Whitening and antioxidant activities of bornyl acetate and nezukol fractionated from Cryptomeria japonica essential oil

Synopsis: Objectives: The aim of this study was to investigate the whitening and antioxidant activities of essential oils from Cryptomeria japonica by determining their tyrosinase inhibition, 1,1‐diphenyl‐2‐picrylhydrazyl (DPPH) radical scavenging and superoxide dismutase (SOD)‐like activities. Methods: Essential oils of C. japonica leaves were extracted with distilled water, and after condensation of volatile constituents, the condensates were extracted with ethyl acetate. Crude essential oils of C. japonica were divided into six fractions by thin layer chromatography and open column chromatography, and their chemical analysis was performed by GC/MS. Major compounds of fractions were composed of kaurene, bornyl acetate, nezukol, (‐)‐4‐terpineol, δ‐cadinene, α‐terpineol, γ‐eudesmol, α‐eudesmol and elemol. Results: For tyrosinase inhibitory activity using two substrates, l‐tyrosine and 3,4‐dihydroxyphenylalanine (l‐DOPA), kaurene, bornyl acetate and nezukol were highly effective. In antioxidant activity, (‐)‐4‐terpinenol and δ‐cadinene showed high DPPH radical scavenging activity, and bornyl acetate and nezukol indicated extremely high SOD‐like activity. Conclusion: Therefore, bornyl acetate and nezukol fractionated from C. japonica essential oil, which showed highly active whitening and antioxidant activities, have potential applications in cosmeceutical materials.

Résumé: Objectifs: Le but de cette étude était d'évaluer les activités éclaircissante et anti‐oxydantes des huiles essentielles obtenues à partir de Cryptomeria japonica en déterminant leur capacité d'inhibition de la tyrosinase, d'inactiver le radical DPPH (1,1‐diphényl‐2‐picrylhydrazyl), et de mimer l'activité superoxyde dismutase (SOD). Methodes: Les huiles essentielles des feuilles de C. japonica ont été extraites avec de l'eau distillée, et après condensation des constituants volatils, les condensats ont été extraits avec de l'acétate d'éthyle. Les huiles essentielles bruts de C. japonica ont été divisées en six fractions par chromatographie sur couche mince et par chromatographie sur colonne ouverte, et leur analyse chimique a été effectuée par GC / MS. Les principaux composés des fractions étaient constitués de kaurene, d'acétate de bornyle, de nezukol, de (‐)‐4‐terpinéol, de δ‐cadinene, d'α‐terpinéol, de γ‐eudesmol, d'α‐eudesmol et d'élémol. Resultats: Pour l'activité inhibitrice de la tyrosinase en utilisant deux substrats, la L‐tyrosine et la 3,4‐dihydroxy‐phénylalanine (L‐DOPA), le kaurene, l'acétate de bornyle et le nezukol étaient très efficaces. En activité antioxydante, le (‐)‐4‐terpinenol et le δ‐cadinene ont montré une activité radicalaire haute de captage du DPPH, et l'acétate de bornyle et le nezukol ont indiqué une activité extrêmement élevée SOD‐like. Conclusion: Par conséquent, l'acétate de bornyle et le nezukol fractionné de l'huile essentielle de C. japonica, qui ont montré des activités antioxydantes et éclaircissantes très fortes ont des applications potentielles dans les formulations cosmétiques.

antioxidant activity; bornyl acetate; Cryptomeria japonica; essential oils; nezukol; whitening activity

Exposure to ultraviolet rays induces many negative effects on skin health like erythema, hyperpigmentation, skin cancer and more [1] . As a result, skin care products have been developed for skin whitening and antioxidant action. These functional products are made using synthetic processes, and the most widely used synthetic chemical compounds in skin care have been suspected to be associated with negative health effects [2] . Because of this, there is a growing interest in the use of natural compounds derived from plant origins [3] .

Melanin induced from tyrosine is one of the important elements in the determination of skin colour. Melanin is synthesized in melanocytes via the oxidation of tyrosine into 3,4‐dihydroxyphenylalanine (DOPA) through the action of tyrosinase, which contains copper mono‐oxygenase [4] . Hyperpigmentation in human skin has been shown to be caused by overproduction of dermal melanin pigment, which is synthesized in melanocytes via the action of tyrosinase. Thus, several researches have been conducted on tyrosinase inhibitors for their depigmenting activity [4] . Plant extracts have been found to promote inhibition of melanin development [5] .

The human body is always under oxidative stress on account of reactive oxygen species (ROS) caused by the exposure of oxygen to ultraviolet rays [6] . Oxygen radicals of reactive oxygen species cause optical damage and accelerate the skin ageing process via antioxidant destruction, lipid peroxidation, and protein and DNA oxidation, resulting in the formation of wrinkles. In addition, melanin results in cutting and abnormal cross‐linkage of connective tissue (collagen, elastin, hyaluronic acid and so on) chains [7] .

Free radicals that alter metabolism, exposure to environmental pollution and ultraviolet rays, and a variety of other stressors promote the skin ageing process via inactivation of antioxidative enzymes and destruction of antioxidants [8] .

There is an interest in developing whitening and antioxidant products using natural compounds from plants to treat and protect against hyperpigmentation and ageing of the skin caused by ultraviolet rays, ROS, free radicals and other insults.

Many researches have been conducted on the whitening effects and antioxidant activity of plants [9] . These studies examined medicinal plants and herbs like aromatic plants, but essential oils from forest resources have not been frequently researched, although it is known that the essential oils of many woody plants have phytonutrients with high antimicrobial activity [10] , [11] , [12] . Thus, it is worthwhile to evaluate whitening effects and antioxidant activities of essential oils derived from forest resources.

Cryptomeria japonica, an evergreen conifer, was widely planted in southern areas and Jeju Island in South Korea. The wood is mostly used as a raw material for production of furniture and other wooden structures. Because of its industrial importance, its components, like terpenoids, have been researched for use in the pharmaceutical or cosmetic industries [12] , [13] , [14] .

Essential oils of C. japonica have been found to have high antifungal, antibacterial and antitermicidal activities [15] , [16] , [17] . Among constituents from essential oils of C. japonica, bornyl acetate has been known as a highly active antimicrobial agent [18] , but nezukol is not very well‐known terpene compound, which is identified in conifer species' leaves and heartwood.

Hence, the aim of the present study was to examine the whitening and antioxidant activity of C. japonica essential oils by determining the tyrosinase inhibitory activity, 1,1‐diphenyl‐2‐picrylhydrazyl (DPPH) radical scavenging activity and superoxide dismutase (SOD)‐like activity.

The leaves of C. japonica grown in Jeju island, South Korea, were collected in 2010. After sorting the leaves, they were stored at −39°C until essential oils were extracted. Tyrosinase, l‐tyrosine and l‐DOPA (3,4‐dihydroxyphenylalanine) were obtained from Sigma‐Aldrich Korea. DPPH and pyrogallol (1,2,3‐benzenetriol) were also obtained from Sigma‐Aldrich Korea.

Essential oils of C. japonica leaves were extracted with distilled water on a lab scale by steam distillation. Volatile compounds in C. japonica leaves (500 g) were condensed for 24 h as the temperature was gradually increased to 100°C. Essential oils in condensate were extracted three times with 100 mL of ethyl acetate as a solvent, and the final essential oils were concentrated by rotary evaporator. The yield of extracted essential oils of C. japonica leaves was 4.7% based on dry mass (369 g) of initial leaves weight. The oils were stored in the freezer at 4°C until analysis.

The first step in determining the bio‐active constituents of C. japonica essential oils was thin layer chromatography (TLC) assay. The second step was preliminary fractionation by open column chromatography. This study was conducted using silica gel 60 (40~100 μm) as the stationary solvent because the components of the essential oils were relatively non‐polar. Silica gel (450 g) mixed with hexane was packed into a column (8 cm in diameter) for 24 h.

Then, 15 g of C. japonica essential oils was loaded into the column, accounting for about 3% of the packed silica. Eluent solvents were mixtures of hexane and ethyl acetate at the rate of 1 : 0, 49 : 1, 30 : 1, 18 : 1, 9 : 1, 4 : 1, 1 : 1 and 0 : 1. Each gross weight of eluent solvents was 2 L. Every eluent solvents was sequentially loaded at flow rate of 20 mL min−1 and fractionated on each 50‐mL flasks. Essential oils of each fractionated flasks were divided into six fractions according to same TLC plate aspects (Fig. [NaN] ). These six fractions of crude oil and the crude oil of C. japonica were used as the samples in this experiment.

The whitening activity of C. japonica essential oils (crude oil and its six fractions) was determined by measuring tyrosinase inhibitory activity which was modified from Yagi's method [19] . l‐tyrosine and l‐DOPA were used as the substrates in this experiment, because they are precursors of melanin synthesis [4] . Ascorbic acid and hydroquinone were used as positive controls. The samples (C. japonica essential oils), ascorbic acid and hydroquinone with final concentrations of 50, 100, 500, 1000 and 2000 ppm, were first dissolved in ethyl alcohol. The assay was performed as a mixture of substrate (450 μL), tyrosinase (100 μL), and sample (100 μL) was incubated at 37°C for 20 min. l‐tyrosine (0.3 mg mL−1) and l‐DOPA (2 mg mL−1) of powder form were dissolved with 0.1 mol L−1 phosphate buffer (Na2HPO4·12H2O, NaH2PO4·2H2O, pH 6.8). The aqueous solution of tyrosinase (l‐tyrosine: 125 units mL−1, l‐DOPA: 25 units mL−1) was also dissolved in the same buffer. The reactant mixtures were then immediately evaluated to determine the initial rate of increase in optical density at 492 nm on the basis of dopachrome formation, using a UV 1601P spectrophotometer (Shimadzu 1601P, Japan). The activity was calculated with the following equation [equation ].

Activity(%)=[1−(As/Ac)]×100

As: absorbance in the presence of the inhibitor; Ac: absorbance of the control reaction (= full reaction, containing no test compound).

The antioxidant activities of the C. japonica essential oils were measured in terms of hydrogen donating or radical scavenging ability, using DPPH as a stable radical [20] . Radical scavenging activity was determined by a spectrophotometric method based on the reduction in a methanol solution of DPPH as a modification of the Blois method [21] . The absorbance decreases as a result of a colour change from purple to yellow as the radical is scavenged by antioxidants through donation of hydrogen to form the stable DPPH‐H molecule [22] .

A 0.2 mL sample of 5000 ppm antioxidant solution in methanol was added to 1.8 mL of methanolic DPPH radical solution, resulting in an initial DPPH radical concentration of 6 × 10−5 mol L−1. The mixtures were stirred for 10 s and were reacted for 30 min at room temperature. The reactant mixtures were then immediately evaluated at 517 nm using the same spectrophotometer. The activity was calculated according to equation.

The activity of the antioxidants in suppressing the reactivity of the superoxide ion was measured according to the SOD assay procedure of Marklund and Marklund [23] . SOD is an important antioxidant defence in nearly all cells exposed to oxygen. Also, SOD very rapidly dismutases univalently reduced oxygen. The antioxidant activity of C. japonica essential oils was measured using pyrogallol (1,2,3‐benzenetriol) with concentration of a 5000 ppm, as reducing agent. Pyrogallol has long been known to autoxidize rapidly, especially in alkaline solutions, and the reaction has been employed for the removal of oxygen. It was an indirect method of measuring superoxide catching activity by achieving lower oxidation velocity of pyrogallol through browning because of pyogallol's autoxidation [24] .

Pyrogallol solution (0.2 mL of 7.2 mmol L−1 in methanol solution) and 0.2 mL of the samples dissolved in methanol were mixed with 2.6 mL of Tris‐HCl buffer solution (50 mmol L−1 Tris + 10 mmol L−1 EDTA, pH 8.8), and the mixtures were reacted for 10 min at 25°C. The reactant mixtures were then immediately evaluated at 420 nm using the spectrophotometer. The activity was calculated according to equation.

Quantitative analysis of the volatile constituents of the active C. japonica essential oil and six fractions of crude oil was performed by GC/MS analysis. The GC column was an HP5 (model: Agilent 6890, Santa Clara, CA, U.S.A.), and the carrier gas was helium. Analytical temperatures were 260 and 280°C in the injector and detector, respectively. Initial oven temperature was kept at 120°C for 5 min and then increased by 4°C min−1 to 280°C, where it was held for 10 min. MS analysis used an Agilent 5973 (Santa Clara, CA, U.S.A.) and was performed in EI mode. The chemical structures of the active compounds were analysed by comparing the mass data of the active compounds peaks with the standard library data (Willy 7th ed).

Normality and homogeneity of each data were examined by the general linear model procedure with the Statistical Analysis System programming package (version 9.1, SAS Institute, Cary, NC, U.S.A.). If a significant effect was found in a variable (P < 0.05), the least significant difference (LSD) values were used to differentiate mean values and significance. Data are presented as the mean ± SD (standard deviation).

Thirty compounds were identified by GC/MS analysis, representing 95.7% of the total oil (Fig. [NaN] ). The essential oils of C. japonica leaves were characterized by a high number of monoterpenes such as α‐pinene (9.84%), (‐)‐4‐terpineol (5.71%), sabinene (5.53%), δ‐carene (3.05%) and α‐limonene (2.02%), although sesquiterpenes and diterpenes were more prevalent. The most abundant compound was kaurene (19.36%), a diterpene. Kaurene and the sesquiterpenes such as α‐eudesmol (12.18%), elemol (10.93%) and γ‐eudesmol (10.61%) were important components comprising of the remaining 54% of the essential oils.

GC/MS results of fractions of C. japonica essential oils are shown in Table [NaN] . Major compounds of fractions were confirmed as kaurene in fraction A, bornyl acetate and nezukol in fraction B, (‐)‐4‐terpineol in fraction C, (‐)‐4‐terpineol and δ‐cadinene in fraction D, α‐terpineol in fraction E, and γ‐eudesmol, α‐eudesmol and elemol in fraction F.

Major constituents of six fractions from Cryptomeria japonica essential oils

Tyrosinase inhibitory activity was determined for crude essential oil of C. japonica and its six fractions. In our routine screening using tyrosinase, the essential oils obtained by steam distillation of C. japonica leaves inhibited l‐tyrosine and l‐DOPA oxidation. The oil concentrations were 50, 100, 500, 1000 and 2000 ppm. The relative tyrosinase inhibitory activities of C. japonica essential oils are shown in Tables [NaN] and [NaN] . The positive controls, ascorbic acid and hydroquinone, demonstrated highly effective activities, almost 100% inhibition at most concentrations, and crude essential oil of C. japonica showed 88.35 ± 0.79% inhibition for l‐tyrosine and 76.77 ± 0.67% for l‐DOPA. Fraction A showed 86.76 ± 0.26% inhibition for l‐tytosine and that of fraction B was 87.53 ± 0.35%. Fractions A and B showed 88.45 ± 0.73% and 85.03 ± 0.63% inhibition for l‐DOPA, respectively. They were significantly not more effective than the positive controls (P < 0.05), but showed increasing inhibition activity compared with other fractions as the concentrations were increased. Unexpectedly, no activity of hydroquinone was detected on tyrosinase inhibitory activity for l‐DOPA, and it only showed 49.21 ± 0.93% inhibition, which was much lower than that of fraction A or B at 2000 ppm statistically (P < 0.05). Of note, hydroquinone has been banned in the cosmetic industry since January 2001 because of concerns of side effects like leukomelanoderma and exogenous ochronosis [25] . l‐DOPA is oxidized from l‐tyrosine by tyrosinase. So, fractions A and B, which affect tyrosinase inhibitory activity for l‐DOPA, appear to be effective agents against ageing and pigmentation.

Tyrosinase inhibitory activity of Cryptomeria japonica essential oils and positive controls using l ‐tyrosine as substrate. Crude essential oil, six fractions and positive controls were tested at concentrations of 50, 100, 500, 1000 and 2000 ppm

1 Values are means (± standard deviation). Different small letters in the same column indicate significant difference values at P < 0.05 (least significance difference test).

2 ND: not detected.

Tyrosinase inhibitory activity of Cryptomeria japonica essential oils and positive controls using l ‐DOPA as substrate. Crude essential oil, six fractions and positive controls were tested at concentrations of 50, 100, 500, 1000 and 2000 ppm. Activities of fractions C and D are not shown because they were not effective for tyrosinase inhibitory activity using l ‐DOPA

• 3 Values are means (± standard deviation). Different small letters in the same column indicate significant difference values at P < 0.05 (least significance difference test).
• 4 ND: not detected.

Therefore, fractions A and B of C. japonica essential oil which majorly contained kaurene, bornyl acetate and nezukol might have been considered as potential natural compounds that can be used whitening, as they are effective in inhibiting tyrosinase activity.

The DPPH radical scavenging activity of C. japonica essential oils was examined by comparing it with the activities of a known antioxidant such as α‐tocopherol (positive control). The results are shown in Fig. [NaN] . Fraction C of C. japonica essential oils was highly effective with 86.91% activity, which was almost as great as that of the 90.19 ± 0.00% of α‐tocopherol. It is considered that fraction C has the greater activity in the antioxidant activity.

Fractions B (44.11 ± 0.40%) and D (44.40 ± 0.34%) were not as effective as the positive control significantly (P < 0.05), but they showed better active DPPH radical scavenging activity compared with other fractions. According to Koleva's work, rapid or slow radical scavenging ability related to a reaction stoichiometry corresponding approximately to the number of electrons available for donation [26] . Also, according to Bondet et al., radical scavenging abilities of some compounds can be influenced by their different kinetic behaviours [27] . For slow reacting compounds, the influence was attributed to the complex reacting mechanism [2] . In our study, bornyl acetate and nezukol of C. japonica may be involved in one or more secondary reactions, so they would result in slower reduction in DPPH radical solutions. Thus, we tested the DPPH radical scavenging activity of bornyl acetate and nezukol by increasing the reaction time to 5 h. Figure [NaN] showed that the activity against α‐tocopherol was steadily increased for 5 h because of the effective constituents and the highly efficient antioxidant activity. Antioxidants control free radicals by donating their own electrons. Free radicals can be produced from a number of sources such as pollution and overexposure to sunlight, and they are responsible for age‐related disease. Previous studies have reported that terpenoids, which are found in essential oils from woody plants, are effective in the treatment of many diseases [28] , [29] . Through this study, bornyl acetate, nezukol and (‐)‐4‐terpineol, major components of fraction B and C, may protect against free radical damage and be natural protectants against ageing.

The SOD‐like activity of C. japonica essential oils was also examined by comparison with α‐tocopherol. The results of SOD‐like activity are shown in Fig. [NaN] . The activity of α‐tocopherol, the positive control, was 40.93 ± 0.00%. In contrast, 5000 ppm of bornyl acetate and nezukol (fraction B) showed higher effective SOD‐like activity than α‐tocopherol at 91.19 ± 0.85% statistically (P < 0.05). Fraction B was considered as the removal reagent of reactive oxygen (O2·−) similarly as in SOD (2 O2·− + 2 H+ → O2 + H2O2). Also, fraction F (49.82 ± 0.40%) and crude oil (43.24 ± 1.37%) were more effective than α‐tocopherol (40.93 ± 0.00%). On the other hand, fraction A, which had the highest tyrosinase inhibitory activity, and fraction C, which had the highest DPPH radical scavenging activity, showed the lowest SOD‐like activity significantly (P < 0.05). Therefore, bornyl acetate and nezukol may be potential antioxidant material because it also showed effective DPPH radical scavenging activity. Fraction E contained α‐terpineol, a stereoisomer of (‐)‐4‐terpineol, which was not highly effective in any tests. Furthermore, although fraction D contained (‐)‐4‐terpineol, it demonstrated low activity because it also contained a large amount of δ‐cadinene. (‐)‐4‐Terpineol and δ‐cadinene were revealed as an antagonist. In previous studies, α‐terpineol and terpinen‐4‐ol from the essential oils of Laurus nobilis seed have shown high antioxidant activity [30] . However, (‐)‐terpinen‐4‐ol as standard material did not show any significant DPPH radical scavenging activity or SOD‐like activity in our study (data not shown). Therefore, we postulate that the high DPPH radical scavenging activity of fraction C was because of the interaction between terpinen‐4‐ol and other constituents present in small quantity.

Because fraction B demonstrated high SOD‐like activity, and fraction C showed high DPPH radical scavenging activity, bornyl acetate, nezukol and (‐)‐4‐terpineol might process strong antioxidant properties. Bornyl acetate is known as a highly active antimicrobial agent [18] , but its whitening and antioxidant activities are unknown. So, we tested it and showed the compound to have relatively low tyrosinase inhibitory activity (l‐tyrosine: 58.56 ± 1.28%, l‐DOPA: 61.73 ± 0.46%), DPPH radical scavenging activity (1.63 ± 0.12%, after 5 h: 1.74%) and SOD‐like activity (10.72 ± 0.18%). So, activities of fraction B were unable to determine the activity of bornyl acetate. The effectiveness of fraction B on whitening and antioxidant activity might be increased by the synergistic activity between bornyl acetate and nezukol or only the effects of nezukol, even though each compound could not be tested for whitening and antioxidant effects because of their commercial unavailability. In the major components of the active fractions A and B, the bornyl acetate is monoterpene, whereas the nezukol and kaurene are classified into diterpenes. The major component of fraction C and E, which are lower activity fractions than the fraction A and B, consists of a monoterpene, terpineol. Therefore, the reason why fraction A and B showed higher activity than the other fractions was because of the presence of diterpenes. It already reported that diterpenes show higher antioxidant effect than monoterpenes.

Essential oils of C. jaoponica are effective for whitening and antioxidant activity. It was concluded that kaurene, bornyl acetate and nezukol, the major compounds of fractions A and B, are especially effective constituents whitening activity because of their high tyrosinase inhibitory activity. Also, because of high SOD‐like activity of fraction B and high DPPH radical scavenging activity of fraction C, bornyl acetate, nezukol and (‐)‐4‐terpineol, the major constituents of them, might possess strong antioxidant properties. Importantly, fraction B was the most effective on this work. Based on our results, bornyl acetate and nezukol from C. japonica essential oils have great potential applications in skin care materials as they contain highly active compounds for whitening and antioxidant activities.

This study was totally funded by a grant from ‘Forest Science and Technology projects (Project No. S120708L1001704C)’ provided by Korea Forest Service.

Graph: Thin layer chromatography of fractions isolated from essential oils of Cryptomeria japonica. (developing solvent; hexane : ethyl acetate = 8 : 1). Left column of each TLC , CO : phases of crude oil of C. japonica , and right column of each TLC : separation phases of fractions.

Graph: GC / MS spectrum of individual compounds from Cryptomeria japonica essential oils. 1: α‐thujene (1.18%), 2: α‐pinene (9.84%), 3: sabinene (5.53%), 4: β‐myrcene (1.64%), 5: δ‐carene (3.05%), 6: α‐terpinene (3.01%), 7: α‐limonene (2.02%), 8: γ‐terpinene (1.97%), 9: α‐terpinolene (1.06%), 10: (‐)‐4‐terpineol (5.71%), 11: α‐terpineol (13.43%), 12: bornyl acetate (0.63%), 13: thujopsene (0.72%), 14: germacrene D (0.38%), 15: δ‐cadinene (0.67%), 16: elemol (10.93%), 17: γ‐eudesmol (10.61%), 18: α‐eudesmol (12.18%), 19: kaurene (19.36%), 20: nezukol (0.37%).

Graph: DPPH radical scavenging activity of Cryptomeria japonica essential oils and positive control. Positive control: α‐tocopherol. Crude essential oil, six fractions and positive controls were used at a concentration of 5000 ppm. Different small letters in the same column indicate significant difference values at P  <   0.05 (least significance difference test).

Graph: DPPH radical scavenging activity of fraction B from Cryptomeria japonica over 5 h. Positive control: α‐tocopherol.

Graph: Superoxide dismutase‐like activity of Cryptomeria japonica essential oils. Positive control: α‐tocopherol. Crude essential oil, six fractions and positive controls were used at a concentration of 5000 ppm. Different small letters in the same column indicate significant difference values at P  <   0.05 (least significance difference test).