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 [
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 [
The human body is always under oxidative stress on account of reactive oxygen species (ROS) caused by the exposure of oxygen to ultraviolet rays [
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 [
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 [
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 [
Essential oils of C. japonica have been found to have high antifungal, antibacterial and antitermicidal activities [
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
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 [
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 [
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
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 [
Pyrogallol solution (0.2 mL of 7.2 mmol L
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
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
Fraction Compounds Relative proportion (%) Retention time Structure A Kaurene 61.08 33.39 B Bornyl acetate 28.07 15.92 Nezukol 26.89 34.97 C (‐)‐4‐Terpineol 39.35 13.02 D (‐)‐4‐Terpineol 61.29 13.04 δ‐Cadinene 38.71 21.99 E α‐Terpineol 73.73 13.43 F γ‐Eudesmol 34.45 24.72 α‐Eudesmol 33.00 25.28 Elemol 27.30 22.74
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 [
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
50 ppm 100 ppm 500 ppm 1000 ppm 2000 ppm Ascorbic acid 27.75 ± 0.78d 45.51 ± 0.41b 96.81 ± 0.24a 97.20 ± 0.39a 97.10 ± 0.39a Hydroquinone 91.77 ± 0.21a 93.87 ± 0.80a 100 ± 0.00a 93.15 ± 0.95a 100 ± 0.00ab Crude essential oil 6.93 ± 0.82e 4.95 ± 0.65f 21.22 ± 0.34d 68.62 ± 0.31b 88.35 ± 0.79bc Fraction A 8.51 ± 0.12e 21.80 ± 0.06ed 40.52 ± 0.49b 66.01 ± 0.31c 86.76 ± 0.26c Fraction B 36.65 ± 0.88b 27.57 ± 0.35d 42.61 ± 0.51b 84.96 ± 0.19b 87.53 ± 0.35bc Fraction C NDg NDf NDf 2.16 ± 0.45f 47.75 ± 0.84e Fraction D 3.45 ± 0.28 fg 3.06 ± 0.61f 13.66 ± 0.79e 20.94 ± 0.25e 24.54 ± 0.57f Fraction E 32.95 ± 0.87cb 38.57 ± 0.77c 32.42 ± 0.68c 46.05 ± 0.51d 43.01 ± 0.58e Fraction F 29.46 ± 0.51 cd 20.00 ± 0.93e 27.85 ± 0.81c 62.60 ± 0.43c 76.60 ± 0.86d
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
50 ppm 100 ppm 500 ppm 1000 ppm 2000 ppm Ascorbic acid 32.72 ± 0.99a 40.47 ± 0.60a 98.99 ± 0.82a 97.87 ± 0.83a 99.22 ± 0.69a Hydroquinone NDd NDf NDf NDd 49.21 ± 0.93e Crude essential oil 1.55 ± 0.32d 2.92 ± 0.62e 6.06 ± 0.34e 31.71 ± 0.97c 76.77 ± 0.67c Fraction A 5.23 ± 078c 11.38 ± 0.96c 51.46 ± 0.64b 72.92 ± 0.89b 88.45 ± 0.73b Fraction B 12.45 ± 0.29b 17.12 ± 0.58b 24.48 ± 0.12c 57.40 ± 0.93b 85.03 ± 0.63b Fraction E NDd NDf 2.82 ± 0.93e 10.19 ± 0.50d 26.77 ± 0.94f Fraction F 7.43 ± 0.96c 7.18 ± 0.92d 13.21 ± 0.79d 56.01 ± 0.89b 65.12 ± 0.91d
- 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 [
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 (O
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 [
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).
By S. H. Kim; S. Y. Lee; C. Y. Hong; K. S. Gwak; M. J. Park; D. Smith and I. G. Choi