Nanoemulsion and Nanogel Containing Cuminum cyminum L Essential Oil: Antioxidant, Anticancer, Antibacterial, and Antilarval Properties

Cuminum cyminum L. is a widespread medicinal plant with a broad spectrum of biological activity. In the present study, the chemical structure of its essential oil was examined utilizing GC-MS analysis (gas chromatography-mass spectrometry). Then, a nanoemulsion dosage form was prepared with a droplet size and droplet size distribution (SPAN) of 121 ± 3 nm and 0.96. After that, the dosage form of the nanogel was prepared; the nanoemulsion was gelified by the addition of 3.0% carboxymethyl cellulose. In addition, the successful loading of the essential oil into the nanoemulsion and nanogel was approved by ATR-FTIR (attenuated total reflection Fourier transform infrared) analysis. The IC50 values (half maximum inhibitory concentration) of the nanoemulsion and nanogel against A-375 human melanoma cells were 369.6 (497–335) and 127.2 (77–210) μg/mL. In addition, they indicated some degrees of an antioxidant activity. Interestingly, after treatment of Pseudomonas aeruginosa with 5000 µg/mL nanogel, bacterial growth was completely (∼100%) inhibited. In addition, the growth of Staphylococcus aureus after treatment with the 5000 μg/ml nanoemulsion was decreased by 80%. In addition, nanoemulsion and nanogel LC50 values for Anopheles stephensi larvae were attained as 43.91 (31–62) and 123.9 (111–137) µg/mL. Given the natural ingredients and promising efficacy, these nanodrugs can be regarded for further research against other pathogens or mosquito larvae.


Introduction
Skin cancer, with more than 1 million new cases, was the ffth most common cancer worldwide in 2020 [1]. Skin cancer is categorized into melanoma and nonmelanoma; melanoma is defned as the uncontrolled proliferation of melanocytes [2]. Melanoma, with ∼55,500 deaths annually, is the most dreadful kind of skin cancer [3]. Te direct and indirect costs of treating this disease are estimated at 3.3-8.1 billion and 3.5 billion US$ in a year [4,5].
Staphylococcus aureus and Pseudomonas aeruginosa are two important Gram-positive and Gram-negative pathogens that can bring about contagious soft tissue and skin infections with diferent symptoms, including color changes, pain, and swelling [6]. Besides, they could enter the body and cause severe and life-threatening infections such as septicemia [7]. Moreover, malaria, which claims about half a million lives every year, is still one of the most feared infectious diseases; it is communicated to humans via bites of infected female Anopheles mosquitoes [8]. Moreover, Anopheles stephensi Liston is among the most signifcant malaria vectors in South Asia and the Middle East [9].
Despite the advantages of EOs as drugs or insecticides, such as biocompatibility and biodegradability, their efectiveness should be enhanced. Te preparation of essential oil-based nanopreparations, including nanoemulsions, nanoparticles, lipid nanovesicles, and nanogels, has recently been presented as a promising method to improve the stability and efcacy of essential oils [21,22]. In this study, nanoemulsion and nanogel containing C. cyminum EO were frst prepared. Teir biological activities were then compared, including anticancer, antioxidant, antibacterial, and larvicidal efects.

GC-MS Analysis.
EO was analyzed using a gas chromatography unit (Agilent 6890, HP-5MS column, USA) linked to a mass spectrometer (Agilent 5973, USA), as explained in our previous study [23]. In brief, the column temperature was set to 40°C (fxed for 1 minute), then ramped up to 250°C at 3°C/minute and kept at this temperature for 60 minutes. Te injection port and detector temperatures were fxed at 250 and 230°C, respectively. Helium (99.999%) was utilized as a carrier gas; other operating conditions included split 25 mL/min, septum purge 6 mL/min, and column fow rate 1 mL/min. Mass spectra were obtained in the full scan mode in the 50-550 m/z range with 70 eV ionization energy. Retention indices were measured using a mixture of n-alkanes (C6-C27) employing the van den Dool and Kratz formula. EO components were determined by the combination of temperatureprogrammed retention indices and mass spectrometry with ADAMS and NIST 17.

Preparation of Nanoemulsions Containing C. cyminum EO.
Spontaneous emulsifcation was utilized to prepare nanoemulsions containing C. cyminum EO [24]. In brief, a defned amount of C. cyminum EO (100 µL) and various levels of Tween 20 and Tween 80 (100-300 µL) were frst mixed for 5 min to form a homogenous solution (2000 rpm, at room temperature). Ten, distilled water was added dropwise to the desired volume (5000 µL) and stirred for 40 min to stabilize (2000 rpm, room temperature).
DLS (dynamic light scattering) type apparatus was used to analyze the prepared nanoemulsions' size. Moreover, the droplet size distribution (SPAN) was measured with the equation D90-D10/D50, where D is the diameter and 90, 10, and 50 are the cumulative percentages of droplets with lower diameters than the specifed values. A nanoemulsion with optimal size characteristics, including a droplet size of <200 nm and SPAN <1 [25], was selected for further investigation. Note, the optimal nanoemulsion contained 2% v/v C. cyminum and 5% v/v Tween 80. A similar method was applied to prepare nanoemulsion (-oil), only C. cyminum EO was not used.
For short-term stability analysis of the nanoemulsion, 5 mL samples were prepared and divided into fve 1 mL microtubes. Te three microtubes were centrifuged at −4, +4, and +25°C (14, 000g, 30 min). Two other microtubes were used for freeze-thaw and heat-cool stability assays. In a freeze-thaw cycle, the microtube was placed at −20°C (freezer) and at room temperature for six consecutive 48hour intervals. For the heating-cooling analysis, the microtube was stored at +45°C (steam bath) and at room temperature for six consecutive intervals of 48 hours. After each test, the samples were examined visually for sedimentation, creaming, or biphasic.
For the long-term analysis, samples were kept at 4 and 26°C for six months and then visually inspected for sedimentation, creaming, or biphasic.

Preparation of Nanogel Containing C. cyminum EO.
Te selected nanoemulsion was gelifed by adding 3% w/v CMC; it was stirred for 4 h in a minor condition (180 rpm). Te viscosity of the nanogels at shear rates of 0.1-100 1/s was examined using a rheometer machine (MCR-302, Anton Paar, Austria). Te prepared nanogels were stored for six months at two temperatures of 4 and 26°C for any sedimentation or biphasic. A similar method was used to prepare nanogel (-oil), only C. cyminum EO was not used.

Investigation of Morphology of Nanoemulsion and
Nanogel. TEM analysis (transmission electron microscopy) was conducted to inspect the morphology of the nanoemulsion and nanogel. One drop of each was poured on a 200-meshcarbon-coated copper grid and utilized in a TEM device (Philips EM208S 100 KV, Max Res 0.2 nm, Netherlands).

Investigation of Loading of the EO in the Nanoemulsion
and Nanogel. ATR-FTIR analysis was conducted to examine the successful loading of the EO in the nanoemulsion and nanogel. Te spectra of the EO, nanoemulsion (-oil), nanoemulsion, nanogel (-oil), and nanogels were recorded at 400-3900 cm −1 by means of an ATR-FTIR spectrometer (Tensor II model, Bruker Co, Germany).

Evaluation of Antioxidant Efects.
A 0.3 mM DPPH solution (394.32 g/mol) was frst prepared using absolute ethanol; 150 µL/well was added to 96-well plates [26]. Ten, serial dilutions of nanoemulsion and nanogel were prepared by means of absolute ethanol. By adding 50 µL/well of each dilution, the antioxidant impacts of nanoemulsion and nanogel at 62.5, 125, 250, 500, and 1000 µg/mL were evaluated. Ten, the treated plate was incubated for 30 min in a dark connection at room temperature to complete the reaction. Finally, the absorbance of wells was read at 517 nm with the use of the plate reader (Synergy HTX multimode reader, BioTek, USA). Te antioxidant efect at each concentration was measured by means of the equation A control−A sample/A control × 100. In the control group, 50 µL/well of absolute ethanol was used. In the negative control groups, 50 µL/well of nanoemulsion (-oil) and nanogel (-oil) was added instead of 50 µL/well of nanoemulsion and nanogel (1000 µg/mL); they contained equal amounts of ingredients but without C. cyminum EO.

Evaluation of Anticancer Efects.
MTT assay was made to examine the cytotoxic impact of the nanoemulsion and nanogel on A-375 cells, as explained in our previous report [27]. Te cells were cultured in RPMI, which contained 10% fetal bovine serum and 1% antibiotics, and seeded (1 × 10 4 cells/well) in a 96-well plate. After the cells reached 80% confuence, the liquid content was substituted with 100 µL/well of fresh media culture. Serial dilutions of nanoemulsion and nanogel were then prepared using RPMI. By adding 100 µL/well of each dilution, the cytotoxic efects of nanoemulsion and nanogel at 62.5, 125, 250, 500, and 1000 µg/mL were assessed. Treated plates were then incubated for 24 h at 37°C with CO 2 . After that, the liquid content was replaced with 100 µL/well MTT solution (0.5 mg/mL; dissolved in RPMI), and plates were incubated in the mentioned condition for four h. Ten, 100 µL/well of dimethyl sulfoxide was added to dissolve the created formazan crystal. Finally, the absorbance of wells was read at 570 nm using the plate reader, and cell viability at each concentration was measured by means of the equation A sample/A control × 100. In the control group, 100 µL/well RPMI was used. In the negative control groups, 100 µL/well of nanoemulsion (-oil) and nanogel (-oil) was added instead of 100 µL/well of nanoemulsion and nanogel (1000 µg/mL); they contained equal amounts of ingredients but without C. cyminum EO.
2.9. Evaluation of the Antibacterial Activity of the Nanoemulsion and Nanogel. Te ATCC100 method with a minor modifcation was utilized to evaluate the antibacterial activity [28]. First, P. aeruginosa and S. aureus were cultured in the Mueller-Hinton broth. Ten, 2 mL of fresh bacterial suspensions (2 × 10 5 CFU/mL) were separately poured into 5 cm plates. Ten, serial dilutions of nanoemulsion and nanogel were prepared using the Mueller-Hinton broth. By adding 2 mL/plate of each dilution, the antibacterial impacts of nanoemulsion and nanogel at 1250, 2500, and 5000 µg/mL were investigated. After that, treated plates were incubated at 37°C for 24 h and 10 µL of each plate's supernatants were cultured on plates containing Mueller-Hinton agar (37°C, 24 h). Te colonies were counted, and bacterial growth (%) was measured using the CFU sample/CFU control × 100. In the control group, 2 mL/plate Mueller-Hinton broth was utilized. In negative control groups, 2 mL/plate of nanoemulsion (-oil) and nanogel (-oil) was added instead of 2 mL/plate nanoemulsion and nanogel (1000 µg/mL); they contained equal amounts of ingredients but without C. cyminum EO.

Evaluation of the Larvicidal Activity of the Nanoemulsion and Nanogel.
Larvicidal bioassays were performed according to the WHO guidelines with a slight modifcation [29]. Beakers that contained 198.5 mL of no chlorine water, including 25 A. stephensi larvae, were frst prepared. Serial dilutions of nanoemulsion and nanogel were then prepared using absolute ethanol. By adding 1.5 mL/baker of each dilution, the larvicidal impacts of nanoemulsion and nanogel at 12.5, 25, 50, 100, and 150 µg/mL were investigated. Larval mortality after 24 h of exposure was counted. In the control group, 1.5 mL/baker absolute ethanol was used. In the negative control groups, 1.5 mL/baker of nanoemulsion (-oil) and nanogel (-oil) was added instead of 1.5 mL/baker of nanoemulsion and nanogel (150 µg/mL); they contained equal amounts of ingredients but without C. cyminum EO.

Statistical Analyses.
Tree replicates were performed for all tests, and the fnal values were presented as the mean ± standard deviation. Means and standard deviations were calculated using Excel software (Microsoft Ofce, version 2010). Final values for all samples were compared by SPSS software using one-way ANOVA with a 95% confdence interval. Te CalcuSyn software (free version, BIO-SOFT, Cambridge, UK) was used to calculate the IC50 and LC50 values of the nanoemulsion and nanogels.
Journal of Tropical Medicine 3

Prepared Nanoemulsions.
Te characteristics of the size and ingredients of 10 prepared nanoemulsions are given in Table 2. Sample no. 9 with a droplet size of 121 ± 3 nm and a droplet size distribution of 0.96 showed the best size characteristics and was chosen as the optimal nanoemulsion. Its DLS profle is shown in Figure 1(a); one sharp peak also confrmed the narrow droplet size distribution. Besides, the TEM image of the optimal nanoemulsion with circular droplets is shown in Figure 1(b). In the DLS analysis, the hydrodynamic radius of the droplets is measured, so the droplet size is always larger than that of the TEM analysis. Moreover, in the DLS analysis, close droplets are identifed as a droplet in concentrated systems (such as the prepared nanoemulsion). Furthermore, no sedimentation, creaming, or phase separation was seen in the selected nanoemulsion (no. 9) after short-term (centrifugation at three temperatures, heating-cooling cycles, and free-taw cycles) and long-term (storage at two temperatures for six months) stability tests. Its stability was thus confrmed.

Prepared Nanogel.
Te optimal nanoemulsion was gelifed by adding 3.0% w/v CMC. As shown in Figure 1(a), the viscosity of the nanogel almost fully fts with Carreau-Yasuda as the well-known regression of non-Newtonian fuids, i.e., the viscosity decreased with a growing shear rate [30]. Besides, the TEM image of the nanogel is shown in Figure 2(b); the nanogel droplets are not detectable due to the network structure. Furthermore, after six months of storage at 4 and 26°C, no sedimentation and biphasic were observed; nanogel's stability was thus confrmed.

Confrming Successful
Loading of the EO. ATR-FTIR spectra of C. cyminum EO, nanoemulsion (-oil), nanoemulsion, nanogel (-oil), and nanogel are shown in Figure 3. Te ATR-FTIR spectrum of C. cyminum EO is presented in Figure 3(a); the broad peak at 3369 cm −1 is allocated to OH. Te bands at 2960, 2925, and 2870 cm −1 showed the CH stretching vibration of Sp 3 in alkanes. Besides, the bands at 2819 and 2721 cm −1 specify C-H aldehyde. Te strong peak at 1702 and 1673 cm −1 corresponds to the stretching vibration of C�O in aldehyde and ketones in the EO. Tese strong peaks represented a high amount of aldehydes in the C. cyminum EO. Te bands at 1575 and 1461 cm −1 can be related to the C�C skeleton vibration of an aromatic matter. Te band at 1074 cm −1 was attributed to C-O stretching vibration. Te band at 986 cm −1 corresponds to C-H bending absorption, and the strong peak at 815 cm −1 is allocated to the benzene ring's C-H vibration absorption. Te band at 687 cm −1 is attributed to the vibration absorption of alkenes.
Te ATR-FTIR spectrum of nanoemulsion (-oil) is displayed in Figure 3(b); the broadband between 3200-3675 cm −1 can be related to hydrogen bonding. Te band at 2923 cm −1 is associated with -CH stretching vibration. Besides, the small peak at 1733 cm −1 can be ascribed to the carbonyl group in Tween. Te band at 1251 cm −1 can be associated with C-OH. Te main strong band at about 1085 cm −1 could be ascribed to C-O stretching vibration.
Te ATR-FTIR spectrum of the nanoemulsion is shown in Figure 3(c); a broad and characteristic band at around 3200-3600 cm −1 can be ascribed to OH because of hydrogen bonding between Tween 80, water, and EO. Te band at 1734 cm −1 is related to carbonyl stretching vibration because of EO and Tween 80. Te main and strong band at 1089 cm −1 corresponds to C-O.
Te ATR-FTIR nanogel (-oil) spectrum is displayed in Figure 3(d). Te characteristic peak at 2923 cm −1 is ascribed to the C-H stretching vibration of alkane. Te band at 1734 cm −1 can be related to the carbonyl group in CMC. Furthermore, the band at 1578 and 1413 cm −1 can be attributed to symmetric and asymmetric carboxylates in CMC. Finally, the main and strong peak at 1080 cm −1 can be related to C-O stretching vibration.
Furthermore, some reports on nanostructures containing C. cyminum EO have been published. For example, the antibacterial impact of its ultrasonicated nanoemulsion was investigated against S. aureus in an agar difusion well assay with an inhabitation zone of 20.3 ± 0.11 mm [35]. However, as EOs contain volatile compounds, spontaneous emulsifcation is preferred in such nanoformulations; droplets with the desired size and droplet size distribution are achieved by optimizing amounts of EO and surfactant (i.e., without any external energy such as ultrasonication) [36]. So, in the current study, the nanoemulsion of C. cyminum EO was prepared using the spontaneous method. Te small droplet size permits the system to stabilize and bypass problems such as creaming or sedimentation. Besides, the low surface and interfacial tensions encourage suitable spreading and penetration of the active compounds [37]. Tese characteristics enhance nanoemulsions' bioavailability and efcacy [38,39].
Despite the advantages of nanoemulsions, their topical application is challenging because of their low viscosity. In recent years, nanoemulsions-based nanogel has received more attention; they have all the benefts of nanoemulsions, and their topical application is also facilitated by enhanced viscosity [40,41]. For instance, chitosan-cafeic acid nanogel containing C. cyminum EO showed more efcacy than nonformulated EO; MIC against Aspergillus favus was reported at 650 and 350 ppm [42]. In another research, the enzymatic degradation in nanogel containing 5Vtriphosphates was 90% less than the nonformulated state of   the enzyme [43]. In the current study, the anticancer efcacy of the nanogel (IC50 127.2 µg/mL) three-folds was more potent than the nanoemulsion (369.6 µg/mL). Free radicals with unpaired electrons damage cellular membranes, lipids, proteins, and DNA directly. It also causes pivotal mechanisms causing skin aging [44,45]. Antioxidants are substances that counteract the impacts of endogenous and exogenous oxidative stresses through scavenging free radicals. Endogenous free radicals are formed naturally through normal human metabolism; however, exogenous species result from sunlight, pollution, harsh chemicals, illness, stress, lack of sleep, and cigarette smoke [46,47]. For topical administration of antioxidants, their stability is important. Tey need to be absorbed into the skin, reach their target tissue in the active form, and stay there long enough to exercise the desired impacts [48,49]. In the current study, both nanoemulsion and nanogel showed some degrees of antioxidant efect; however, further investigation is needed.
Te current study used CMC (anionic cellulose derivative) as the gelling agent, a water-soluble polymer with linear polysaccharides of anhydrous glucose [50]. Due to proper characteristics, such as mechanical resistance, viscous properties, and low cost, it has received more attention in drug delivery systems [51,52]. Tis study showed that nanogel are more efective on P. aeruginosa than on S. aureus. Te cell surface of S. aureus has a negative net charge [53]. As CMC is negatively charged, it seems proper interaction between nanogel and the bacterial membrane did not occur; however, it should be checked in future studies. On the other hand, Gram-negative bacteria such as P. aeruginosa have an outer membrane that envelope bacteria, improve their stability, and protect them from antibiotics [54]. However, the outer membrane is a selective permeation barrier for the hydrophilic material (i.e., nanogel) that easily enters the bacteria [55].
Excessive use of insecticides to control vector-borne diseases such as malaria has brought about environmental pollution (soil and water) and resistance against synthetic insecticides [56,57]. Terefore, many attempts have been made to develop plant-derived insecticides, especially EO. For instance, the larvicidal efect of C. cyminum 0.5% crude extracts against A. stephensi and Culex quinquefasciatus was reported as 16% and 15% [58]. Te present study examined the larvicidal impacts of nanoemulsion and nanogel containing C. cyminum EO. Interestingly, no report was found on the larvicidal impacts of a nanogel containing EO.

Data Availability
Te data generated or analyzed during this study are available from the corresponding author upon reasonable request.

Ethical Approval
Tis study has been approved by the Ethics Committee of Fasa University of Medical Sciences, Fasa, Iran (IR.FUMS.REC.1400.164).

Consent
Not applicable.

Conflicts of Interest
Te authors declare that they have no conficts of interest.

Authors' Contributions
RR contributed to the nanoemulsion and nanogel preparation and performed MTT and DPPH assays. EZ interpreted ATR-FTIR. AA performed antibacterial tests. MN contributed to the preparation of the nanoemulsion and nanogel. SF performed the larvicidal test. NN reviewed the literature. MO designed the study, analyzed data, and drafted the manuscript. All authors contributed to drafting the manuscript. All authors have read and approved the fnal version of the manuscript.