) in an equal ratio, and the mixture (30% w/w) was added to EO-GMA. Finally water (61% w/w) was dropped in order to obtain an apparent and clear NE [18]. The prepared JhEO-NE and JgEO-NE formula were subjected to thermodynamic stability studies and self-nanoemulsification efficiency tests according to the International Conference for Harmonization (ICH) guidelines. Freshly prepared formula were packaged in glass bottles and subjected to different storage conditions of refrigeration at (5 ± 3 • C) and different ambient conditions over a period of 3 months. Physical assessment of the samples was achieved by visual inspection of phase separation, color and/or odor change and pH measurements at zero time (freshly prepared formula), as well as after storage periods of 1, 2 and 3 months. The particle size, polydispersity index (PDI) and zeta potential (ZP) were also measured (using Malvern Zetasizer Nanoseries, Malvern Instruments Limited, UK).

MTT Protocol
A 96 well tissue culture plate was inoculated with 1 × 10 5 cells/ml (100 µL/well) and incubated at 37 • C for 24 h to develop a complete monolayer sheet. Growth medium was decanted from 96 well micro titer plates after confluent sheet of cells were formed, cell monolayer was washed twice with wash media. Two-fold dilutions of tested samples were made in RPMI medium with 2% serum (maintenance medium). 0.1 mL of each dilution was tested in different wells leaving 3 wells as control, receiving only maintenance medium. The plate was incubated at 37 • C and examined. Cells were checked for any physical signs of toxicity e.g., partial or complete loss of the monolayer, rounding, shrinkage, or cell granulation. The cytotoxic potential was investigated utilizing doses of (1000, 500, 250, 125, 62.5, 31.25, and 15.62 µg/mL), while the antiviral activity was assessed by applying (31.25, 15.62, and 7.81 µg/mL) of each sample.
MTT solution was prepared (5 mg/mL in PBS) (Bio Basic Inc.; Markham, Ontario, Canada) and 20 µL MTT solution were added to each well. A shaking table was used (150 rpm for 5 min) to thoroughly mix the MTT into the media and incubated (37 • C, 5% CO 2 ) for 1-5 h to allow the MTT to be metabolized. Media was dumped off and the plate was dried on paper towels-if necessary-to remove residues. Formazan (MTT metabolic product) was resuspended in 200 µL DMSO, then placed on a shaking table −150 rpm for 5 min-to thoroughly mix the formazan into the solvent. Optical density was measured at 560 nm (and subtract background at 620 nm), which is directly correlated with cell quantity.

Calculation of the Selectivity Index (SI)
Selectivity indices (SI) were obtained after dividing CC 50 (the half maximal inhibitory concentration of normal Vero cells) by the specific IC 50 of cancer cells and viral infected cells. Selectivity indices are used to evaluate the cytotoxic potential and antiviral activity relative to the normal cells toxicity; where high (SI) indicates high potency and low cellular toxicity [21][22][23].

Statistical Analysis
All statistical analyses were performed using GraphPad Prism version 9.2.0 to calculate the half-maximal inhibitory concentration (IC 50 ) and the half-maximal cytotoxic concentration (CC 50 ) where the level of significance was set at (p > 0.05). Quantitative data were expressed as mean ± standard deviation (SD). GraphPad Prism version 9.2.0 was also used to create multiple bar charts of the antiviral activity and cell viability.

Characterization of Nanoemuslion Formulations (Physical Characterization)
JhEO-NE and JgEO-NE formula passed thermodynamic stability as well as selfnanoemulsification efficiency tests. There was no observed phase separation or physical changes, in color, odor, or pH, over the storage period of 3 months at refrigeration conditions (5 ± 3 • C). They had negative ZP charges of (−18.63 and −229.67), nanometric sizes of (8.03 ± 0.45 and 12.09 ± 0.81 nm) and low PDI values of (0.30 ± 0.04 and 0.27 ± 0.03), respectively.

Cytotoxic Concentrations of Tested Samples on Normal Cells
The half maximal cytotoxic concentration (CC 50 ) for the essential oils (EOs) and the nanoemuslion formulatios of Jasminum grandiflorum L. (JgEO-NE) and Jasminum humile L. (JhEO-NE) was assessed on VERO cells using MTT assay (Tables 2 and 3). Dose-response curves are shown in (Figure 1

Cytotoxic Activity of Tested Samples
The cytotoxic activity was evaluated for the two (EOs) as well as their nanoemulsion formulations against three cell lines; HepG-2, MCF-7 and THP-1. The Vero cell line is a non-tumorigenic cell line established from kidney cells of the African green monkey (Cercopitbecus aetbiops). It can be used for investigating cell growth, differentiation, and cytotoxicity against non-cancerous cells. This cell line is an excellent in vitro model for investigating cytotoxicity and carcinogenesis due to its well-defined growth pattern and behavior in culture [24][25][26]. Human hepatocellular carcinoma cells (HepG-2) are usually used as an in vitro model for assessing cytotoxicity against liver cancer cells. They preserve several genotypic and phenotypic features of hepatocytes. Their cytotoxicity assay is highly sensitive and specific [27]. MCF-7 is a classic in vitro model usually used for determining the cytotoxicity against breast cancer cells for having several mammary epithelium characteristics, e.g., estradiol processing, and presence of estrogen receptors (ER) in their cytoplasm [28]. THP-1 is a monocytic-derived cell line; commonly used to investigate immune system disorders, and cytotoxicity. Many reports investigated the cytotoxic potential of different drugs on THP-1 leukemia cells; as a model for acute myeloid leukemia [29][30][31][32][33][34][35]. Dose-response curves are shown in (Figures 2-4). Both (EOs) showed weak cytotoxic activity against the examined cell lines. JgEO showed IC 50 value of 324.90 µg/mL against HepG-2, 327.53 µg/mL against MCF-7 and 286.37 µg/mL against THP-1 cells, while JhEO showed IC 50 value of 289.10 µg/mL against HepG-2, 304.13 µg/mL against MCF-7 and 265.60 µg/mL against THP-1 cells. Yet, the prepared nanoemuslion formulations of the two (EOs) induced significant cytotoxic activity against all cell lines in a concentrationdependent manner ( Figure S5-S7), and the results are depicted in (Table 2). showed IC50 value of 289.10 μg/mL against HepG-2, 304.13 μg/mL against MCF-7 and 265.60 μg/mL against THP-1 cells. Yet, the prepared nanoemuslion formulations of the two (EOs) induced significant cytotoxic activity against all cell lines in a concentrationdependent manner ( Figures S5-S7), and the results are depicted in (Table 2).

Antiviral Activity of Tested Samples
The antiviral activity was evaluated at the maximum non-toxic concentration (MNTC) against 100 tissue culture infectious dose TCID50/mL of HAV and HSV-1 viruses. Doseresponse curves are shown in (Figures 5 and 6). The examined (EOs) showed very weak antiviral activity against both viruses. JgEO revealed an IC 50 value of 275.57 µg/mL against HAV and 299.63 µg/mL against HSV-1, while JhEO showed an IC 50 value of 265.70 µg/mL against HAV and 275.33 µg/mL against HSV-1. However, the prepared nanoemuslion formulations of the two (EOs) induced significant antiviral activity against both viruses in a concentration-dependent manner ( Figures S6 and S7), and the results are depicted in (Table 3). Statistical significance was determined for their antiviral activities in comparison with the standard drug acyclovir after using various concentrations ( Figure S2).

Antiviral Activity of Tested Samples
The antiviral activity was evaluated at the maximum non-toxic concentration (MNTC) against 100 tissue culture infectious dose TCID50/mL of HAV and HSV-1 viruses. Dose-response curves are shown in (Figures 5 and 6). The examined (EOs) showed very weak antiviral activity against both viruses. JgEO revealed an IC50 value of 275.57 μg/mL against HAV and 299.63 μg/mL against HSV-1, while JhEO showed an IC50 value of 265.70 μg/mL against HAV and 275.33 μg/mL against HSV-1. However, the prepared nanoemuslion formulations of the two (EOs) induced significant antiviral activity against both viruses in a concentration-dependent manner ( Figures S6 and S7), and the results are depicted in (Table 3). Statistical significance was determined for their antiviral activities in comparison with the standard drug acyclovir after using various concentrations ( Figure  S2). (JhEO-NE) demonstrated a lower IC50 i.e., a higher antiviral activity against both viruses. It exhibited an IC50 value of 21.80 μg/mL against HAV and 18.49 μg/mL against HSV-1, while (JgEO-NE) showed IC50 value of 25.37 μg/mL against HAV and 12.49 μg/mL against HSV-1. Plain-NE was also examined, showing IC50 values of 45.24 μg/mL against HAV and 42.11 μg/mL against HSV-1. Several constituents in the chemical composition of both EOs were reported to have antiviral activity, such as linalool [41], nerolidol [42], and straight chain hydrocarbon compounds (acyclic alkanes) e.g., tetracosane [43], and nonacosane [44].

Conclusions
GC-MS profiling showed the presence of twenty four compounds in the EO of J. humile and seventeen compounds in the EO of J. grandiflorum. A stable unprecedented nanoemulsion formulation was prepared for each EO. Biological investigation was performed for the pure EOs and NE formulations of both plants, examining their antiviral activity against HAV and HSV-1 viruses, as well as their cytotoxicity against HepG-2, MCF-7 and THP-1 cell lines.
As cytotoxic agents, both NE formula revealed much more activity and selectivity against solid tumor cell lines; HepG-2 and MCF-7 than the liquid tumor THP-1 cells. Their potency was higher and their selectivity index was comparative to that of the standard cytotoxic drug; doxorubicin. A Flow cytometric cell cycle analysis would be recommended; to measure the cells' distribution in different stages of the cell cycle and evaluate cell proliferation in more detail. On the other hand; although their antiviral activity was enhanced (than the pure EOs), the standard antiviral drug -acyclovir-was more active than both nanoformulations against HAV and HSV-1.
Results showed clearly that NE formulations had much greater potency than pure EOs. Nanoemulsification highly increased the cytotoxic and antiviral activities of the pure EOs of the two plants which provides an excellent way to improve the biological activity of liposoluble natural agents. Moreover, NE formula could be a promising carrier for the drug administration of anticancer agents; which could enhance their efficacy, and reduce both dose and cost. These encouraging in-vitro results also recommend further in-vivo and clinical investigations of the phytopharmaceutical NE formulations as promising approach to enhance the essential oils cytotoxic and antiviral activities. (JhEO-NE) demonstrated a lower IC 50 i.e., a higher antiviral activity against both viruses. It exhibited an IC 50 value of 21.80 µg/mL against HAV and 18.49 µg/mL against HSV-1, while (JgEO-NE) showed IC 50 value of 25.37 µg/mL against HAV and 12.49 µg/mL against HSV-1. Plain-NE was also examined, showing IC 50 values of 45.24 µg/mL against HAV and 42.11 µg/mL against HSV-1. Several constituents in the chemical composition of both EOs were reported to have antiviral activity, such as linalool [41], nerolidol [42], and straight chain hydrocarbon compounds (acyclic alkanes) e.g., tetracosane [43], and nonacosane [44].

Conclusions
GC-MS profiling showed the presence of twenty four compounds in the EO of J. humile and seventeen compounds in the EO of J. grandiflorum. A stable unprecedented nanoemulsion formulation was prepared for each EO. Biological investigation was performed for the pure EOs and NE formulations of both plants, examining their antiviral activity against HAV and HSV-1 viruses, as well as their cytotoxicity against HepG-2, MCF-7 and THP-1 cell lines.
As cytotoxic agents, both NE formula revealed much more activity and selectivity against solid tumor cell lines; HepG-2 and MCF-7 than the liquid tumor THP-1 cells. Their potency was higher and their selectivity index was comparative to that of the standard cytotoxic drug; doxorubicin. A Flow cytometric cell cycle analysis would be recommended; to measure the cells' distribution in different stages of the cell cycle and evaluate cell proliferation in more detail. On the other hand; although their antiviral activity was enhanced (than the pure EOs), the standard antiviral drug -acyclovir-was more active than both nanoformulations against HAV and HSV-1.
Results showed clearly that NE formulations had much greater potency than pure EOs. Nanoemulsification highly increased the cytotoxic and antiviral activities of the pure EOs of the two plants which provides an excellent way to improve the biological activity of liposoluble natural agents. Moreover, NE formula could be a promising carrier for the drug administration of anticancer agents; which could enhance their efficacy, and reduce both dose and cost. These encouraging in-vitro results also recommend further in-vivo and clinical investigations of the phytopharmaceutical NE formulations as promising approach to enhance the essential oils cytotoxic and antiviral activities.