Essential Oil and the Preparation of Formulations Containing EO

Litsea cubeba (May Chang) fruit essential oil was purchased from Nanga (Złotów, Poland), while other components of formulations were from Zrób Sobie Krem Kosmetyki Naturalne Katarzyna Damętka (Prochowice, Poland). The cream formulation was composed of: Butyrospermum parkii butter (60 parts), Cocos nucifera oil (27.5 parts), sunflower oil (12 parts), Lisea cubeba essential oil (0.5 parts). Liquid formulation was composed of sunflower oil (99.5 parts) and Lisea cubeba essential oil (0.5 parts).

Determination of the Chemical Composition of EO and Formulations

The phytochemical profile of the used EO and profiles of the obtained formulations were checked by means of gas chromatography-mass spectrometry (GC–MS). The analyses were performed on Shimadzu GC–2010 Plus coupled to a QP2010 Ultra mass spectrometer with Phenomenex capillary column ZB–5 MS (30 m, 0.25mm inside diameter, and 0.25 μm coating thickness). The method followed the conditions described previously by Sieniawska et al. [15]. The initial column temperature was set at 50 °C, held for 3 min, and then heated to 250 °C at a rate of 8 °C/min; this temperature was held for 2 min. The injector temperature was 250 °C. Helium was used as the carrier gas with a flow rate of 1 mL/min. Split ratio was set at 1:20. Ionization was performed by electron impact at 70 eV. The interface and ion source temperatures were 250 and 220 °C, respectively. Mass spectral data were acquired in the scan mode in the m/z range 40–500 with the scan rate of 0.20 s per scan. Volatiles were identified based on their MS spectra and relative retention indices determined with reference to a homologous series of C8–C24 n-alkanes using a computer-supported spectral library (NIST, National Institute of Standards and Technology). Samples were dissolved in hexane prior to the analysis.

Determination of the Safety Profile of EO and Formulations

The cell lines were acquired from the American Type Cell Culture Collection. VERO (ATCC, CCL-81, Cercopithecus aethiops, monkey kidney) cells were cultured using Dulbecco Modified Eagle Medium (DMEM, Corning, Tewksbury, MA, USA). In contrast, CCD-1059Sk (ATCC, CRL-2072, Homo sapiens, human skin fibroblast) were cultured in Modified Eagle Medium (MEM, Corning). Cell media were supplemented with penicillin and streptomycin (Penicillin-Streptomycin Solution, Corning) and fetal bovine serum (FBS, Capricorn). 10% of FBS was used for cell passaging, while 2% of FBS in the medium was used for cell maintenance and experiments. The phosphate-buffered saline (PBS) was purchased from Corning, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and dimethyl sulfoxide (DMSO) were acquired from Sigma (Sigma-Aldrich). Cell lines were incubated at 37^°C in a 5% CO₂ atmosphere (CO₂ incubator, Panasonic). The Litsea cubeba essential oil (LcEO) and preparations were dissolved in dimethyl sulfoxide and stored at 8^°C when not used in experiments.

The cytotoxicity was tested using an MTT-based colorimetric assay. Briefly, the cells were passaged (VERO 1.5 x 105 cells/mL, CCD-1059Sk 5.0 x 105 cells/mL) into 96-well plates, and after overnight incubation, the cell media was removed, and the cells were treated with serial dilutions of LcEO or LcEO-loaded preparations in culture media. Cytotoxicity of DMSO used as a solvent was also tested. After incubation (6h, 12h, 24h, or 48h), the cell media was removed, cells were washed with PBS, and 100 μL/well of 10% MTT solution (5 mg/mL) in FBS-free media was added incubation continued for 4 h. Afterwards, 100 μL/well of SDS/DMF/PBS (14% SDS, 36% DMF, 50% PBS) mixture was added to dissolve the formazan crystals, and after overnight incubation, the Synergy H1 Multi-Mode Microplate Reader (BioTek Instruments) equipped with Gen5 software (ver. 3.09.07; BioTek Instruments) was used to measure absorbance at 540 and 620 nm. Data was then exported to GraphPad Prism to calculate CC₅₀ (concentration decreasing cell viability by 50% in comparison to control cells), CC₁₀ (concentration decreasing cell viability by 10% in comparison to control cells) and CC₉₀ (concentration decreasing cell viability by 90% in comparison to control cells) from dose-response curves (non-linear regression). The GraphPad Prism was also used for statistical evaluation (two-way ANOVA, Tukey multiple comparisons test).

Determination of Antioxidant and Anti-tyrosinase Activity of EO and Formulations

In the current work, the antioxidant assays used were cupric ion reduction antioxidant capacity (CUPRAC), ferric ion reduction antioxidant power (FRAP), metal chelating ability (MCA), 1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,2′-azino-bis(3-ethylbenzothiazoline) 6-sulfonic acid (ABTS), and the phosphomolybdenum (PBD) assay. The details of the assays were explained in our previous paper, and the results were determined as the equivalents of Trolox (TE) or EDTA (EDTAE) (in the MCA assay) [16]. Regarding the tyrosinase assay, the inhibition was expressed as standard equivalents of kojic acid (KAE). The experimental details of the enzyme inhibition assays were given in our earlier studies [16, 17]

The Chemical Composition of EO and the Chemical Profiles of Formulations

The GC-MS analysis confirmed the authenticity of the purchased LcEO. Its chemical constituents are presented in Table 1. The chemical profiles of all tested samples are shown in Figure 1 and are in agreement with the profile of EO. The differences stem from the base used for preparing the formulations. The three major constituents, comprising 84.98% of the EO, were D-limonene, neral, and geranial, and they also dominate in formulations. Other volatiles were present in minor amounts. The compounds contributing more than 1% to the sum of all ingredients were alpha-pinene, sabinene, eucalyptol, linalool, citronellal, geraniol, and caryophyllene.

Table 1: The chemical composition of Litsea cubeba essential oil.

Figure 1: The chemical profiles of Litsea cubeba essential oil (a) and its formulations (b – cream; c – liquid). Numbers from A correspond to compounds described in Table 1.

The Safety Profile of EO and Formulations

The results of cytotoxicity studies after 6h, 12h, 24h, or 48h of LcEO incubation with VERO cells are presented in Figure 2a. A time-dependent increase of LcEO cytotoxicity can be observed, with the highest toxicity observed after 48h of incubation. Table 2 presents the influence of incubation time on the CC₁₀, CC₅₀, and CC₉₀ values. These parameters have been calculated from dose-response curves obtained during LcEO incubation with VERO cells (Fig. 2a) and are often used to assess the cytotoxicity of natural products and synthetic compounds. Despite the observed increase of cytotoxicity with incubation time, not all differences in CC₁₀, CC_50, and CC₉₀ values were statistically significant. Statistical evaluation (Fig. 2b) showed significant differences in all tested parameters between 12h and 24h of incubation and, in the case of CC90, between 6h and 12h of incubation. That is why the 24-hour incubation period was used for experiments with CCD-1059Sk. The LcEO showed higher cytotoxicity towards CCD-1059Sk than VERO, with a CC50 of 14.74 ± 1.6 μg/mL. Notably, the crème and liquid formulations showed low toxicity on CCD-1059Sk, with the CC50 of 221.75 ± 25.53 and 354.3 ± 9.19 μg/mL, respectively (Fig. 3). The DMSO, used as a solvent for stock solutions, was non-toxic in the concentrations used in the presented research.

Figure 2: Evaluation of Litsea cubeba essential oil cytotoxicity towards VERO cells (a – dose-response effect of LcEO on VERO cells after 6, 12, 24, and 48h incubation, b – evaluation of statistical significance of cytotoxicity parameters after 6, 12, 24, and 48h incubation; two-way ANOVA, Tukey multiple comparisons test, * p<0.05, ** p<0.001).

Table 2: Influence of incubation time on the CC10, CC50 and CC90values.

Figure 3: Evaluation of cytotoxicity of Litsea cubeba essential oil and cosmetic formulations towards CCD-1059Sk cells.

Table 3: Antioxidant and anti-tyrosinase activity of Litsea cubeba essential oil and its formulations.

The Antioxidant and Anti-tyrosinase Activity of LcEO and Formulations

A number of tests were performed to understand the antioxidant and anti-tyrosine activity of L. cubeba essential oil and its formulations, the results of which are presented in Table 3. L. cubeba EO shows the highest activity in CUPRAC test (80.63 mg TE/g) and also significant activity in FRAP assay (50.09 mg TE/g), Anti-tyrosinase assay (38.40 mg KAE/g), and Phosphomolybdenum (27.66 mmol TE/g). In radical scavenging and reducing power assays, the essential oil exhibited more activity than the used cream and liquid formulations. However, the highest metal chelating ability was detected in liquid formulation with 29.32 mg EDTAE/g, while the essential oil was not active in the assay. When comparing liquid and cream formulations, the liquid formulation showed more potent antioxidant activity across all assays tested, including ABTS, CUPRAC, FRAP, phosphomolybdenum, and metal chelation. Notably, its CUPRAC reducing ability was nearly threefold higher. Regarding tyrosinase inhibition, the liquid formulation was most effective (43.06 mg KAE/g), outperforming both the essential oil (38.40 mg KAE/g) and cream formulation (20.65 mg KAE/g). These findings suggest that liquid formulation is more potent than the cream form in both antioxidant and anti-tyrosinase activities.

DISCUSSION

To evaluate the potential safety of LcEO for topical applications, we assessed its cytotoxic effects on normal cells. The cytotoxicity results on Vero cells demonstrated that both higher concentrations of LcEO and longer exposure times significantly increased cytotoxicity (Figs 2a and 2b). Therefore, concentrated or undiluted essential oil preparations should not be applied directly to the skin. A highly statistically significant difference in CC50 values was observed between 12 and 24 hours of exposure, indicating that a 12-hour application is within a safe range. Based on this finding, and considering that skincare products are typically applied in the morning and evening, remaining on the skin for approximately 12 hours, we selected a 24-hour exposure period for subsequent experiments. It is also important to note that the concentration of LcEO in the tested formulations was only 0.5%, which may result in different biological activity compared to pure essential oil.

To ensure the integrity of the essential oil in the final product, we analyzed the chemical composition of the prepared formulations. The results showed that the formulations maintained a chemical profile similar to that of pure LcEO, confirming that the essential oil components dissolved well and preserved their relative proportions. The primary constituents of LcEO were D-limonene (12.75%), neral (30.57%), and geranial (41.66%).

Based on the results, the antioxidant effects of the formulations were somewhat reduced compared to pure LcEO, although not in direct proportion to concentration. Interestingly, the metal-chelating capacity was enhanced in the formulations. The cream maintained a comparable level of tyrosinase inhibition to that of the pure essential oil, suggesting that other formulation ingredients may contribute positively to this activity.

Antioxidant and anti-tyrosinase assays revealed that the liquid formulation was more effective than the cream. Notably, the essential oil’s antioxidant properties were largely preserved in the liquid formulation, as demonstrated by the CUPRAC and FRAP assays. Moreover, the liquid formulation exhibited enhanced tyrosinase inhibitory activity compared to the pure essential oil. This improved efficacy may be attributed to the formulation’s ability to stabilize and protect key active compounds such as geranial, neral, and D-limonene.

For instance, geranial—the primary component of the tested essential oil—has been shown to exhibit strong radical scavenging activity in ABTS and DPPH assays, along with significant reducing power in the FRAP assay, as previously reported by Sharopov et al. [18]. The presence of conjugated double bonds in geranial’s structure likely contributes to its antioxidant capabilities [19]. Similarly, Mimica-Dukic et al. also reported strong DPPH radical scavenging activity for geranial [20]. In addition to its antioxidant activity, geranial has demonstrated potent inhibitory effects on tyrosinase. For example, Capetti et al. evaluated citral-rich essential oils and found that Litsea cubeba essential oil exhibited the highest tyrosinase inhibition due to its high citral content, a mixture of geranial and neral [21]. Similar findings were also reported by Matsuura et al [22]. More recently, Capetti et al. reported that a blend of essential oils, including L. cubeba, Pinus mugo, and Cymbopogon winterianus, showed strong anti-tyrosinase activity, with citral identified as the key active component [23].

Additionally, D-limonene, a monocyclic monoterpene, is widely recognized for its therapeutic properties. It protects cells from oxidative damage by reducing oxidative stress markers such as TBARS, lipid hydroperoxides, and conjugated dienes, while simultaneously enhancing the activity of antioxidant enzymes including SOD, CAT, GPx, GST, and GSH. D-limonene also exerts anti-inflammatory effects by lowering levels of inflammatory mediators such as NO, PGE2, IL-1β, IL-6, and TNF-α [24]. Beyond its antioxidant and anti-inflammatory roles, D-limonene is also a significant tyrosinase inhibitor. In a study by El Hachlafi et al., D-limonene (IC₅₀: 86.07 μg/mL) exhibited stronger tyrosinase inhibition than quercetin (IC₅₀: 111.03 μg/mL) [25]. Additionally, Forestrania et al. demonstrated that D-limonene has a high binding affinity for the tyrosinase active site, supporting its potential as an effective skin-protective agent [26].

Due to these multifunctional properties, geranial, neral, and D-limonene are commonly used not only in anti-inflammatory and antimicrobial products but also in cosmetics. Their pleasant lemon-like scent, along with their bioactive benefits, makes them ideal ingredients for perfumes, soaps, and lotions. Furthermore, L. cubeba essential oil contains various minor components which, although present in smaller amounts, may also contribute to the oil’s overall biological activity.