Characterization of N. sativa essential oil
Table (3) showed the chemical composition of N. sativa EO which is evaluated in vitro as a natural plant extract against some HCC cells. Three major volatile components represented 84.0% of the total identified compounds were reported. These components include α-thujene (12.8%), p-cymene (40.0%) and thymoquinone (TQ, 31.2%). In addition, there are some other identified components which are less abundant compared to the three major ones (Table 3). In general, the identified components are considered to be qualitatively classical and conforming with previous studies (Edris et al. 2018; Shaaban et al. 2015) with no new components to report. However, from the quantitative view point, there is a difference in the percentage of these components from those reported in the previous studies. For instance, the content of the three major constituents α-thujene, p-cymene and TQ were previously found to be 0.7%, 20.0% and 68.1%, respectively (Hamed et al. 2017). Such significant difference in the composition of the main constituents of N. sativa EO is commonly experienced by different researchers. That is due to some factors related to genetic diversity of the seeds and/or the climatic variations from one geographical location to another. Generally, the content of TQ in the essential oil participate significantly to the anticancer activity of N. sativa EO due to the role of this component in apoptosis, as previously reviewed in the introduction section.
Formulation, characterization and stability of the nanoemulsions
The EO of N. sativa was formulated in two water-based nanoemulsions (F1 & F2) using the low energy spontaneous emulsification method. This method is simple and can be scaled up easily to the industrial level without obligation to use expensive high energy equipment. Nanoemulsions (F1 & F2) differ from one another in the composition of the surfactants (Table1). Nanoemulsion (F1) was fabricated using a mixture of surfactants including Tween 80 plus Tween 20 at (2:1) weight ratio. This mixture was selected based on the fact that combination of surfactants can affect better emulsification, stability and smaller particle size compared to single surfactant (Galindo-Alvarez et al. 2011). The ratio of (2:1) was optimized after different trials by the authors aiming to find the most appropriate weight fractions that give smaller and stable nanoparticles.
On the other hand, nanoemulsion (F2) was fabricated using a single surfactant (Tween 80), which is widely used as drug formulation vehicle for anticancer drugs like the taxane anticancer agents that include paclitaxel and docetaxel (Tije et al. 2003).
Figures (1-3) shows the different approaches which were used to characterize the formulated nanoemulsions regarding the appearance, particles morphology and particle size distribution.
Figure (1) indicates that the appearance of both formulae is transparent or translucent but not cloudy, giving a preliminary impression of small nanoparticles that does not scatter light. The nanoemulsion formulated using a mixture of surfactants (F1) looked transparent (Fig. 1, right) while that formulated using single surfactant (F2) appeared translucent (Fig. 1, left). That indicate more light scattering and relatively larger particles compared to (F1). UV-vis absorption at 600nm confirmed the visual appearance where the absorption value of (F1) was 0.023 which is lower than that of (F2) which recorded 0.194. These spectroscopic data indicate two nanoemulsions in which the EO is dispersed homogenous as nanoparticles in the approximate range of 10-100 nm, as predicted from the low visible light absorption and the minimally scatter light.
Figure (2) shows the particles’ morphology of (F1) and (F2) using the negative staining transmission electron microscope (TEM). The figure indicates that both nanoemulsions have a spherical-shaped particles which have a diameter in the targeted nano-size range (~ 100 nm, Fig. 2). However, TEM analysis just gave an approximation on the particle size of the nanoemulsion. That is because it is hard to find a field that cover all particle size populations in the nanoemulsion due to the low oil droplets concentration (1.0%). In addition, artifact formation due to negative staining of the nanoemulsions and the high vacuum of operation may also alter the real size of the particles.
Therefore, for more comprehensive data about the particle size diameter of both nanoemulsions, dynamic light scattering analysis was conducted (Fig. 3). The figure shows that nanoemulsion (F1) has two particles populations, the first has droplets of 9.4 nm (intensity 84.6%) and the second has droplets of 72.3 nm (intensity 15.4%). Similarly, nanoemulsion (F2) also showed two relatively larger particles populations, the first is 11.6 nm (65.5%) and the second is 119.7nm (34.5%). These data correlated well with the appearance of both nanoemulsions illustrated in Fig. (1), especially (F2) which appeared translucent with more light scattering due to the second population of particles that have 119.7nm.
From the above-mentioned findings (Fig. 1-3) we can confirm the formulation of N. sativa EO in two nanoemulsion (F1 &F2) which differ slightly from one another in their average particle size due to surfactants’ composition.
These nanoemulsions were also found to be physically stable against gravitational separation after 6-month storage period at 25ºC. Visual observation of the nanoemulsions at regular intervals during that storage period did not detect any opacity, oil separation or creaming, which indicate high physical stability of both formulae. What was remarkable is that nanoemulsion (F2) which looked translucent right after formulation (Fig. 1, left) became more transparent after storage. The measured UV-vis absorbance at λ600 nm confirmed that visual observation in which the value changed from 0.194 right after formulation (zero time) to become 0.02 after 6-months storage. This indicate that the particles of the formulated nanoemulsions are dynamic and tend to change their size continuously to the smaller during storage till they reach a final equilibrium state. This is considered to be an advantage of the formulated nanoemulsions which proved to be both thermodynamically and kinetically stable. That is due to the meticulous choice of the ingredients and their ratios in both nanoemulsions (Table 1).
After formulation, characterization and stability evaluation of the nanoemulsions, all formulation including the pure distilled unformulated EO (F0) were subjected to the different anticancer evaluations as will be tackled in the next passages.
Assessment of cytotoxicity of N. sativa essential oil and its nanoemulsions
The cytotoxic effect of the distilled N. Sativa EO (F0) as well as its two nanoemulsions (F1 & F2) was evaluated against some hepatocellular carcinoma (HCC) cell lines, namely, HepG2 and Huh-7 cells. These cell lines are commonly used as models for in vitro evaluation of cytotoxicity of chemotherapeutic drugs which is used in advanced liver cancer (Dubbelboer et al. 2019). The cytotoxic effect of the different N. sativa EO formulations was evaluated in two terms, cell proliferation percentage and IC50 values.
Cytotoxicity in terms of cell proliferation percentage
In this assay, the percentage of HCC cells which remained alive after treatment with gradient doses (20-100 µg/ml) of each N. sativa EO formulation was evaluated and illustrated in Figure (4). The results revealed that there is a dose-dependent decrease in HCC cell proliferation percentage upon treatment with the different formulations.
In details, the unformulated distilled EO of N. sativa (F0) inhibited HCC cell proliferation to 47.2% and 42.1% for HepG2 and Huh-7 cells, respectively after treatment with 100 µg EO/ml (Fig. 4). This means that the inactivation of both HCC cells reached 52.8% and 57.9% respectively after treatment with the highest tested dose of (F0).
The antiproliferative activity of (F0) is likely to be attributed to the presence of thymoquinone (TQ, 31.0%) among the chemical constituents of N. sativa EO (Table 3). This compound per se is an active anticancer agent which manifested its own antiproliferative and protective activity against different cancer cell models (Ha et al. 2020; Bhattacharjee et al. 2020, including HCC (Bimonte et al. 2019). Generally, TQ has been found to modulate a battery of signaling pathways that prevents tumor progression (Zhang et al. 2020; Afrose et al. 2020; Fatfat et al. 2019; Schneider-Stock et al. 2014). In more specific discussion regarding HCC, an earlier study by Ashour et al. (2014) indicated that TQ can cause G2M cell cycle arrest and inhibit expression of NF-jB and suppressed IL-8 and its receptors, beside upregulation of TRAIL death receptors. More recently, Aslan et al. (2021) added that TQ showed antiproliferative activity against HepG2 cells by inducing ceramide accumulation and ER stress in conjunction with decreasing S1P, C1P and NF-κB mediated cell survival. All these effects can promote HepG2 cancer cell death by triggering apoptosis. In addition, TQ can sensitize HepG2 cells to TRAIL-induced apoptosis via oxidative DNA damage and by inducing the death receptor pathway (Zhang et al. 2021).
Based on the above mentioned, we should link the antiproliferative activity of the EO (F0) to its content of TQ which was found to be only 31.2% of the constituents of N. sativa EO (Table 3). This percentage is considered to be low compared with other values (56.2% and 68.1%) which was previously reported by our group (Hamed et al. 2017) using different seeds of N. sativa. That illustrates the importance of screening for the seeds that bear high content of TQ. In addition, the method of extracting the EO from the seeds also has significant effect on the content of TQ in the oil (Edris et al. 2018; Edris 2010).
So, we preliminary presume that the previously illustrated example of EO with high TQ content (68.1%, Hamed et al. 2017) would have shown about double the antiproliferative activity compared to the EO evaluated in the current study which contained only 31.0 % TQ. However, we should be careful regarding this statement because the relationship between TQ content in EO and antiproliferative activity may not be linear as we presume. Therefore, it will be interesting in the future to examine this statement using different seeds that bear variable percentages of TQ among their EO. In this regard, seeds from Nigella hispanicais recommended as a negative control for comparison, because its EO has 0.0% TQ (Kokoska et al. 2012), with no other antiproliferative active ingredients in that EO.
Due to the diversity of volatile components in the EO of N. sativa (Table 3), it could be possible that other components participate together with TQ in the overall antiproliferative activity of the EO. For instance, the EO contain considerable amounts of terpenes like p-cymene (40.0%) and trans-a-thujene (12.8%) in combination with other minor components (Table 3). To our knowledge, we did not find reports which proclaim cytotoxic effect of each individual components of these terpenes. However, Silva et al. (2020) emphasis on the important role of these terpenic compounds (all together, without TQ) on the overall cytotoxic activity of EO of N. sativa against MCF-7 cells.
One of the interesting findings that we can extract from Fig. 4 is that Huh-7 cells are more sensitive to the EO of N. sativa (F0) than HepG2. The same observation was previously reported in another study (Ashour et al. 2014) where pure TQ was used as an antiproliferative agent. The reason of that phenomena is not known so far, but we assume that it is related to the receptors on the surface of the cell wall of both HepG2 and Huh-7 cells. It worth indicating that one of the main differences between these cells is on p53 expression. HepG- cells carry wild-type p53, whereas Huh-7 cells have null and point mutations at p53 codon 220, respectively.
Effect of nanoemulsions on the cell antiproliferation percentage
The EO of N. sativa was formulated in two water-based nanoemulsion formulae (F1 & F2) which differ from one another in the composition of the surfactants (Table 1). The structure of the nanoemulsion in the current study is typically composed of a continuous pool of water which bears nanoparticle composed of spherical layers of surfactant(s) encapsulating the EO of N. sativa in its inner lipophilic core. This nanoemulsion is described as one of the phytomedicine-based delivery systems which is used for therapeutic strategies in liver cancer (Kumar et al. 2020). It is also suitable for oral or parenteral administration routs due to its water-based nature.
Figure 4 showed that both nanoemulsion systems (F1 & F2) inhibited the proliferative activity of HCC cells more than the non-formulated EO (F0). For instance, nanoemulsion (F1), showed inhibition in proliferative activity to become only 28.7% and 15.5 % against HepG2 and Huh-7 cells, respectively. Interestingly, nanoemulsion (F2) showed the same trend with even more proliferation inhibition activity which was further dropped to 21.9% and 9.2% against HepG2 and Huh-7 cells, respectively. From these data it is clear that the activity of N. sativa EO (F0) is greatly enhanced upon formulation in nanoemulsion. That, can be attributed to the better diffusion and enhanced permeability and retention of nanoparticles into the cancer cells (Yao et al. 2020; Zhu et al. 2015). That in turn can lead to increase tumor cell response to the model drug (Karasulu et al. 2009).
Fig. 4 also indicates that nanoemulsion (F2) has better inhibition of HCC cells proliferation than (F1). That confirms clearly the effect of nanocarrier material, i.e. the surfactant, on the activity of cancer drug models formulated in nanoemulsion. So far, we do not have enough clear evidence to justify why nanocarrier made of 100% Tween 80, as nanoemulsion F2, gave better inhibition in proliferation compared to (F1) which is made of a mixture of Tween 80 (66.6%) and Tween 20 (33.3%). However, that can be due to the inherent cytotoxic effect of Tween 80 on some cancer cells (Kubis et al. 1979; Kay 1965. It could be also be due to the difference in the carbon chain length of the fatty acid moiety of both surfactant nanocarriers which host the EO in the inner core. Tween 80 have 18 carbon atoms while Tween 20 have only 12 carbon atoms. This difference in the volume faction of the inner fatty acid part of the nanocarrier may differentially effect on the interaction and hence the release of the EO to HCC cells. More work is still needed to justify this issue on an evidence-based background.
Cytotoxicity in terms of IC50
IC50 is another manifestation of cytotoxicity of certain drug model against cancer cells. It is defined as the concentration of the drug necessary to kill half of the cancerous cells, therefore, the smaller the value of IC50 the more cytotoxic is the drug. Results in Table (4) shows that the different formulations of N. sativa EO (F0, F1 & F2) have lower IC50values compared to the reference drug Doxorubicin (100 µg/ml). The lowest IC50 compared to that of Doxorubicin was found for nanoemulsion (F2) against Huh-7 cells.
The ratio of IC50 of the drug relative to that of F0, F1 and F2 is denoted as fold change (a), which shows the magnitude of enhanced activity of the EO formulations relative to Doxorubicin (Table 4). This value is always > 1 indicating that all formulations are more cytotoxic than the reference drug, especially nanoemulsion (F2) against Huh-7 cells where the fold change is 2.81.
From Table (4) it is also evident that the nanoemulsions (F1 & F2) have lower IC50 value, i.e. higher cytotoxicity, relative to (F0). That confirms the potentials of nanoparticles for enhancing the cytotoxicity and inhibitory activity of N. sativa EO. The ratio of IC50 of (F0) relative to that (F1) and (F2) is denoted as fold change (b), which was also always > 1, indicating higher IC50 values for (F0). The fold change (b) reached its highest value for nanoemulsion (F2) against Huh-7 (2.54), indicating that (F0) is much lower in cytotoxicity compared to nanoemulsion (F2).
Comparing our results with previous investigations we found that the IC50 of pure TQ against HepG2 was 6.16 µg/mL (El-Najjar et al. 2010; Fröhlich et al. 2017). In addition, a plant extract isolated from Indigofera zollingeriana also showed low IC50 values against HepG2 (6.8 ug/ml) and Huh-7 cells (8.7 ug/ml) (Vo et al. 2020). However, we can justify their lower IC50 values compared to ours based on the prolonged contact time between the HCC cells and the plant extract, which was double (48h) the contact time that we applied (24h) in the current study. One should also bear in mind that the wide spread cultivation of N. sativa and its abundance all over the world make it more available for practical pharmaceutical applications on large scale.
After studying the cytotoxic effect of N. Sativa EO and its two nanoemulsions against HCC cells, this section investigates the mechanistic approach of killing cancerous cells. The tools used for that purpose include flow cytometric analysis and genetic expressions of pro-apoptotic (Bax) and the anti-apoptotic (Bcl-2) gene markers.
Flow cytometric analysis is one of the most popular applications of studying apoptosis. It offers the ability to study large numbers of cells individually rather than a mixed population. The technique can also be used to detect and quantify the level of apoptosis in a population of cells at static points or in a time course. Figure (5) showed the apoptotic diagram of the two HCC cells and normal WI-38 cells after treatment with the different formulations of N. sativa EO. From the figure it is evident that the count percent of HCC cells in the quadrant of negative annexin V/negative PI were decreased gradually in a significant manner after treatment with F0, F1, and F2, respectively. The same treatment shifted the cells into positive annexin V/positive PI quadrants leading to apoptotic cells. From Fig. 5 it is clear that nanoemulsion (F2) caused the highest significant induction rate of apoptosis compared to (F0) and (F1). In addition, Huh-7 cells showed less count percent than HepG2 cell line, indicating more sensitivity toward the treatment. These two conclusions came in accordance with our previous finding in the results concerning cytotoxicity evaluations. Figure (6) represents the graphs of the flow cytometric apoptotic screening diagram which was presented previously in Fig. (5). Cell viability percentage (Fig 6a) and apoptotic cell percentage (Fig. 6b) elicit in detailed the same results that we previously discussed in Fig. (5).
Gene expression of Bax and Bcl-2 genes
Genetic expressions of pro-apoptotic (Bax) and the anti-apoptotic (Bcl-2) gene markers where evaluated in the current study (Fig. 7) to verify (support) the results obtained from the previously discussed apoptotic diagrams (Fig. 5). Data in Fig. (7a) indicate that F0, F1 & F2 showed upregulation of Bax gene expression compared to the untreated normal cells (control). Nanoemulsion (F2) showed the highest upregulating activity, and Huh-7 cells was the most responding to the treatment, as previously shown in the cytotoxic and apoptotic evaluations. In addition, nanoemulsion (F2) caused the highest significant downregulation of Bcl-2 genetic expression especially in Huh-7 compared to the normal cells (Fig. 7b).
The ratio of genetic expressions of pro-apoptotic (Bax) and anti-apoptotic (Bcl-2) is very important to confirm the apoptotic effect of any new treatment. Therefore, this ratio was calculated in the current study and shown in Table 5. The data indicates that nanoemulsion (F2) has the highest Bax/Bcl-2 ratios especially for Huh-7 (69) followed by HepG2 (18) compared with normal cells (1.56). Nanoemulsion (F1) also showed high Bax/Bcl-2 ratio, but next to (F2) in activity, in which the ratio was 9.25 and 7.0 for HepG2 and Huh-7, respectively. On the other hand, the unformulated pure N. sativa EO (F0) recorded the least Bax/Bcl-2 ratios for HepG2 (2.25) and Huh-7 (2.71) compared with normal cells (1.09). This result indicates the significance of specially tailored N. sativa EO nanoemulsion (F2) for modulating the pro-apoptotic and the anti-apoptotic genes for the favor of apoptosis.
Safety of N. sativa and its nanoemulsions
One of the common draw backs of anticancer chemotherapeutic drugs is its lack of selectivity (non-specificity), in their cytotoxic effect. This means that they could be cytotoxic to malignant as well as normal body cells resulting in little improvement on patient case. Therefore, the authors in the current study were keen to include a model of normal cells like WI-38 beside HCC cells in the evaluation of the cytotoxic and apoptotic activity of the different formulae of N. sativa EO. Data illustrated in Figs. (4-7) and Table (5) showed that the EO and its nanoemulsions have a minimal harmful effect on the normal WI-38 cells. These figures reflect the specificity of N. sativa EO formulae against HCC cells, while sparing normal WI-38 cells without harmful effects. That could be due to the reported selectivity of TQ, (31.2% of EO composition) for induction of HCC to apoptosis (Jehan et al. 2020).