Antioxidant Activities of Essential Oils from Citrus × natsudaidai (Yu. Tanaka) Hayata Peels at Different Ripening Stage

Citrus × natsudaidai (Yu. Tanaka) Hayata peels used in this study were collected from Citrus Research Institute (National Institute of Horticultural and Herbal Science) in Jeju Island, Korea. The samples were collected thrice (March, September, and December) according to ripening stages.

To extract the essential oil from the Citrus × natsudaidai peel with that of moisture content from 80.06% to 81.37%, we used the previously used extraction method (Yang et al., 2021a). C. × natsudaidai peels (2 × 2 cm) were hydro-distillated at atmospheric pressure using a Clevenger-type apparatus. A 10 L round-bottom flask containing 1.0 kg of peels was placed in a digital heating mantle (MS-DM 608, MTOPS^®, Yangju, Korea), and 5 L of distilled water was poured into it, which was then connected to the Clevenger-type apparatus. The peel was extracted for 30 h. The collected essential oils were dried over anhydrous sodium sulfate (98.5%, Cat No. 834605125, Samchun, Seoul, Korea) and filtered using a 0.45 μm membrane disk filter. The essential oils were transferred to dark vials and stored at 4°C for further analysis. The yield of the essential oils was calculated using Equation (1):

To identify the volatile components therein, the essential oils during fruit ripening were analyzed using GC-MS equipped with a VF-5MS capillary column (Table 1). These analysis conditions were based on our prior research (Yang et al., 2021b).

The components of essential oils according to maturity stage were identified on the basis of the peaks with the highest spectral matching when the S/N ratio reached ≥ 50 in total-ion chromatography using the NIST library search program (version 11; National Institute of Standards and Technology, Gaithersburg, MD, USA). The Kovats retention index (KI) values of the individual compounds were determined based on the comparison of their relative retention times with an n-alkanes mixture (C₈-C₂₀, Cat No. 04071, Merck KGaA, Darmstadt, Germany) using the VF-5MS column. The essential oils’ components were assigned based on the comparison of their calculated KI values to literature values (e.g., NIST Chemistry WebBook).

The TPC of the essential oils was determined by the Folin-Ciocalteu method (Singleton and Rossi, 1965). Using a standard curve as a reference, the TPC was determined and represented as mg gallic acid equivalents (mg GAE/100 g).

Ethanol (≥ 99.5%, Cat No. 459836, Merck KGaA) was used to dilute the essential oils to be analyzed. Afterward, in a 10 mL volumetric flask, 500 μL of oil was added to 2.5 mL of 2N Folin-Ciocalteu reagent (eCat No. F9252, Merck KGaA) alongside 2 mL Na₂CO₃ (7.5%, Cat No. 222321, Merck KGaA). After incubation for 60 min at 37°C, the absorbance versus prepared blank was read at 760 nm. The standard reference curve for gallic acid was made for the following concentrations: 1, 2, 4, 6, 8, and 10 mg/mL, respectively. The correlation coefficient and regression equation were determined and expressed in mg GAE/100 g. All measurements were performed in triplicates.

The free-radical scavenging assay of 2,2-diphenyl-1-picrylhydrazyl (DPPH, Cat No. D9132, Merck KGaA) was used to examine the antioxidant properties of the essential oils. The measurement was based on the method described in (Sayuti et al., 2018). The essential oils were prepared by diluting 100 mg of peel oil in 1 mL of ethanol, producing a concentration of 1 mg/mL. The stock solution was sonicated to ensure sample homogeneity. Six other concentrations were prepared at 1, 2, 4, 6, 8, and 10 mg/mL, diluted from the 100 mg/mL stock solution. Approximately 1 mL of 0.2 mM solution of DPPH in ethanol was each added into six series of prepared concentrations (1, 2, 4, 6, 8, and 10 mg/mL) of the sample solution (4 mL). The solution was vigorously mixed and allowed to stand at room temperature for 30 minutes in the dark, after which its absorbance was measured spectrophotometrically at 517 nm using a Micro-plate spectrophotometer. Ethanol was used as blank (only ethanol) and negative control (4 mL ethanol mixed with 1 mL DPPH), while ascorbic acid was used as the standard.

The scavenging activity of ABTS⁺ was conducted to determine the radical scavenger activity. The ABTS [2,2’-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid), Cat No. 10102946001, Merck KGaA] radical scavenging activity of the essential oils was determined using Re et al. (1999) method. ABTS⁺ was generated by reacting an ABTS⁺ aqueous solution (7 mM) with K₂S₂O₈ (2.45 mM, final concentration, Cat No. 216224, Merck KGaA) in the dark for 16 h and adjusting the absorbance of 734 nm to 0.7 ± 0.02 with ethanol. The sample with different concentrations (1, 2, 4, 6, 8, and 10 mg/mL) was diluted with 0.2 mL of the required amount to react with the ABTS⁺ solution (2 mL). After 30 min, the absorbance was measured at 734 nm. All measurements were conducted in triplicate.

The total antioxidant potential of the samples was determined using the ferric reducing capacity of plasma FRAP assay by Fernandes et al. (2016) as a measure of antioxidant capacity. The analysis was based on the compound’s reducing power. A potential antioxidant can reduce the ferric ions (Fe³⁺) to ferrous ions (Fe²⁺). The latter forms a blue complex (Fe²⁺/TPTZ) that promotes absorption at 593 nm. The FRAP reagent was prepared by mixing a solution of acetate buffer (300 mM, pH 3.6), 10 mM TPTZ (≥ 98%, Cat No. T1253, Merck KGaA) in 40 mM HCl (1 M, Cat No. H1202, TCI, Tokyo, Japan), and 20 mM FeCl³⁺ (97%, Cat No. 236489, Merck KGaA) at 10:1:1 (v/v/v). Reagent and sample solution were added to each well and mixed thoroughly. After 30 min, absorbance was measured at 593 nm. A standard curve was generated using different concentrations of Trolox. All solutions were used on the day of manufacture. The results were expressed as mg Trolox equivalent/100 g. Analysis was conducted in triplicate for each essential oil.

The results of the tests were repeated in triplicate, and results were presented as mean ± standard deviations. Statistical analysis was conducted using R x64 (ver.4.0.3).

The yield of the essential oils largely varied with the ripening stages. Among the essential oils, immaturity showed the highest yield (5.88 ± 0.23%) followed by overripe (3.62 ± 0.12%) and maturity (2.06 ± 0.09%). According to a previous study, the yield of immature C. × natsudaidai was 0.6%, and that of matured C. × natsudaidai peel was 1.7% (Baik et al., 2008; Oh et al., 2007). Despite the same ripening stage, the essential oil was extracted using the steam distillation method for only 6 hours, resulting in a big difference in yield. This difference in yield is due to the extraction time (about 30 h).

The chemical composition of the essential oils during fruit maturation was identified by GC-MS. As shown in Table 2, the chemical composition at different stages of ripening was identified by GC-MS.

As a result, the proportion of the constituents in the essential oil was also different. D-Limonene was the major constituent in all oil samples (86.44% to 90.45%), followed by γ-terpinene and β-myrcene ranged between 4.75% to 6.32% and 1.09% to 1.30%, respectively. According to Vekiari et al. (2002), harvesting time is a critical parameter influencing the chemical compositions of the essential oil significantly. As C. × natsudaidai matured, the contents of β-pinene, cymene, and d-limonene were increased. On the other hand, the contents of α-terpinene, γ-terpinene, terpinolene, linalool, β-terpineol, terpinene-4-ol, α-terpineol, and octanal, were gradually decreased. Dugo et al. investigated the seasonal variation of the chemical composition of the essential oil extracted from two cultivars, Sicilian mandarin (Citrus × deliciosa Tenore. cv Avana and Tardivo di Ciaculli), and reported an increase of limonene content from September to February (from immature stage to mature stage) (Dugo et al., 2011). In contrast to the behavior observed from October to February, limonene content decreased at the overripe stage (from February to March). This result showed a similar tendency to our study result. According to Dugo et al., limonene is compensated by the other constituents of the monoterpene hydrocarbons, so it decreases sharply in March when each monoterpene hydrocarbon exhibits the same decreased behavior. Furthermore, it was found that the yield and composition of these essential oils were different depending on soil properties (Moretti et al., 1998).

Phenolic compounds are related to several biological roles, such as activities of antioxidant free radical scavenging (Salar and Seasotiya, 2011). We were determined to compare the TPC of essential oils according to the ripening stages.

The results, as shown in Table 3 indicated that TPC in all essential oils was seen to be significantly different amounts. Among them, the highest TPC was found in immaturity (1011.25 ± 57.15 mg/100 g) followed by overripe (961.76 ± 124.55 mg/100 g) and maturity (895.77 ± 69.99 mg/100 g). One-way ANOVA analysis was performed to prove whether TPCs of essential oils according to the ripening stage were significantly different. As a result, the ripening stages do not make significant differences in TPC (P = 0.4503). The TPC contents decreased in the ripening stage but increased in overripe. That can be related to a shift in the biosynthetic pathway of secondary metabolites toward the production of essential oils in the plant. When the plant encounters oxidative stress due to a sudden change of temperature in summer and early winter, the biosynthetic pathway of secondary metabolites in the plant is directed toward the production of essential oils rather than phenolic compounds (Boveiri Dehsheikh et al., 2019). These phytochemical compounds support bioactivity in medicinal plants and are thus responsible for the antioxidant activities of the essential oils extracted from C. × natsudaidai peels according to ripening stages (Altemimi et al., 2017).

In this analysis, the abilities of the test compounds to donate hydrogen atoms or electrons were measured spectrophotometrically. Table 4 shows the results of the DPPH inhibition assay using different essential oils and control (Ascorbic acid).

It showed that all the essential oils can reduce the pink-colored free radical to yellow-colored diphenyl picrylhydrazine, indicating that these essential oils possessed DPPH radical scavenging activity. The EC₅₀ values of DPPH free-radical scavenging capacity were 15.91 ± 0.38, 16.98 ± 0.40, and 16.21 ± 0.32 mg/mL for immaturity, maturity, and overripe, respectively. The mechanism of the DPPH assay depends on the structural conformation of the antioxidant. Some compounds react very quickly with DPPH·, reducing the number of DPPH· molecules equal to the hydroxyl groups (Bondet et al., 1997). In Table 2, the proportion of oxygenated terpene components in the essential oil at the immaturity period is higher than in other ripening stages. In particular, the proportion of α-terpineol was high at 2.16% in the immaturity stage, but it significantly reduced as the maturation stage progressed. The proportion of other oxygenated monoterpene-based components also gradually decreased as the C. × natsudaidai matured. This was consistent with the DPPH radical scavenging ability. Also, this tendency was consistent with the change in the proportion of monoterpene components (α-pinene, β-ocimene, and linalool oxide). Choi et al. (2000) were investigated thirty-four species of citrus essential oils and their components for radical scavenging activities using DPPH assay (Choi et al., 2000). Citrus ichangensis, Citrus × aurantiifolia, and Citrus limon showed higher radical-scavenging activities than other citrus cultivars. These excellent radical scavenging activities were found to be γ-terpinene and terpinolene. Therefore, the high radical scavenging ability in the immature stage is due to the higher proportion of γ-terpinene and terpinolene than in other stages.

Although the DPPH· free radical is ubiquitously used to estimate the potential free radical scavenging activity of natural products, the ABTS⁺ free radical is commonly used when issues of solubility of interference arise, and the use of DPPH based assay becomes inappropriate (Medini et al., 2011). In Table 5, the results of ABTS radical scavenging activity of peel essential oils according to ripening stages.

In the determination of ABTS radical scavenging capacity, the EC₅₀ values of immaturity (22.43 ± 0.45 mg/mL), maturity (26.82 ± 0.48 mg/mL), and overripe (20.43 ± 0.37 mg/mL) were much higher than those of ascorbic acid (0.066 ± 0.001 mg/mL). The overripe stage showed the highest ABTS radical scavenging activity compared to the other stages. For ABTS, no correlation between EC₅₀ and total phenols was found. However, the antioxidant activity was low in the maturity stage, as shown in the DPPH results. The ABTS radical scavenging activity was similar to the change of β-myrcene, and it is judged that β-myrcene affects the ABTS radical scavenging activity. In a previous study, rosemary essential oil had great free radical scavenging capacity; it was confirmed that myrcene contributed significantly to the free radical scavenging ability of rosemary essential oil (Ojeda-Sana et al., 2013). In vivoβ-myrcene demonstrate antioxidant effect (Bonamin et al., 2014). The antioxidant effects of orally administered β-myrcene against ethanol-induced gastric ulcers in male Wistar rats were confirmed. The β-myrcene showed antioxidant enzyme activity from the glutathione reductase system as evidenced by the decreased activity of superoxide dismutase (SOD) and increased levels of glutathione peroxidase, glutathione reductase, and total glutathione in gastric tissue. Future studies investigating antioxidant activity of β-myrcene, need to conduct appropriate dosage levels that have therapeutic effects in humans.

The FRAP assay, which provides fast reproducible results, measures the reducing potentials of an antioxidant reacting with a ferric tripyridyltriazine (Fe³⁺-TPTZ) complex to produce colored ferrous tripyridyltriazine (Fe²⁺-TPTZ). The results of the FRAP assay using different essential oils are shown in Table 6.

According to ripening stages, most essential oils showed reducing power from 815.21 ± 29.39 mg Fe(II)/100 g in maturity stage to 1,417.68 ± 0.00 mg Fe(II)/100 g in the overripe stage. The highest reducing power (FRAP) was shown in overripe and the reducing power in immaturity is comparable but less than the reducing power in overripe. This FRAP away tendency was similar as shown in the ABT results. In a previous study, the antioxidant activities of different 15 species flavonoids were measured by the FRAP assay (Firuzi et al., 2005). As a result, quercetin, fisetin, myricetin, and kaempferol were the highest reducing power. It has been shown that hydroxyl groups and especially catechol moiety, 3-OH, and 2,3-double bonds are the most important factors in determining high antioxidant activity. The proportion of β-myrcene is a constituent that shows a similar tendency compared to reducing power among the components in essential oils for each maturation stage. Since β-myrcene has a 2,3-double bond structure, it is considered a component is contributing to the high antioxidant activity of essential oils in the overripe.